@article{khan_walden_theil_goss_2017, title={Thermodynamic and Kinetic Analyses of Iron Response Element (IRE)-mRNA Binding to Iron Regulatory Protein, IRP1}, volume={7}, ISSN={["2045-2322"]}, DOI={10.1038/s41598-017-09093-5}, abstractNote={Comparison of kinetic and thermodynamic properties of IRP1 (iron regulatory protein1) binding to FRT (ferritin) and ACO2 (aconitase2) IRE-RNAs, with or without Mn2+, revealed differences specific to each IRE-RNA. Conserved among animal mRNAs, IRE-RNA structures are noncoding and bind Fe2+ to regulate biosynthesis rates of the encoded, iron homeostatic proteins. IRP1 protein binds IRE-RNA, inhibiting mRNA activity; Fe2+ decreases IRE-mRNA/IRP1 binding, increasing encoded protein synthesis. Here, we observed heat, 5 °C to 30 °C, increased IRP1 binding to IRE-RNA 4-fold (FRT IRE-RNA) or 3-fold (ACO2 IRE-RNA), which was enthalpy driven and entropy favorable. Mn2+ (50 µM, 25 °C) increased IRE-RNA/IRP1 binding (K d) 12-fold (FRT IRE-RNA) or 6-fold (ACO2 IRE-RNA); enthalpic contributions decreased ~61% (FRT) or ~32% (ACO2), and entropic contributions increased ~39% (FRT) or ~68% (ACO2). IRE-RNA/IRP1 binding changed activation energies: FRT IRE-RNA 47.0 ± 2.5 kJ/mol, ACO2 IRE-RNA 35.0 ± 2.0 kJ/mol. Mn2+ (50 µM) decreased the activation energy of RNA-IRP1 binding for both IRE-RNAs. The observations suggest decreased RNA hydrogen bonding and changed RNA conformation upon IRP1 binding and illustrate how small, conserved, sequence differences among IRE-mRNAs selectively influence thermodynamic and kinetic selectivity of the protein/RNA interactions.}, journal={SCIENTIFIC REPORTS}, author={Khan, Mateen A. and Walden, William E. and Theil, Elizabeth C. and Goss, Dixie J.}, year={2017}, month={Aug} }
@article{bernacchioni_ghini_theil_turano_2016, title={Modulating the permeability of ferritin channels}, volume={6}, ISSN={["2046-2069"]}, DOI={10.1039/c5ra25056k}, abstractNote={Electric field gradients across the C3 and C4 ferritin channels controls the directional Fe2+fluxes towards the catalytic ferroxidase center.}, number={25}, journal={RSC ADVANCES}, author={Bernacchioni, C. and Ghini, V. and Theil, E. C. and Turano, P.}, year={2016}, pages={21219–21227} }
@misc{theil_tosha_beherat_2016, title={Solving Biology's Iron Chemistry Problem with Ferritin Protein Nanocages}, volume={49}, ISSN={["1520-4898"]}, DOI={10.1021/ar500469e}, abstractNote={Ferritins reversibly synthesize iron-oxy(ferrihydrite) biominerals inside large, hollow protein nanocages (10-12 nm, ∼480 000 g/mol); the iron biominerals are metabolic iron concentrates for iron protein biosyntheses. Protein cages of 12- or 24-folded ferritin subunits (4-α-helix polypeptide bundles) self-assemble, experimentally. Ferritin biomineral structures differ among animals and plants or bacteria. The basic ferritin mineral structure is ferrihydrite (Fe2O3·H2O) with either low phosphate in the highly ordered animal ferritin biominerals, Fe/PO4 ∼ 8:1, or Fe/PO4 ∼ 1:1 in the more amorphous ferritin biominerals of plants and bacteria. While different ferritin environments, plant bacterial-like plastid organelles and animal cytoplasm, might explain ferritin biomineral differences, investigation is required. Currently, the physiological significance of plant-specific and animal-specific ferritin iron minerals is unknown. The iron content of ferritin in living tissues ranges from zero in "apoferritin" to as high as ∼4500 iron atoms. Ferritin biomineralization begins with the reaction of Fe(2+) with O2 at ferritin enzyme (Fe(2+)/O oxidoreductase) sites. The product of ferritin enzyme activity, diferric oxy complexes, is also the precursor of ferritin biomineral. Concentrations of Fe(3+) equivalent to 2.0 × 10(-1) M are maintained in ferritin solutions, contrasting with the Fe(3+) Ks ∼ 10(-18) M. Iron ions move into, through, and out of ferritin protein cages in structural subdomains containing conserved amino acids. Cage subdomains include (1) ion channels for Fe(2+) entry/exit, (2) enzyme (oxidoreductase) site for coupling Fe(2+) and O yielding diferric oxy biomineral precursors, and (3) ferric oxy nucleation channels, where diferric oxy products from up to three enzyme sites interact while moving toward the central, biomineral growth cavity (12 nm diameter) where ferric oxy species, now 48-mers, grow in ferric oxy biomineral. High ferritin protein cage symmetry (3-fold and 4-fold axes) and amino acid conservation coincide with function, shown by amino acid substitution effects. 3-Fold symmetry axes control Fe(2+) entry (enzyme catalysis of Fe(2+)/O2 oxidoreduction) and Fe(2+) exit (reductive ferritin mineral dissolution); 3-fold symmetry axes influence Fe(2+)exit from dissolved mineral; bacterial ferritins diverge slightly in Fe/O2 reaction mechanisms and intracage paths of iron-oxy complexes. Biosynthesis rates of ferritin protein change with Fe(2+) and O2 concentrations, dependent on DNA-binding, and heme binding protein, Bach 1. Increased cellular O2 indirectly stabilizes ferritin DNA/Bach 1 interactions. Heme, Fe-protoporphyrin IX, decreases ferritin DNA-Bach 1 binding, causing increased ferritin mRNA biosynthesis (transcription). Direct Fe(2+) binding to ferritin mRNA decreases binding of an inhibitory protein, IRP, causing increased ferritin mRNA translation (protein biosynthesis). Newly synthesized ferritin protein consumes Fe(2+) in biomineral, decreasing Fe(2)(+) and creating a regulatory feedback loop. Ferritin without iron is "apoferritin". Iron removal from ferritin, experimentally, uses biological reductants, for example, NADH + FMN, or chemical reductants, for example, thioglycolic acid, with Fe(2+) chelators; physiological mechanism(s) are murky. Clear, however, is the necessity of ferritin for terrestrial life by conferring oxidant protection (plants, animals, and bacteria), virulence (bacteria), and embryonic survival (mammals). Future studies of ferritin structure/function and Fe(2+)/O2 chemistry will lead to new ferritin uses in medicine, nutrition, and nanochemistry.}, number={5}, journal={ACCOUNTS OF CHEMICAL RESEARCH}, author={Theil, Elizabeth C. and Tosha, Takehiko and Beherat, Rabindra K.}, year={2016}, month={May}, pages={784–791} }
@article{behera_torres_tosha_bradley_goulding_theil_2015, title={Fe2+ substrate transport through ferritin protein cage ion channels influences enzyme activity and biomineralization}, volume={20}, ISSN={["1432-1327"]}, DOI={10.1007/s00775-015-1279-x}, abstractNote={Ferritins, complex protein nanocages, form internal iron-oxy minerals (Fe2O3·H2O), by moving cytoplasmic Fe(2+) through intracage ion channels to cage-embedded enzyme (2Fe(2+)/O2 oxidoreductase) sites where ferritin biomineralization is initiated. The products of ferritin enzyme activity are diferric oxy complexes that are mineral precursors. Conserved, carboxylate amino acid side chains of D127 from each of three cage subunits project into ferritin ion channels near the interior ion channel exits and, thus, could direct Fe(2+) movement to the internal enzyme sites. Ferritin D127E was designed and analyzed to probe properties of ion channel size and carboxylate crowding near the internal ion channel opening. Glu side chains are chemically equivalent to, but longer by one -CH2 than Asp, side chains. Ferritin D127E assembled into normal protein cages, but diferric peroxo formation (enzyme activity) was not observed, when measured at 650 nm (DFP λ max). The caged biomineral formation, measured at 350 nm in the middle of the broad, nonspecific Fe(3+)-O absorption band, was slower. Structural differences (protein X-ray crystallography), between ion channels in wild type and ferritin D127E, which correlate with the inhibition of ferritin D127E enzyme activity include: (1) narrower interior ion channel openings/pores; (2) increased numbers of ion channel protein-metal binding sites, and (3) a change in ion channel electrostatics due to carboxylate crowding. The contributions of ion channel size and structure to ferritin activity reflect metal ion transport in ion channels are precisely regulated both in ferritin protein nanocages and membranes of living cells.}, number={6}, journal={JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY}, author={Behera, Rabindra K. and Torres, Rodrigo and Tosha, Takehiko and Bradley, Justin M. and Goulding, Celia W. and Theil, Elizabeth C.}, year={2015}, month={Sep}, pages={957–969} }
@misc{theil_2015, title={IRE mRNA riboregulators use metabolic iron (Fe2+) to control mRNA activity and iron chemistry in animals}, volume={7}, ISSN={["1756-591X"]}, DOI={10.1039/c4mt00136b}, abstractNote={A family of noncoding RNAs bind Fe2+to change protein synthesis.}, number={1}, journal={METALLOMICS}, author={Theil, Elizabeth C.}, year={2015}, pages={15–24} }
@article{bernacchioni_ciambellotti_theil_turano_2015, title={Is His54 a gating residue for the ferritin ferroxidase site?}, volume={1854}, ISSN={["1878-1454"]}, DOI={10.1016/j.bbapap.2015.02.011}, abstractNote={Ferritin is a ubiquitous iron concentrating nanocage protein that functions through the enzymatic oxidation of ferrous iron and the reversible synthesis of a caged ferric-oxo biomineral. Among vertebrate ferritins, the bullfrog M homopolymer ferritin is a frequent model for analyzing the role of specific amino acids in the enzymatic reaction and translocation of iron species within the protein cage. X-ray crystal structures of ferritin in the presence of metal ions have revealed His54 binding to iron(II) and other divalent cations, with its imidazole ring proposed as "gate" that influences iron movement to/from the active site. To investigate its role, His54 was mutated to Ala. The H54A ferritin variant was expressed and its reactivity studied via UV-vis stopped-flow kinetics. The H54A variant exhibited a 20% increase in the initial reaction rate of formation of ferric products with 2 or 4 Fe²⁺/subunit and higher than 200% with 20 Fe²⁺/subunit. The possible meaning of the increased efficiency of the ferritin reaction induced by this mutation is proposed taking advantage of the comparative sequence analysis of other ferritins. The data here reported are consistent with a role for His54 as a metal ion trap that maintains the correct levels of access of iron to the active site. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.}, number={9}, journal={BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS}, author={Bernacchioni, Caterina and Ciambellotti, Silvia and Theil, Elizabeth C. and Turano, Paola}, year={2015}, month={Sep}, pages={1118–1122} }
@article{schweitzer_zheng_cleland_goodwin_boatman_theil_marcus_fakra_2014, title={A role for iron and oxygen chemistry in preserving soft tissues, cells and molecules from deep time}, volume={281}, number={1775}, journal={Proceedings of the Royal Society of London. Series B}, author={Schweitzer, M. H. and Zheng, W. X. and Cleland, T. P. and Goodwin, M. B. and Boatman, E. and Theil, E. and Marcus, M. A. and Fakra, S. C.}, year={2014} }
@article{theil_turano_ghini_allegrozzi_bernacchioni_2014, title={Coordinating subdomains of ferritin protein cages with catalysis and biomineralization viewed from the C (4) cage axes}, volume={19}, ISSN={["1432-1327"]}, DOI={10.1007/s00775-014-1103-z}, abstractNote={Integrated ferritin protein cage function is the reversible synthesis of protein-caged, solid Fe2O3·H2O minerals from Fe2+ for metabolic iron concentrates and oxidant protection; biomineral order differs in different ferritin proteins. The conserved 432 geometric symmetry of ferritin protein cages parallels the subunit dimer, trimer, and tetramer interfaces, and coincides with function at several cage axes. Multiple subdomains distributed in the self-assembling ferritin nanocages have functional relationships to cage symmetry such as Fe2+ transport though ion channels (threefold symmetry), biomineral nucleation/order (fourfold symmetry), and mineral dissolution (threefold symmetry) studied in ferritin variants. On the basis of the effects of natural or synthetic subunit dimer cross-links, cage subunit dimers (twofold symmetry) influence iron oxidation and mineral dissolution. 2Fe2+/O2 catalysis in ferritin occurs in single subunits, but with cooperativity (n = 3) that is possibly related to the structure/function of the ion channels, which are constructed from segments of three subunits. Here, we study 2Fe2+ + O2 protein catalysis (diferric peroxo formation) and dissolution of ferritin Fe2O3·H2O biominerals in variants with altered subunit interfaces for trimers (ion channels), E130I, and external dimer surfaces (E88A) as controls, and altered tetramer subunit interfaces (L165I and H169F). The results extend observations on the functional importance of structure at ferritin protein twofold and threefold cage axes to show function at ferritin fourfold cage axes. Here, conserved amino acids facilitate dissolution of ferritin-protein-caged iron biominerals. Biological and nanotechnological uses of ferritin protein cage fourfold symmetry and solid-state mineral properties remain largely unexplored.}, number={4-5}, journal={JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY}, author={Theil, Elizabeth C. and Turano, Paola and Ghini, Veronica and Allegrozzi, Marco and Bernacchioni, Caterina}, year={2014}, month={Jun}, pages={615–622} }
@article{bernacchioni_ghini_pozzi_di pisa_theil_turano_2014, title={Loop Electrostatics Modulates the Intersubunit Interactions in Ferritin}, volume={9}, ISSN={["1554-8937"]}, DOI={10.1021/cb500431r}, abstractNote={Functional ferritins are 24-mer nanocages that self-assemble with extended contacts between pairs of 4-helix bundle subunits coupled in an antiparallel fashion along the C2 axes. The largest intersubunit interaction surface in the ferritin nanocage involves helices, but contacts also occur between groups of three residues midway in the long, solvent-exposed L-loops of facing subunits. The anchor points between intersubunit L-loop pairs are the salt bridges between the symmetry-related, conserved residues Asp80 and Lys82. The resulting quaternary structure of the cage is highly soluble and thermostable. Substitution of negatively charged Asp80 with a positively charged Lys in homopolymeric M ferritin introduces electrostatic repulsions that inhibit the oligomerization of the ferritin subunits. D80K ferritin was present in inclusion bodies under standard overexpressing conditions in E. coli, contrasting with the wild type protein. Small amounts of fully functional D80K nanocages formed when expression was slowed. The more positively charged surface results in a different solubility profile and D80K crystallized in a crystal form with a low density packing. The 3D structure of D80K variant is the same as wild type except for the side chain orientations of Lys80 and facing Lys82. When three contiguous Lys groups are introduced in D80KI81K ferritin variant the nanocage assembly is further inhibited leading to lower solubility and reduced thermal stability. Here, we demonstrate that the electrostatic pairing at the center of the L-loops has a specific kinetic role in the self-assembly of ferritin nanocages.}, number={11}, journal={ACS CHEMICAL BIOLOGY}, author={Bernacchioni, Caterina and Ghini, Veronica and Pozzi, Cecilia and Di Pisa, Flavio and Theil, Elizabeth C. and Turano, Paola}, year={2014}, month={Nov}, pages={2517–2525} }
@article{behera_theil_2014, title={Moving Fe2+ from ferritin ion channels to catalytic OH centers depends on conserved protein cage carboxylates}, volume={111}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1318417111}, abstractNote={Ferritin biominerals are protein-caged metabolic iron concentrates used for iron-protein cofactors and oxidant protection (Fe(2+) and O2 sequestration). Fe(2+) passage through ion channels in the protein cages, like membrane ion channels, required for ferritin biomineral synthesis, is followed by Fe(2+) substrate movement to ferritin enzyme (Fox) sites. Fe(2+) and O2 substrates are coupled via a diferric peroxo (DFP) intermediate, λmax 650 nm, which decays to [Fe(3+)-O-Fe(3+)] precursors of caged ferritin biominerals. Structural studies show multiple conformations for conserved, carboxylate residues E136 and E57, which are between ferritin ion channel exits and enzymatic sites, suggesting functional connections. Here we show that E136 and E57 are required for ferritin enzyme activity and thus are functional links between ferritin ion channels and enzymatic sites. DFP formation (Kcat and kcat/Km), DFP decay, and protein-caged hydrated ferric oxide accumulation decreased in ferritin E57A and E136A; saturation required higher Fe(2+) concentrations. Divalent cations (both ion channel and intracage binding) selectively inhibit ferritin enzyme activity (block Fe(2+) access), Mn(2+) << Co(2+) < Cu(2+) < Zn(2+), reflecting metal ion-protein binding stabilities. Fe(2+)-Cys126 binding in ferritin ion channels, observed as Cu(2+)-S-Cys126 charge-transfer bands in ferritin E130D UV-vis spectra and resistance to Cu(2+) inhibition in ferritin C126S, was unpredicted. Identifying E57 and E136 links in Fe(2+) movement from ferritin ion channels to ferritin enzyme sites completes a bucket brigade that moves external Fe(2+) into ferritin enzymatic sites. The results clarify Fe(2+) transport within ferritin and model molecular links between membrane ion channels and cytoplasmic destinations.}, number={22}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Behera, Rabindra K. and Theil, Elizabeth C.}, year={2014}, month={Jun}, pages={7925–7930} }
@article{khan_ma_walden_merrick_theil_goss_2014, title={Rapid kinetics of iron responsive element (IRE) RNA/iron regulatory protein 1 and IRE-RNA/eIF4F complexes respond differently to metal ions}, volume={42}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gku248}, abstractNote={Metal ion binding was previously shown to destabilize IRE-RNA/IRP1 equilibria and enhanced IRE-RNA/eIF4F equilibria. In order to understand the relative importance of kinetics and stability, we now report rapid rates of protein/RNA complex assembly and dissociation for two IRE-RNAs with IRP1, and quantitatively different metal ion response kinetics that coincide with the different iron responses in vivo. kon, for FRT IRE-RNA binding to IRP1 was eight times faster than ACO2 IRE-RNA. Mn2+ decreased kon and increased koff for IRP1 binding to both FRT and ACO2 IRE-RNA, with a larger effect for FRT IRE-RNA. In order to further understand IRE-mRNA regulation in terms of kinetics and stability, eIF4F kinetics with FRT IRE-RNA were determined. kon for eIF4F binding to FRT IRE-RNA in the absence of metal ions was 5-times slower than the IRP1 binding to FRT IRE-RNA. Mn2+ increased the association rate for eIF4F binding to FRT IRE-RNA, so that at 50 µM Mn2+ eIF4F bound more than 3-times faster than IRP1. IRP1/IRE-RNA complex has a much shorter life-time than the eIF4F/IRE-RNA complex, which suggests that both rate of assembly and stability of the complexes are important, and that allows this regulatory system to respond rapidly to change in cellular iron.}, number={10}, journal={NUCLEIC ACIDS RESEARCH}, author={Khan, Mateen A. and Ma, Jia and Walden, William E. and Merrick, William C. and Theil, Elizabeth C. and Goss, Dixie J.}, year={2014}, pages={6567–6577} }
@article{kwak_schwartz_haldar_behera_tosha_theil_solomon_2014, title={Spectroscopic Studies of Single and Double Variants of M Ferritin: Lack of Conversion of a Biferrous Substrate Site into a Cofactor Site for O-2 Activation}, volume={53}, ISSN={["0006-2960"]}, DOI={10.1021/bi4013726}, abstractNote={Ferritin has a binuclear non-heme iron active site that functions to oxidize iron as a substrate for formation of an iron mineral core. Other enzymes of this class have tightly bound diiron cofactor sites that activate O2 to react with substrate. Ferritin has an active site ligand set with 1-His/4-carboxylate/1-Gln rather than the 2-His/4-carboxylate set of the cofactor site. This ligand variation has been thought to make a major contribution to this biferrous substrate rather than cofactor site reactivity. However, the Q137E/D140H double variant of M ferritin, has a ligand set that is equivalent to most of the diiron cofactor sites, yet did not rapidly react with O2 or generate the peroxy intermediate observed in the cofactor sites. Therefore, in this study, a combined spectroscopic methodology of circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD has been applied to evaluate the factors required for the rapid O2 activation observed in cofactor sites. This methodology defines the coordination environment of each iron and the bridging ligation of the biferrous active sites in the double and corresponding single variants of frog M ferritin. Based on spectral changes, the D140H single variant has the new His ligand binding, and the Q137E variant has the new carboxylate forming a μ-1,3 bridge. The spectra for the Q137E/D140H double variant, which has the cofactor ligand set, however, reflects a site that is more coordinately saturated than the cofactor sites in other enzymes including ribonucleotide reductase, indicating the presence of additional water ligation. Correlation of this double variant and the cofactor sites to their O2 reactivities indicates that electrostatic and steric changes in the active site and, in particular, the hydrophobic nature of a cofactor site associated with its second sphere protein environment, make important contributions to the activation of O2 by the binuclear non-heme iron enzymes.}, number={3}, journal={BIOCHEMISTRY}, author={Kwak, Yeonju and Schwartz, Jennifer K. and Haldar, Suranjana and Behera, Rabindra K. and Tosha, Takehiko and Theil, Elizabeth C. and Solomon, Edward I.}, year={2014}, month={Jan}, pages={473–482} }
@article{theil_2013, title={Ferritin: The Protein Nanocage and Iron Biomineral in Health and in Disease}, volume={52}, ISSN={["1520-510X"]}, DOI={10.1021/ic400484n}, abstractNote={At the center of iron and oxidant metabolism is the ferritin superfamily: protein cages with Fe2+ ion channels and two catalytic Fe/O redox centers that initiate the formation of caged Fe2O3·H2O. Ferritin nanominerals, initiated within the protein cage, grow inside the cage cavity (5 or 8 nm in diameter). Ferritins contribute to normal iron flow, maintenance of iron concentrates for iron cofactor syntheses, sequestration of iron from invading pathogens, oxidant protection, oxidative stress recovery, and, in diseases where iron accumulates excessively, iron chelation strategies. In eukaryotic ferritins, biomineral order/crystallinity is influenced by nucleation channels between active sites and the mineral growth cavity. Animal ferritin cages contain, uniquely, mixtures of catalytically active (H) and inactive (L) polypeptide subunits with varied rates of Fe2+/O2 catalysis and mineral crystallinity. The relatively low mineral order in liver ferritin, for example, coincides with a high percentage of L subunits and, thus, a low percentage of catalytic sites and nucleation channels. Low mineral order facilitates rapid iron turnover and the physiological role of liver ferritin as a general iron source for other tissues. Here, current concepts of ferritin structure/function/genetic regulation are discussed and related to possible therapeutic targets such as mini-ferritin/Dps protein active sites (selective pathogen inhibition in infection), nanocage pores (iron chelation in therapeutic hypertransfusion), mRNA noncoding, IRE riboregulator (normalizing the ferritin iron content after therapeutic hypertransfusion), and protein nanovessels to deliver medicinal or sensor cargo.}, number={21}, journal={INORGANIC CHEMISTRY}, author={Theil, Elizabeth C.}, year={2013}, month={Nov}, pages={12223–12233} }
@article{theil_turano_2013, title={METALLOENZYMES Cage redesign explains assembly}, volume={9}, number={3}, journal={Nature Chemical Biology}, author={Theil, E. C. and Turano, P.}, year={2013}, pages={143–144} }
@article{theil_westhof_2011, title={New Dimensions of RNA in Molecular Recognition and Catalysis}, volume={44}, ISSN={["0001-4842"]}, DOI={10.1021/ar200261e}, abstractNote={ADVERTISEMENT RETURN TO ISSUEPREVEditorialNEXTNew Dimensions of RNA in Molecular Recognition and CatalysisElizabeth C. Theil and Eric WesthofView Author Information CHORI (Children’s Hospital Oakland Research Institute), UC-Berkeley, and North Carolina State University IBMC-CNRS and University of StrasbourgCite this: Acc. Chem. Res. 2011, 44, 12, 1255–1256Publication Date (Web):December 20, 2011Publication History Published online20 December 2011Published inissue 20 December 2011https://doi.org/10.1021/ar200261eCopyright © 2011 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views1750Altmetric-Citations2LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (560 KB) Get e-AlertsSUBJECTS:Catalysis,Genetics,Molecular structure,Monomers,Peptides and proteins Get e-Alerts}, number={12}, journal={ACCOUNTS OF CHEMICAL RESEARCH}, author={Theil, Elizabeth C. and Westhof, Eric}, year={2011}, month={Dec}, pages={1255–1256} }
@article{wei_theil_2000, title={Identification and characterization of the iron regulatory element in the ferritin gene of a plant (soybean)}, volume={275}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M910334199}, abstractNote={Iron increases ferritin synthesis, targeting plant DNA and animal mRNA. The ferritin promoter in plants has not been identified, in contrast to the ferritin promoter and mRNA iron-responsive element (IRE) in animals. The soybean leaf, a natural tissue for ferritin expression, and DNA, with promoter deletions and luciferase or glucuronidase reporters, delivered with particle bombardment, were used to show that an 86-base pair fragment (iron regulatory element (FRE)) controlled iron-mediated derepression of the ferritin gene. Mutagenesis with linkers of random sequence detected two subdomains separated by 21 base pairs. FRE has no detectable homology to the animal IRE or to known promoters in DNA and bound a trans-acting factor in leaf cell extracts. FRE/factor binding was abrogated by increased tissue iron, in analogy to mRNA (IRE)/iron regulatory protein in animals. Maximum ferritin derepression was obtained with 50 microm iron citrate (1:10) or 500 microm iron citrate (1:1) but Fe-EDTA was ineffective, although the leaf iron concentration was increased; manganese, zinc, and copper had no effect. The basis for different responses in ferritin expression to different iron complexes, as well as the significance of using DNA but not mRNA as an iron regulatory target in plants, remain unknown.}, number={23}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Wei, JZ and Theil, EC}, year={2000}, month={Jun}, pages={17488–17493} }
@article{ke_sierzputowska-gracz_gdaniec_theil_2000, title={Internal loop/bulge and hairpin loop of the iron-responsive element of ferritin mRNA contribute to maximal iron regulatory protein 2 binding and translational regulation in the iso-iron-responsive element/iso-iron regulatory protein family}, volume={39}, ISSN={["0006-2960"]}, DOI={10.1021/bi9924765}, abstractNote={Iron-responsive elements (IREs), a natural group of mRNA-specific sequences, bind iron regulatory proteins (IRPs) differentially and fold into hairpins [with a hexaloop (HL) CAGUGX] with helical distortions: an internal loop/bulge (IL/B) (UGC/C) or C-bulge. C-bulge iso-IREs bind IRP2 more poorly, as oligomers (n = 28−30), and have a weaker signal response in vivo. Two trans-loop GC base pairs occur in the ferritin IRE (IL/B and HL) but only one in C-bulge iso-IREs (HL); metal ions and protons perturb the IL/B [Gdaniec et al. (1998) Biochemistry 37, 1505−1512]. IRE function (translation) and physical properties (Tm and accessibility to nucleases) are now compared for IL/B and C-bulge IREs and for HL mutants. Conversion of the IL/B into a C-bulge by a single deletion in the IL/B or by substituting the HL CG base pair with UA both derepressed ferritin synthesis 4-fold in rabbit reticulocyte lysates (IRP1 + IRP2), confirming differences in IRP2 binding observed for the oligomers. Since the engineered C-bulge IRE was more helical near the IL/B [Cu(phen)2 resistant] and more stable (Tm increased) and the HL mutant was less helical near the IL/B (ribonuclease T1 sensitive) and less stable (Tm decreased), both CG trans-loop base pairs contribute to maximum IRP2 binding and translational regulation. The 1H NMR spectrum of the Mg−IRE complex revealed, in contrast to the localized IL/B effects of Co(III) hexaammine observed previously, perturbation of the IL/B plus HL and interloop helix. The lower stability and greater helix distortion in the ferritin IL/B-IRE compared to the C-bulge iso-IREs create a combinatorial set of RNA/protein interactions that control protein synthesis rates with a range of signal sensitivities.}, number={20}, journal={BIOCHEMISTRY}, author={Ke, YH and Sierzputowska-Gracz, H and Gdaniec, Z and Theil, EC}, year={2000}, month={May}, pages={6235–6242} }
@article{theil_takagi_small_he_tipton_danger_2000, title={The ferritin iron entry and exit problem}, volume={297}, ISSN={["1873-3255"]}, DOI={10.1016/S0020-1693(99)00375-8}, abstractNote={The entry and exit of Fe(II) ions in ferritin are endpoints in the process which concentrates iron as a solid (hydrated ferric oxide) to be used by living cells. Ferritin is a response to the trillion fold mismatch between the solubility of iron in neutral, aqueous, aerated solutions and the requirements for protein biosynthesis. The supramolecular structure of 24 polypeptides (subunits) joined by non-covalent bonds in a highly symmetrical (4, 3, 2), large (12 nm diameter) protein with a cavity (8 nm diameter) is found in plants, animals and microorganisms. Of the five types of iron sites which can be defined, iron entry (site 1) is likely at the junction of three subunits. A ferroxidase site (site 2), present in H-type ferritins, binds Fe(II) which reacts with dioxygen to form an initial, μ-1,2 diferric peroxo complex. The peroxo complex decays into hydrogen peroxide and the multiple diferric oxo complexes that are mineral precursors (sites 3a, 3b, 3x) in transit across the protein to the cavity; the ferroxidase site is sensitive to both natural and engineered variations in Fe ligands and ‘second shell amino’ acids such as tyrosine 30 and leucine 134. Studies of ferritin with subunits which lack the ferroxidase site (L-type) show that the mineral anchor sites of clustered R-COOH (site 4) are sensitive to RCH3 and RSH replacements. Iron exit (site 5), studied by adding NADH/FMN as a trigger, is at or near the entry site. Iron exit can be enhanced by localized unfolding of the polypeptide at the junction of three subunits, suggesting a regulation-sensitive biological signal for iron exit. The entry and exit of iron to and from the mineral, unique to ferritin has steps which parallel those in channel ion transport, and ion transport to and from biomineral such as tooth and bone. Iron entry involves oxidation at non-heme iron catalytic centers, similar to ribonucleotide reductase and methane monoxygenase, but diverging in the role of iron as a substrate rather than a cofactor. Sorting out the evolutionary and mechanistic relationships of ferritin and other proteins which use metal ions, as well as fully characterizing all five types of the functional iron sites are challenges which will keep biological inorganic chemists occupied for some time to come.}, number={1-2}, journal={INORGANICA CHIMICA ACTA}, author={Theil, EC and Takagi, H and Small, GW and He, L and Tipton, AR and Danger, D}, year={2000}, month={Jan}, pages={242–251} }
@article{ha_shi_small_theil_allewell_1999, title={Crystal structure of bullfrog M ferritin at 2.8 angstrom resolution: analysis of subunit interactions and the binuclear metal center}, volume={4}, ISSN={["1432-1327"]}, DOI={10.1007/s007750050310}, number={3}, journal={JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY}, author={Ha, Y and Shi, DS and Small, GW and Theil, EC and Allewell, NM}, year={1999}, month={Jun}, pages={243–256} }
@article{gdaniec_sierzputowska-gracz_theil_1999, title={Iron regulatory element and internal loop/bulge structure for ferritin mRNA studied by cobalt(III) hexammine binding, molecular modeling, and NMR spectroscopy (vol 37, pg 1505, 1998)}, volume={38}, ISSN={["0006-2960"]}, DOI={10.1021/bi9950746}, abstractNote={ADVERTISEMENT RETURN TO ISSUEPREVAddition/CorrectionORIGINAL ARTICLEThis notice is a correctionIron Regulatory Element and Internal Loop/Bulge Structure for Ferritin mRNA Studied by Cobalt(III) Hexammine Binding, Molecular Modeling, and NMR SpectroscopyZofia Gdaniec, Hanna Sierzputowska-Gracz, and Elizabeth C. TheilCite this: Biochemistry 1999, 38, 17, 5676Publication Date (Web):April 8, 1999Publication History Published online8 April 1999Published inissue 1 April 1999https://doi.org/10.1021/bi9950746Copyright © 1999 American Chemical SocietyRequest reuse permissions This publication is free to access through this site. Learn MoreArticle Views286Altmetric-Citations5LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (5 KB) Get e-Alertsclose Get e-Alerts}, number={17}, journal={BIOCHEMISTRY}, author={Gdaniec, Z and Sierzputowska-Gracz, H and Theil, EC}, year={1999}, month={Apr}, pages={5676–5676} }
@article{pereira_small_krebs_tavares_edmondson_theil_huynh_1998, title={Direct spectroscopic and kinetic evidence for the involvement of a peroxodiferric intermediate during the ferroxidase reaction in fast ferritin mineralization}, volume={37}, ISSN={["0006-2960"]}, DOI={10.1021/bi980847w}, abstractNote={Rapid freeze-quench (RFQ) Mössbauer and stopped-flow absorption spectroscopy were used to monitor the ferritin ferroxidase reaction using recombinant (apo) frog M ferritin; the initial transient ferric species could be trapped by the RFQ method using low iron loading (36 Fe2+/ferritin molecule). Biphasic kinetics of ferroxidation were observed and measured directly by the Mössbauer method; a majority (85%) of the ferrous ions was oxidized at a fast rate of approximately 80 s-1 and the remainder at a much slower rate of approximately 1.7 s-1. In parallel with the fast phase oxidation of the Fe2+ ions, a single transient iron species is formed which exhibits magnetic properties (diamagnetic ground state) and Mössbauer parameters (DeltaEQ = 1.08 +/- 0.03 mm/s and delta = 0.62 +/- 0.02 mm/s) indicative of an antiferromagnetically coupled peroxodiferric complex. The formation and decay rates of this transient diiron species measured by the RFQ Mössbauer method match those of a transient blue species (lambdamax = 650 nm) determined by the stopped-flow absorbance measurement. Thus, the transient colored species is assigned to the same peroxodiferric intermediate. Similar transient colored species have been detected by other investigators in several other fast ferritins (H and M subunit types), such as the human H ferritin and the Escherichia coli ferritin, suggesting a similar mechanism for the ferritin ferroxidase step in all fast ferritins. Peroxodiferric complexes are also formed as early intermediates in the reaction of O2 with the catalytic diiron centers in the hydroxylase component of soluble methane monooxygenase (MMOH) and in the D84E mutant of the R2 subunit of E. coli ribonucleotide reductase. The proposal that a single protein site, with a structure homologous to the diiron centers in MMOH and R2, is involved in the ferritin ferroxidation step is confirmed by the observed kinetics, spectroscopic properties, and purity of the initial peroxodiferric species formed in the frog M ferritin.}, number={28}, journal={BIOCHEMISTRY}, author={Pereira, AS and Small, W and Krebs, C and Tavares, P and Edmondson, DE and Theil, EC and Huynh, BH}, year={1998}, month={Jul}, pages={9871–9876} }
@article{burton_harlow_theil_1998, title={Evidence for reutilization of nodule iron in soybean seed development}, volume={21}, ISSN={["1532-4087"]}, DOI={10.1080/01904169809365453}, abstractNote={Abstract Iron (Fe) is required in plants for the function of the important processes of photosynthesis, respiration, DNA synthesis, and nitrogen (N) fixation. Concentrations of Fe show tissue specific changes during development. In soybean seeds, Fe accumulates through the linear phase of seed development, but the source of seed Fe, whether remobilized from other tissues or taken from the root environment, is not known. Root nodules of legumes have higher concentrations of Fe than other vegetative organs. To examine whether nodules could provide Fe to the seeds, two cultivars (Tokyo and Arksoy), differing in seed ferritin and Fe content were grown in a phytotron and given a single dose of 59Fe‐EDTA early in development [15 days after inoculation with Bradyrhizobium (DAI)]. The 59Fe distribution as well as immunoreactive ferritin were examined throughout development in nodule, leaf, and seed tissue. Leaves, nodules, and seeds accounted for 75 to 87% of the total plant 59Fe throughout the reproductive period with seeds increasing from 0 to 35–46% at maturity. The largest decrease in 59Fe occurred in nodules. If all 59Fe lost from nodules were translocated to seeds, then 40–59% of 59Fe in seeds could have come from nodules for Tokyo or Arksoy, respectively. The remaining seed Fe came from vegetative tissue and from the rhizosphere. Seed 59Fe in Tokyo was 2.5 times that of Arksoy. In both cultivars, 59Fe, soluble Fe, and ferritin concentrations in seed decreased from 39 DAI until maturity, suggesting that dry weight accumulation in seeds proceeds at a faster rate than Fe accumulation. Nodule ferritin remained constant suggesting a role in concentrating Fe for reutilization as nodules begin to senesce and decline in function.}, number={5}, journal={JOURNAL OF PLANT NUTRITION}, author={Burton, JW and Harlow, C and Theil, EC}, year={1998}, pages={913–927} }
@article{gdaniec_sierzputowska-gracz_theil_1998, title={Iron regulatory element and internal loop/bulge structure for ferritin mRNA studied by cobalt(III) hexammine binding, molecular modeling, and NMR spectroscopy}, volume={37}, ISSN={["0006-2960"]}, DOI={10.1021/bi9719814}, abstractNote={The ferritin IRE, a highly conserved (96-99% in vertebrates) mRNA translation regulatory element in animal mRNA, was studied by molecular modeling (using MC-SYM and DOCKING) and by NMR spectroscopy. Cobalt(III) hexammine was used to model hydrated Mg2+. IRE isoforms in other mRNAs regulate mRNA translation or stability; all IREs bind IRPs (iron regulatory proteins). A G.C base pair, conserved in ferritin IREs, spans an internal loop/bulge in the middle of an A-helix and, combined with a dynamic G.U base pair, formed a pocket suitable for Co(III) hexammine binding. On the basis of the effects of Co(III) hexammine on the 1H NMR spectrum and results of automatic docking into the IRE model, the IRE bound Co(III) hexammine at the pocket in the major groove; Mg2+ may bind to the IRE at the same site on the basis of an analogy to Co(III) hexammine and on the Mg2+ inhibition of Cu-(phen)2 cleavage at the site. Distortion of the IRE helix by the internal loop/bulge near a conserved unpaired C required for IRP binding and adjacent to an IRP cross-linking site suggests a role for the pocket in ferritin IRE/IRP interactions.}, number={6}, journal={BIOCHEMISTRY}, author={Gdaniec, Z and Sierzputowska-Gracz, H and Theil, EC}, year={1998}, month={Feb}, pages={1505–1512} }
@article{takagi_shi_ha_allewell_theil_1998, title={Localized unfolding at the junction of three ferritin subunits - A mechanism for iron release?}, volume={273}, ISSN={["0021-9258"]}, DOI={10.1074/jbc.273.30.18685}, abstractNote={How and where iron exits from ferritin for cellular use is unknown. Twenty-four protein subunits create a cavity in ferritin where iron is concentrated >1011-fold as a mineral. Proline substitution for conserved leucine 134 (L134P) allowed normal assembly but increased iron exit rates. X-ray crystallography of H-L134P ferritin revealed localized unfolding at the 3-fold axis, also iron entry sites, consistent with shared use sites for iron exit and entry. The junction of three ferritin subunits appears to be a dynamic aperture with a “shutter” that cytoplasmic factors might open or close to regulate iron release in vivo. How and where iron exits from ferritin for cellular use is unknown. Twenty-four protein subunits create a cavity in ferritin where iron is concentrated >1011-fold as a mineral. Proline substitution for conserved leucine 134 (L134P) allowed normal assembly but increased iron exit rates. X-ray crystallography of H-L134P ferritin revealed localized unfolding at the 3-fold axis, also iron entry sites, consistent with shared use sites for iron exit and entry. The junction of three ferritin subunits appears to be a dynamic aperture with a “shutter” that cytoplasmic factors might open or close to regulate iron release in vivo. Ferritins are vesicle-like assemblies of 24 polypeptide (4-helix bundle) subunits that concentrate iron in cells by directing the formation of a ferric mineral in the hollow protein interior (8 nm diameter) (1Waldo G.S. Theil E.C. Suslick K.S. Comprehensive Supramolecular Chemistry. 5. Pergamon, Oxford1996: 65-89Google Scholar, 2Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar, 3Pereira A.S. Tavares P. Lloyd S.G. Danger D. Edmondson D.E. Theil E.C. Huynh B.H. Biochemistry. 1997; 36: 7917-7927Crossref PubMed Scopus (53) Google Scholar). Effective cellular iron concentrations >1011 times the solubility of the ferric ion are achieved by ferritins, which are found in microorganisms, plants, and animals. The complexity and the sophistication of the genetic regulation of the ferritins, involving both DNA and mRNA (4Theil E.C. Biochem. J. 1994; 304: 1-11Crossref PubMed Scopus (183) Google Scholar, 5Hentze M.W. Kühn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1137) Google Scholar, 6Proudhon D. Wei J. Briat J.F. Theil E.C. J. Mol. Evol. 1996; 42: 325-336Crossref PubMed Scopus (55) Google Scholar, 7Rouault T.A. Klausner R.D. J. Biol. Inorg. Chem. 1996; 1: 494-499Crossref Scopus (40) Google Scholar), emphasize the central role of iron and ferritin in life. Rates of Fe(II) oxidation, translocation of Fe(II) and Fe(III) (1.0–2.0 nm), and mineralization are all controlled by the protein (1Waldo G.S. Theil E.C. Suslick K.S. Comprehensive Supramolecular Chemistry. 5. Pergamon, Oxford1996: 65-89Google Scholar, 2Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar). Fe(II) release from ferritin following reduction of the mineral is slow and poorly understood (8Watt G.D. Frankel R.B. Papaefthymiou G.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3640-3643Crossref PubMed Scopus (152) Google Scholar, 9Watt G.D. Jacobs D. Frankel R.B. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7457-7461Crossref PubMed Scopus (93) Google Scholar) but is important for the biosynthesis of iron-proteins, such as those required in respiration, photosynthesis, nitrogen fixation, and cell division, (1Waldo G.S. Theil E.C. Suslick K.S. Comprehensive Supramolecular Chemistry. 5. Pergamon, Oxford1996: 65-89Google Scholar, 2Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar) and as dietary iron (10Beard J.L. Burton J.W. Theil E.C. J. Nutr. 1996; 126: 154-160Crossref PubMed Scopus (102) Google Scholar). How and where the iron exits from ferritin in vivo is not known. We now show that localized unfolding in the assembled protein, at sites of cooperative subunit interactions, can increase the rate of exit of iron from ferritin. When conserved leucine 134 was replaced by proline (L134P), the protein assembled, oxidized Fe(II), and mineralized Fe(III), but the time for complete dissolution of mineral (480 iron)in vitro was greatly decreased (5 min compared with 150 min for the parent protein). X-ray diffraction studies of crystals of H-L134P ferritin showed a flexible region localized near the termini of two subunit helices (C, D), which form the interfaces of subunit trimers and a channel. The results indicate that iron can exit from ferritin at the trimer subunit junction. A possible mechanism for regulated iron release in vivo could be localized disorder in the assembled protein, enhanced by cytoplasmic changes with effects analogous to the effect of H-L134P. The coding sequence for H ferritin, H-L134P, K82Q, and H-L134P, R86Q were obtained by the mutagenesis of PJD5F12L134P sequence (12Waldo G.S. Ling J. Sanders-Loehr J. Theil E.C. Science. 1993; 259: 796-798Crossref PubMed Scopus (79) Google Scholar) with a ChameleonTM double-stranded, site-directed mutagenesis kit (Stratagene). The oligonucleotides, 5F12P134L (5′-CACCTGTTCCTCCAGGTATTCAGTCTCC-3′), 5F12K82Q (5′-CGCTCTGGTTTCTGGACATCCTGCAG-3′), and 5F12R86Q (5′-CCCCATTCATCCTGCTCTGGTTTCTTGACATCC-3′), were used as the mutagenic primers. The coding sequences for H-L134P, K82Q or H-L134P, R86Q were subcloned into a pET-3a vector (Novagen). The coding sequence for H ferritin was subcloned into a pET-9a vector (Novagen) to enhance expression, which was low in the pET-3a vector (Novagen). All of the recombinant ferritin proteins were expressed and purified as described previously (11Waldo G.S. Theil E.C. Biochemistry. 1993; 32: 13262-13269Crossref PubMed Scopus (90) Google Scholar, 12Waldo G.S. Ling J. Sanders-Loehr J. Theil E.C. Science. 1993; 259: 796-798Crossref PubMed Scopus (79) Google Scholar, 13Fetter J. Cohen J. Danger D. Sanders-Loehr J. Theil E.C. J. Biol. Inorg. Chem. 1997; 2: 652-661Crossref Scopus (44) Google Scholar). The method for iron uptake was described previously (13Fetter J. Cohen J. Danger D. Sanders-Loehr J. Theil E.C. J. Biol. Inorg. Chem. 1997; 2: 652-661Crossref Scopus (44) Google Scholar). In iron release experiments, apoferritins (2.08 μm) were mineralized by the addition of a solution of ferrous sulfate at the iron/protein = 480 in 0.1m MOPS 1The abbreviation used is: MOPS, 4-morpholinepropanesulfonic acid. (pH 7.0) and 0.2 m NaCl, followed by incubation for 2 h at room temperature and then incubation overnight at 4 °C (11Waldo G.S. Theil E.C. Biochemistry. 1993; 32: 13262-13269Crossref PubMed Scopus (90) Google Scholar, 12Waldo G.S. Ling J. Sanders-Loehr J. Theil E.C. Science. 1993; 259: 796-798Crossref PubMed Scopus (79) Google Scholar, 13Fetter J. Cohen J. Danger D. Sanders-Loehr J. Theil E.C. J. Biol. Inorg. Chem. 1997; 2: 652-661Crossref Scopus (44) Google Scholar). Iron release was initiated by the addition of 2.5 mm bipyridyl, 2.5 mm FMN, and 2.5 mm NADH to reconstituted ferritin in 0.1 m MOPS (pH 7.0) and 0.2 m NaCl (14Jones T. Spencer R. Walsh C. Biochemistry. 1978; 17: 4011-4017Crossref PubMed Scopus (159) Google Scholar, 15Mertz J.R. Theil E.C. J. Biol. Chem. 1983; 258: 11719-11726Abstract Full Text PDF PubMed Google Scholar). The amount of iron released from ferritin was monitored at room temperature by the absorbance at 522 nm of the Fe(II)-bipyridyl complex. Crystals of H-L134P, K82Q or H-L134P, R86Q ferritin were obtained by the hanging drop method. The crystallization conditions were optimized, beginning with the sparse-matrix sampling method (16Jancanil J. Kim S.H. J. Appl. Crystallogr. 1991; 24: 409-411Crossref Scopus (2076) Google Scholar), with the best H-L134P, K82Q crystals being obtained by mixing 5 μl of a 10 mg/ml protein solution with an equal volume of 15% 2-methyl-2,4-pentanediol, 0.02m CaCl2, 0.1 m sodium acetate buffer, pH 4.6. The best H-L134P, R86Q crystals were obtained in 25% isopropyl alcohol, 0.1 m sodium cacodylate, 0.2mMgCl2. 2K. Yang and D. Shi, unpublished results. Bipyramid-shaped crystals, ∼0.5 × 0.2 mm, formed within 2 weeks. Diffraction data were collected both with a conventional rotating anode x-ray generator and a Siemens area detector and on beamline X-12C at National Synchrotron Light Source, Brookhaven National Laboratory. The data were processed by XENGEN and DENZO, and refinements were carried out with X-PLOR (17Brünger A.T. X-PLOR Manual Version 3.1. Yale University, New Haven, CT1992Google Scholar). Data statistics and final refinement statistics are listed in TableI. Program O (18Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) was used for model building and fitting to the electron density map.Table IData and refinement statistics for frog H-L134P, K82Q ferritinParametersValueSpace groupF432Cell dimensions (Å)a =b = c = 182.8Resolution limits (Å)40–2.4Number of reflections (unique)142,013 (10,541)R merge (%)8.7〈I〉/ς6.2Solvent content (%)62Number of atoms in the final model1345Number of reflection involved in refinement8382R value (%)20.1R free value (%)24.1r.m.s.d. ideal bond lengthar.m.s.d., root mean square deviation.0.012r.m.s.d. ideal bond angle2.61r.m.s.d. ideal dihedral angle21.18r.m.s.d. ideal improper angle0.90Data processing used XENGEN and DENZO, and refinements used X-PLOR (17Brünger A.T. X-PLOR Manual Version 3.1. Yale University, New Haven, CT1992Google Scholar). Fitting to the electron density map and model building used program O (18Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar).a r.m.s.d., root mean square deviation. Open table in a new tab Data processing used XENGEN and DENZO, and refinements used X-PLOR (17Brünger A.T. X-PLOR Manual Version 3.1. Yale University, New Haven, CT1992Google Scholar). Fitting to the electron density map and model building used program O (18Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). Ferritin subunits fold and assemble spontaneously as iron-free proteins (1Waldo G.S. Theil E.C. Suslick K.S. Comprehensive Supramolecular Chemistry. 5. Pergamon, Oxford1996: 65-89Google Scholar, 2Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar, 19Treffry A. Zhao Z. Quail M.A. Guest J.R. Harrison P.M. Biochemistry. 1995; 34: 15204-15213Crossref PubMed Scopus (92) Google Scholar, 20Theil E.C. Annu. Rev. Biochem. 1987; 56: 289-315Crossref PubMed Scopus (1124) Google Scholar, 21Lawson D.M. Artymiuk P.J. Yewdall S.J. Smith J.M.A. Livingstone J.C. Treffry A. Luzzago A. Levi S. Arosio P. Cesareni G. Thomas C.D. Shaw W.V. Harrison P.M. Nature. 1991; 349: 541-544Crossref PubMed Scopus (669) Google Scholar, 22Trikha J. Waldo G.S. Lewandowski F.A. Ha Y. Theil E.C. Weber P.C. Allewell N.M. Proteins. 1994; 18: 107-118Crossref PubMed Scopus (48) Google Scholar, 23Trikha J. Theil E.C. Allewell N.M. J. Mol. Biol. 1995; 248: 949-967Crossref PubMed Scopus (119) Google Scholar, 24Hempstead P.D. Yewdall S.J. Fernie A.R. Lawson D.M. Artymiuk P.J. Rice D.W. Ford G.C. Harrison P.M. J. Mol. Biol. 1997; 268: 424-428Crossref PubMed Scopus (281) Google Scholar) with buffer in the cavity (22Trikha J. Waldo G.S. Lewandowski F.A. Ha Y. Theil E.C. Weber P.C. Allewell N.M. Proteins. 1994; 18: 107-118Crossref PubMed Scopus (48) Google Scholar,23Trikha J. Theil E.C. Allewell N.M. J. Mol. Biol. 1995; 248: 949-967Crossref PubMed Scopus (119) Google Scholar). 3In animal tissues, ferritins are usually found as mixtures with fast (H/M) and slow (L) ferritin subunits in varying ratios under strict genetic control; H/L ratios differ, for example, in ferritin from blood cells, hepatocytes, and macrophages (1Waldo G.S. Theil E.C. Suslick K.S. Comprehensive Supramolecular Chemistry. 5. Pergamon, Oxford1996: 65-89Google Scholar, 2Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar, 20Theil E.C. Annu. Rev. Biochem. 1987; 56: 289-315Crossref PubMed Scopus (1124) Google Scholar). H, M, L, or mixtures of H, M, and L subunits spontaneously fold and assemble into the 24-mer. In nature, the iron minerals in ferritin range in average size from 800 to 2500 iron/molecule and from microcrystalline to amorphous, particularly when the phosphate content is high (1Waldo G.S. Theil E.C. Suslick K.S. Comprehensive Supramolecular Chemistry. 5. Pergamon, Oxford1996: 65-89Google Scholar, 2Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar). The heterodispersity of mineral size in natural ferritin varies from ∼10 to 4000 iron/molecule and is much greater than in minerals reconstituted to 480 iron/molecule from the iron-free protein (15Mertz J.R. Theil E.C. J. Biol. Chem. 1983; 258: 11719-11726Abstract Full Text PDF PubMed Google Scholar). To study iron release, Fe(II) was added to the recombinant ferritin proteins to form iron minerals of constant average size (480 iron/molecule). The role of ferritin in iron release was compared for proteins that differed over 1000-fold in iron uptake rates and in the mechanism of iron oxidation (fast-ferroxidase sites and nucleation sites present, slow-only nucleation sites present). The initial rates of iron oxidation by proteins with ferroxidase sites were 0.99 ± 0.02 (A 650/s) for H ferritin, 0.081 ± 0.038 (A 550/s) for H-L134P ferritin (13Fetter J. Cohen J. Danger D. Sanders-Loehr J. Theil E.C. J. Biol. Inorg. Chem. 1997; 2: 652-661Crossref Scopus (44) Google Scholar), and 1.55 ± 0.22 (A 650/s) for M ferritin (13Fetter J. Cohen J. Danger D. Sanders-Loehr J. Theil E.C. J. Biol. Inorg. Chem. 1997; 2: 652-661Crossref Scopus (44) Google Scholar). The slower rate of iron uptake/ferroxidation for the H-L134P protein is coupled to a shift in the absorbance maximum of the initial Fe(III) complex from 650 to 550 nm (Fig. 1) and a slower decay (12Waldo G.S. Ling J. Sanders-Loehr J. Theil E.C. Science. 1993; 259: 796-798Crossref PubMed Scopus (79) Google Scholar, 13Fetter J. Cohen J. Danger D. Sanders-Loehr J. Theil E.C. J. Biol. Inorg. Chem. 1997; 2: 652-661Crossref Scopus (44) Google Scholar), but the formation rate is still within the range for fast ferritins and 100-fold faster than L ferritin (13Fetter J. Cohen J. Danger D. Sanders-Loehr J. Theil E.C. J. Biol. Inorg. Chem. 1997; 2: 652-661Crossref Scopus (44) Google Scholar). L ferritin has no specific ferroxidation site, and the initial rate of oxidation is 0.0037 ± 0.0017 (A 350/s) (11Waldo G.S. Theil E.C. Biochemistry. 1993; 32: 13262-13269Crossref PubMed Scopus (90) Google Scholar). Iron exit from the mineralized recombinant ferritins was triggered by reduction of Fe(III) with FMNH2/NADH and trapping the Fe(II) as the Fe(II)-bipyridyl complex. No Fe(II)-bipyridyl complex was detected until the reductant was added, and essentially all (96%) of the Fe(II)-bipyridyl complex that formed could be separated from the protein by ultrafiltration (data not shown). Previous studies have shown that the reductive release of iron is independent of reductant/chelator size (8Watt G.D. Frankel R.B. Papaefthymiou G.C. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3640-3643Crossref PubMed Scopus (152) Google Scholar, 9Watt G.D. Jacobs D. Frankel R.B. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7457-7461Crossref PubMed Scopus (93) Google Scholar). Rates of iron release from recombinant ferritins were biphasic (Fig. 2 A). There was little difference in iron release rates among the recombinant ferritins with natural sequences. In contrast to the wild type proteins (H, M, L), substitution of leucine 134, conserved in all ferritins (1Waldo G.S. Theil E.C. Suslick K.S. Comprehensive Supramolecular Chemistry. 5. Pergamon, Oxford1996: 65-89Google Scholar, 2Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar), with proline had a faster initial rate of iron release (Fig. 2) that was essentially monophasic. Complete dissolution of a ferritin mineral of 480 iron and release as Fe(II)-bipyridyl only required 5 min in the H-L134P protein compared with 150 min for the parent protein with L134. Crystals formed by frog H ferritin with L134P showed localized changes in the subunit packing at the junction of three subunits (Figs. 3 and 4). Because the crystals of H-L134P had a very large and complex unit cell, also observed for frog M ferritin (25Ha Y. Theil E.C. Allewell N.M. Acta Crystallogr. D. 1997; 53: 513-523Crossref PubMed Scopus (9) Google Scholar), glutamine was substituted for lysine at position 82 (K82Q) or arginine at position 86 (R86Q) in the BC loop to allow formation of cubic crystals as previously observed for human H-K82Q ferritin (21Lawson D.M. Artymiuk P.J. Yewdall S.J. Smith J.M.A. Livingstone J.C. Treffry A. Luzzago A. Levi S. Arosio P. Cesareni G. Thomas C.D. Shaw W.V. Harrison P.M. Nature. 1991; 349: 541-544Crossref PubMed Scopus (669) Google Scholar). 4Two numbering systems have been used for ferritin sequences. The initial numbering scheme, based on the horse spleen ferritin sequence (34Banyard S.H. Stammers D.K. Harrison P.M. Nature. 1978; 271: 282-284Crossref PubMed Scopus (201) Google Scholar), can be used for all sequences (1Waldo G.S. Theil E.C. Suslick K.S. Comprehensive Supramolecular Chemistry. 5. Pergamon, Oxford1996: 65-89Google Scholar, 2Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar), but separate numbering for human ferritin sequences has also been used (21Lawson D.M. Artymiuk P.J. Yewdall S.J. Smith J.M.A. Livingstone J.C. Treffry A. Luzzago A. Levi S. Arosio P. Cesareni G. Thomas C.D. Shaw W.V. Harrison P.M. Nature. 1991; 349: 541-544Crossref PubMed Scopus (669) Google Scholar, 24Hempstead P.D. Yewdall S.J. Fernie A.R. Lawson D.M. Artymiuk P.J. Rice D.W. Ford G.C. Harrison P.M. J. Mol. Biol. 1997; 268: 424-428Crossref PubMed Scopus (281) Google Scholar). The uniform numbering system is used here; thus 4 must be added from residue numbering for the human H ferritin sequence to relate to the H/L equivalent numbering system. This crystal form of frog H-L134P, K82Q was isomorphous to human H-K82Q (21Lawson D.M. Artymiuk P.J. Yewdall S.J. Smith J.M.A. Livingstone J.C. Treffry A. Luzzago A. Levi S. Arosio P. Cesareni G. Thomas C.D. Shaw W.V. Harrison P.M. Nature. 1991; 349: 541-544Crossref PubMed Scopus (669) Google Scholar) and frog L ferritin crystals, making the structure solution straightforward. The polyalanine model of human H-K82Q ferritin (Protein Data Bank reference 1fha) was used as a starting model. The electron density maps for both frog H-L134P, K82Q and H-L134P, R86Q ferritins are of high quality except at the ends of helices C and D and CD loop, residues 114–133. Despite thorough efforts to model this region using different conformers from the Protein Data Bank, the weak density remained throughout refinement, in contrast to all other high resolution ferritin structures determined to date (21Lawson D.M. Artymiuk P.J. Yewdall S.J. Smith J.M.A. Livingstone J.C. Treffry A. Luzzago A. Levi S. Arosio P. Cesareni G. Thomas C.D. Shaw W.V. Harrison P.M. Nature. 1991; 349: 541-544Crossref PubMed Scopus (669) Google Scholar, 22Trikha J. Waldo G.S. Lewandowski F.A. Ha Y. Theil E.C. Weber P.C. Allewell N.M. Proteins. 1994; 18: 107-118Crossref PubMed Scopus (48) Google Scholar, 23Trikha J. Theil E.C. Allewell N.M. J. Mol. Biol. 1995; 248: 949-967Crossref PubMed Scopus (119) Google Scholar, 24Hempstead P.D. Yewdall S.J. Fernie A.R. Lawson D.M. Artymiuk P.J. Rice D.W. Ford G.C. Harrison P.M. J. Mol. Biol. 1997; 268: 424-428Crossref PubMed Scopus (281) Google Scholar). 1.8-Å resolution synchrotron data collected at Brookhaven National Laboratory showed the same weak density region, and as expected for a truly disordered region, the better the data quality, the weaker the density in this region. The data from this region were excluded from the refinement.Figure 42Fo-Fc map from residue 108 to 138 in crystals of recombinant frog ferritin. The density was contoured at 1ς level, and the map was produced by O (18Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). Left, electron density in the disordered region of H-L134P, K82Q;right, electron density in the same region of L ferritin (22Trikha J. Waldo G.S. Lewandowski F.A. Ha Y. Theil E.C. Weber P.C. Allewell N.M. Proteins. 1994; 18: 107-118Crossref PubMed Scopus (48) Google Scholar), which is representative of ferritins studied (e.g.Refs. 21Lawson D.M. Artymiuk P.J. Yewdall S.J. Smith J.M.A. Livingstone J.C. Treffry A. Luzzago A. Levi S. Arosio P. Cesareni G. Thomas C.D. Shaw W.V. Harrison P.M. Nature. 1991; 349: 541-544Crossref PubMed Scopus (669) Google Scholar, 22Trikha J. Waldo G.S. Lewandowski F.A. Ha Y. Theil E.C. Weber P.C. Allewell N.M. Proteins. 1994; 18: 107-118Crossref PubMed Scopus (48) Google Scholar, 23Trikha J. Theil E.C. Allewell N.M. J. Mol. Biol. 1995; 248: 949-967Crossref PubMed Scopus (119) Google Scholar, 24Hempstead P.D. Yewdall S.J. Fernie A.R. Lawson D.M. Artymiuk P.J. Rice D.W. Ford G.C. Harrison P.M. J. Mol. Biol. 1997; 268: 424-428Crossref PubMed Scopus (281) Google Scholar).View Large Image Figure ViewerDownload (PPT) The sites in the ferritin structure for reductive iron release are different from the rapid oxidation sites: iron release rates differed little in fast (H, M) and slow (L) ferritins with natural sequences (Fig. 2), but oxidation rates varied over a wide range (11Waldo G.S. Theil E.C. Biochemistry. 1993; 32: 13262-13269Crossref PubMed Scopus (90) Google Scholar, 12Waldo G.S. Ling J. Sanders-Loehr J. Theil E.C. Science. 1993; 259: 796-798Crossref PubMed Scopus (79) Google Scholar, 13Fetter J. Cohen J. Danger D. Sanders-Loehr J. Theil E.C. J. Biol. Inorg. Chem. 1997; 2: 652-661Crossref Scopus (44) Google Scholar). Rapid oxidation occurs in the center of the 4-helix bundle that forms the ferritin subunit and involves residues from helices A, B, and C (1Waldo G.S. Theil E.C. Suslick K.S. Comprehensive Supramolecular Chemistry. 5. Pergamon, Oxford1996: 65-89Google Scholar, 2Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar,21Lawson D.M. Artymiuk P.J. Yewdall S.J. Smith J.M.A. Livingstone J.C. Treffry A. Luzzago A. Levi S. Arosio P. Cesareni G. Thomas C.D. Shaw W.V. Harrison P.M. Nature. 1991; 349: 541-544Crossref PubMed Scopus (669) Google Scholar, 24Hempstead P.D. Yewdall S.J. Fernie A.R. Lawson D.M. Artymiuk P.J. Rice D.W. Ford G.C. Harrison P.M. J. Mol. Biol. 1997; 268: 424-428Crossref PubMed Scopus (281) Google Scholar). In contrast residue 134, which when changed to proline altered rates of iron exit, is near the N terminus of helix D (21Lawson D.M. Artymiuk P.J. Yewdall S.J. Smith J.M.A. Livingstone J.C. Treffry A. Luzzago A. Levi S. Arosio P. Cesareni G. Thomas C.D. Shaw W.V. Harrison P.M. Nature. 1991; 349: 541-544Crossref PubMed Scopus (669) Google Scholar, 22Trikha J. Waldo G.S. Lewandowski F.A. Ha Y. Theil E.C. Weber P.C. Allewell N.M. Proteins. 1994; 18: 107-118Crossref PubMed Scopus (48) Google Scholar, 23Trikha J. Theil E.C. Allewell N.M. J. Mol. Biol. 1995; 248: 949-967Crossref PubMed Scopus (119) Google Scholar, 24Hempstead P.D. Yewdall S.J. Fernie A.R. Lawson D.M. Artymiuk P.J. Rice D.W. Ford G.C. Harrison P.M. J. Mol. Biol. 1997; 268: 424-428Crossref PubMed Scopus (281) Google Scholar). The backbone nitrogen of Leu-134 is hydrogen-bonded to the carbonyl oxygen of Leu-129, whereas the side chain of Leu-134 has a hydrophobic interaction with Leu-110 in helix C. A kink in the backbone of the helix D adjacent to residue 134, produced by deviations from the standard dihedral angles at positions 132 and 133 (24Hempstead P.D. Yewdall S.J. Fernie A.R. Lawson D.M. Artymiuk P.J. Rice D.W. Ford G.C. Harrison P.M. J. Mol. Biol. 1997; 268: 424-428Crossref PubMed Scopus (281) Google Scholar), determines precise positioning of the interhelical and intersubunit interactions of the D helix near the subunit trimer interface. The L134P protein will have changed an intrasubunit hydrophobic interaction, hydrogen bonds, and intersubunit interactions. H-L134P ferritin assembled from 24 subunits will have eight regions of local flexibility distributed symmetrically around the surface of the molecule caused by the changes at each junction of three subunits (Fig. 3). In other proteins, introduction of proline into a peptide can be without functional effect (26Sauer U.H. San D.P. Matthews B.W. J. Biol. Chem. 1992; 267: 2393-2399Abstract Full Text PDF PubMed Google Scholar), the proteins adjusting conformation to accommodate the change (26Sauer U.H. San D.P. Matthews B.W. J. Biol. Chem. 1992; 267: 2393-2399Abstract Full Text PDF PubMed Google Scholar, 27Kim P.S. Baldwin R.L. Annu. Rev. Biochem. 1990; 59: 631-660Crossref PubMed Scopus (1203) Google Scholar, 28Dill K.A. Shortle D. Annu. Rev. Biochem. 1991; 60: 795-825Crossref PubMed Scopus (911) Google Scholar). However, in ferritin, the effect of the proline substitution is amplified by disrupting both cooperative interhelical and intersubunit interactions (Fig. 3). The unfolded structure at the 3-fold axes can alter the behavior of assembled ferritin H-L134P in solution. For example, the flexibility of the structure will propagate to the channels for iron entry; the mutation led to a shift in the absorbance maximum of the initial Fe(III) complex (Fig. 1), a decreased rate of oxidation (13Fetter J. Cohen J. Danger D. Sanders-Loehr J. Theil E.C. J. Biol. Inorg. Chem. 1997; 2: 652-661Crossref Scopus (44) Google Scholar), and a decreased decay rate of the initial Fe(III) complex that fortuitously permitted its identification as Fe(III)-tyrosine (12Waldo G.S. Ling J. Sanders-Loehr J. Theil E.C. Science. 1993; 259: 796-798Crossref PubMed Scopus (79) Google Scholar). Whether Fe(III)-tyrosine is specific to H-L134P ferritin protein or simply more readily detected in H-L134P protein because of the slower decay is not yet clear (see Ref. 3Pereira A.S. Tavares P. Lloyd S.G. Danger D. Edmondson D.E. Theil E.C. Huynh B.H. Biochemistry. 1997; 36: 7917-7927Crossref PubMed Scopus (53) Google Scholar). 5A. S. Pereira, E. C. Theil, and B. H. Huynh, unpublished results. Disorder at the 3-fold axis of the assembled H-L134P protein appeared to increase the accessibility of the mineral core to solutes such as reductants and chelators (Fig. 2). The structure of ferritin H-L134P contrasts with the high degree of order in the same region of crystals of recombinant H or L ferritins from frog, horse, and human wild type (Fig. 4) (21Lawson D.M. Artymiuk P.J. Yewdall S.J. Smith J.M.A. Livingstone J.C. Treffry A. Luzzago A. Levi S. Arosio P. Cesareni G. Thomas C.D. Shaw W.V. Harrison P.M. Nature. 1991; 349: 541-544Crossref PubMed Scopus (669) Google Scholar, 22Trikha J. Waldo G.S. Lewandowski F.A. Ha Y. Theil E.C. Weber P.C. Allewell N.M. Proteins. 1994; 18: 107-118Crossref PubMed Scopus (48) Google Scholar, 23Trikha J. Theil E.C. Allewell N.M. J. Mol. Biol. 1995; 248: 949-967Crossref PubMed Scopus (119) Google Scholar, 24Hempstead P.D. Yewdall S.J. Fernie A.R. Lawson D.M. Artymiuk P.J. Rice D.W. Ford G.C. Harrison P.M. J. Mol. Biol. 1997; 268: 424-428Crossref PubMed Scopus (281) Google Scholar) or with amino acid substitutions in the A or B helices (22Trikha J. Waldo G.S. Lewandowski F.A. Ha Y. Theil E.C. Weber P.C. Allewell N.M. Proteins. 1994; 18: 107-118Crossref PubMed Scopus (48) Google Scholar, 23Trikha J. Theil E.C. Allewell N.M. J. Mol. Biol. 1995; 248: 949-967Crossref PubMed Scopus (119) Google Scholar). Conserved residues with carboxylate side chains contributed from three subunits line the channel at the 3-fold axis (1Waldo G.S. Theil E.C. Suslick K.S. Comprehensive Supramolecular Chemistry. 5. Pergamon, Oxford1996: 65-89Google Scholar,3Pereira A.S. Tavares P. Lloyd S.G. Danger D. Edmondson D.E. Theil E.C. Huynh B.H. Biochemistry. 1997; 36: 7917-7927Crossref PubMed Scopus (53) Google Scholar, 21Lawson D.M. Artymiuk P.J. Yewdall S.J. Smith J.M.A. Livingstone J.C. Treffry A. Luzzago A. Levi S. Arosio P. Cesareni G. Thomas C.D. Shaw W.V. Harrison P.M. Nature. 1991; 349: 541-544Crossref PubMed Scopus (669) Google Scholar, 22Trikha J. Waldo G.S. Lewandowski F.A. Ha Y. Theil E.C. Weber P.C. Allewell N.M. Proteins. 1994; 18: 107-118Crossref PubMed Scopus (48) Google Scholar, 23Trikha J. Theil E.C. Allewell N.M. J. Mol. Biol. 1995; 248: 949-967Crossref PubMed Scopus (119) Google Scholar, 24Hempstead P.D. Yewdall S.J. Fernie A.R. Lawson D.M. Artymiuk P.J. Rice D.W. Ford G.C. Harrison P.M. J. Mol. Biol. 1997; 268: 424-428Crossref PubMed Scopus (281) Google Scholar, 34Banyard S.H. Stammers D.K. Harrison P.M. Nature. 1978; 271: 282-284Crossref PubMed Scopus (201) Google Scholar), which is the site for both iron entry (1Waldo G.S. Theil E.C. Suslick K.S. Comprehensive Supramolecular Chemistry. 5. Pergamon, Oxford1996: 65-89Google Scholar, 2Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar, 29Bauminger E.R. Harrison P.M. Hechel D. Hodson N.W. Nowik I. Treffry A. Yewdall S.J. 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Subunit interactions in ferritin occur between dimers and tetramers, as well as trimers (21Lawson D.M. Artymiuk P.J. Yewdall S.J. Smith J.M.A. Livingstone J.C. Treffry A. Luzzago A. Levi S. Arosio P. Cesareni G. Thomas C.D. Shaw W.V. Harrison P.M. Nature. 1991; 349: 541-544Crossref PubMed Scopus (669) Google Scholar, 22Trikha J. Waldo G.S. Lewandowski F.A. Ha Y. Theil E.C. Weber P.C. Allewell N.M. Proteins. 1994; 18: 107-118Crossref PubMed Scopus (48) Google Scholar, 23Trikha J. Theil E.C. Allewell N.M. J. Mol. Biol. 1995; 248: 949-967Crossref PubMed Scopus (119) Google Scholar, 24Hempstead P.D. Yewdall S.J. Fernie A.R. Lawson D.M. Artymiuk P.J. Rice D.W. Ford G.C. Harrison P.M. J. Mol. Biol. 1997; 268: 424-428Crossref PubMed Scopus (281) Google Scholar, 25Ha Y. Theil E.C. Allewell N.M. Acta Crystallogr. D. 1997; 53: 513-523Crossref PubMed Scopus (9) Google Scholar, 34Banyard S.H. Stammers D.K. Harrison P.M. Nature. 1978; 271: 282-284Crossref PubMed Scopus (201) Google Scholar). The assembly of H-L134P ferritin subunits into the typical supramolecular ferritin structure, except at the subunit trimer junctions, emphasizes the contributions of the subunit dimer and tetramer interactions to the structure (1Waldo G.S. Theil E.C. Suslick K.S. Comprehensive Supramolecular Chemistry. 5. Pergamon, Oxford1996: 65-89Google Scholar, 2Harrison P.M. Arosio P. Biochim. Biophys. Acta. 1996; 1275: 161-203Crossref PubMed Scopus (2278) Google Scholar, 36Santambrogio P. Pinto P. Levi S. Cozzi A. Rovida E. Albertini A. Artymiuk P. Harrison P.M. Arosio P. Biochem. J. 1997; 322: 461-468Crossref PubMed Scopus (46) Google Scholar); subunit trimer interactions contribute more to entry and exit of iron. Localized flexibility of ferritin at the subunit trimer junctions, caused by substitution of proline for conserved leucine 134, acts like a camera shutter increasing the aperture. Regulated iron releasein vivo could result from cytoplasmic molecules causing similar conformational changes in ferritin. We are grateful to Luming He and Jennifer Edwards for cloning the frog H, H-L134P, K82Q, and H-L134P, R86Q ferritin sequences and to Dr. G. W. Small for the initial rate measurement on L ferritin. We also thank Dr. Robert Sweet for assistance during data collection at beamline X12C at the National Synchrotron Light Source at Brookhaven National Laboratory. This facility is supported by the United States Department of Energy Offices of Health and Environmental Research and of Basic Energy Science and by the National Science Foundation.}, number={30}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Takagi, H and Shi, DS and Ha, Y and Allewell, NM and Theil, EC}, year={1998}, month={Jul}, pages={18685–18688} }
@article{ke_wu_leibold_walden_theil_1998, title={Loops and bulge/loops in iron-responsive element isoforms influence iron regulatory protein binding - Fine-tuning of mRNA regulation?}, volume={273}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.273.37.23637}, abstractNote={A family of noncoding mRNA sequences, iron-responsive elements (IREs), coordinately regulate several mRNAs through binding a family of mRNA-specific proteins, iron regulatory proteins (IRPs). IREs are hairpins with a constant terminal loop and base-paired stems interrupted by an internal loop/bulge (in ferritin mRNA) or a C-bulge (in m-aconitase, erythroid aminolevulinate synthase, and transferrin receptor mRNAs). IRP2 binding requires the conserved C-G base pair in the terminal loop, whereas IRP1 binding occurs with the C-G or engineered U-A. Here we show the contribution of the IRE internal loop/bulge to IRP2 binding by comparing natural and engineered IRE variants. Conversion of the internal loop/bulge in the ferritin-IRE to a C-bulge, by deletion of U, decreased IRP2 binding by >95%, whereas IRP1 binding changed only 13%. Moreover, IRP2 binding to natural IREs with the C-bulge was similar to the ΔU6 ferritin-IRE: >90% lower than the ferritin-IRE. The results predict mRNA-specific variation in IRE-dependent regulation in vivo and may relate to previously observed differences in iron-induced ferritin and m-aconitase synthesis in liver and cultured cells. Variations in IRE structure and cellular IRP1/IRP2 ratios can provide a range of finely tuned, mRNA-specific responses to the same (iron) signal. A family of noncoding mRNA sequences, iron-responsive elements (IREs), coordinately regulate several mRNAs through binding a family of mRNA-specific proteins, iron regulatory proteins (IRPs). IREs are hairpins with a constant terminal loop and base-paired stems interrupted by an internal loop/bulge (in ferritin mRNA) or a C-bulge (in m-aconitase, erythroid aminolevulinate synthase, and transferrin receptor mRNAs). IRP2 binding requires the conserved C-G base pair in the terminal loop, whereas IRP1 binding occurs with the C-G or engineered U-A. Here we show the contribution of the IRE internal loop/bulge to IRP2 binding by comparing natural and engineered IRE variants. Conversion of the internal loop/bulge in the ferritin-IRE to a C-bulge, by deletion of U, decreased IRP2 binding by >95%, whereas IRP1 binding changed only 13%. Moreover, IRP2 binding to natural IREs with the C-bulge was similar to the ΔU6 ferritin-IRE: >90% lower than the ferritin-IRE. The results predict mRNA-specific variation in IRE-dependent regulation in vivo and may relate to previously observed differences in iron-induced ferritin and m-aconitase synthesis in liver and cultured cells. Variations in IRE structure and cellular IRP1/IRP2 ratios can provide a range of finely tuned, mRNA-specific responses to the same (iron) signal. iron-responsive element(s) iron regulatory protein(s) mitochondrial aconitase transferrin receptor erythroid aminolevulinate synthase. The iron-responsive element (IRE),1 present in the 5′- or 3′-noncoding regions of animal mRNAs encoding proteins of iron and oxidative metabolism, regulates synthesis of the encoded proteins posttranscriptionally. Iron regulatory proteins (IRPs) bind to the IREs to inhibit ribosome binding or protect mRNA from ribonuclease cleavage (1Theil E.C. Sigel A. Signel H. Metal Ions in Biological Systems. Marcel Dekker, New York1998: 403-434Google Scholar, 2Theil E.C. Metal Ions in Gene Regulation.in: Silver S. Walden W. International Thomson Publishing, New York1997Google Scholar, 3Rouault T.A. Klausner R.D. J. Biol. Inorg. Chem. 1996; 1: 494-499Crossref Scopus (40) Google Scholar, 4Hentze M.W. Kuhn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1141) Google Scholar, 5Leibold E.A. Guo B. Annu. Rev. Nutr. 1992; 12: 345-368Crossref PubMed Scopus (120) Google Scholar). The predicted secondary structures of the IRE family are hairpins with a six-nucleotide terminal loop (CAGUGN*, N* = A, C, or U), interrupted by an internal loop/bulge (UGC/C) (ferritin-IRE) or a C-bulge (TfR, eALAS, and m-aconitase IREs), that is generally supported by enzymatic cleavage and chemical probing (6Wang Y.-H. Sczekan S.R. Theil E.C. Nucleic Acids Res. 1990; 18: 4463-4468Crossref PubMed Scopus (55) Google Scholar, 7Wang Y.-H. Lin P.-N. Sczekan S.R. McKenzie R.A. Theil E.C. Biol. Metals. 1991; 4: 56-61Crossref PubMed Scopus (15) Google Scholar, 8Bettany A.J.E. Eisenstein R.S. Munro H.M. J. Biol. Chem. 1992; 267: 16531-16537Abstract Full Text PDF PubMed Google Scholar); NMR spectroscopy shows a G-C base pair in the hairpin loop and in the internal loop/bulge (9Sierzputowska-Gracz H. McKenzie R.A. Theil E.C. Nucleic Acids Res. 1995; 23: 145-152Crossref Scopus (48) Google Scholar, 10Liang L.G. Hall K.B. Biochemistry. 1996; 35: 13585-13596Google Scholar, 11Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (171) Google Scholar, 12Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (77) Google Scholar). Two IRE-binding proteins, IRP1 and IRP2, have a high sequence identity except for a 73-amino acid insertion unique to IRP2, and each of them has 30% sequence identity to m-aconitase; IRP1 can have aconitase activity (13Samaniego F. Chin J. Iwai K. Rouault T.A. Klausner R.D. J. Biol. Chem. 1994; 269: 30904-30910Abstract Full Text PDF PubMed Google Scholar, 14Iwai K. Klausner R.D. Rouault T.A. EMBO J. 1995; 14: 5350-5357Crossref PubMed Scopus (193) Google Scholar, 15Hentze W.H. Argos P. Nucleic Acids Res. 1991; 19: 1739-1740Crossref PubMed Scopus (132) Google Scholar, 16Hirling H. Henderson B.R. Kuhn L.C. EMBO J. 1994; 13: 453-461Crossref PubMed Scopus (153) Google Scholar, 17Kennedy M.C. Mende-Mueller L. Blondin G.A. Beinert H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11730-11734Crossref PubMed Scopus (301) Google Scholar). IRP1 and IRP2 binding to IREs in iron-depleted cells is abrogated when iron is in excess, with IRP1 forming an [4Fe-4S] cluster (16Hirling H. Henderson B.R. Kuhn L.C. EMBO J. 1994; 13: 453-461Crossref PubMed Scopus (153) Google Scholar, 17Kennedy M.C. Mende-Mueller L. Blondin G.A. Beinert H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11730-11734Crossref PubMed Scopus (301) Google Scholar, 18Haile D.J. Rouault R.A. Harford J.B. Kennedy M.C. Blondin G.A. Beinert H. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11735-11739Crossref PubMed Scopus (266) Google Scholar, 19Philpott C.C. Klausner R.D. Rouault T.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7321-7325Crossref PubMed Scopus (117) Google Scholar), and IRP2 being degraded (14Iwai K. Klausner R.D. Rouault T.A. EMBO J. 1995; 14: 5350-5357Crossref PubMed Scopus (193) Google Scholar, 20Guo B., Yu, Y. Leibold E.A. J. Biol. Chem. 1994; 269: 24252-24260Abstract Full Text PDF PubMed Google Scholar, 21Guo B. Brown F.M. Phillips J.D., Yu, Y. Leibold E.A. J. Biol. Chem. 1995; 270: 16529-16535Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 22Guo B. Phillips J.D., Yu, Y. Leibold E.A. J. Biol. Chem. 1995; 270: 21645-21651Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). IRP phosphorylation (23Eisenstein R.L. Tuazon P.T. Schalinske K.L. Anderson S.A. Traugh J.A. J. Biol. Chem. 1993; 268: 27363-27370Abstract Full Text PDF PubMed Google Scholar, 24Schalinske K.L. Eisenstein R.S. J. Biol. Chem. 1996; 271: 7168-7175Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), indicates that IRP functions may be integrated with more general metabolic signals. The significance of two IRPs, apparently equivalent in terms of RNA binding and posttranscriptional regulation, is a puzzle, since exclusivity of IRP1 or IRP2 binding for one or another natural IRE sequence has not yet been observed (25Henderson B.R. Menotti E. Bonnard C. Kuhn L.C. J. Biol. Chem. 1994; 269: 17481-17489Abstract Full Text PDF PubMed Google Scholar, 26Henderson B.R. Menotti E. Kuhn L.C. J. Biol. Chem. 1996; 271: 4900-4908Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 27Menotti E. Henderson B.R. Kuhn L.C. J. Biol. Chem. 1998; 273: 1821-1824Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 28Butt J. Kim H.-Y. Basilion J.P. Cohen S. Iwai K. Philpott C.C. Altschul S. Klausner R.D. Rouault T.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4345-4349Crossref PubMed Scopus (129) Google Scholar). IRP binding specificity for the internal loop/bulge and C-bulge of IREs examined in this study, showed that conversion of the ferritin-IRE internal loop/bulge to a C-bulge, by deletion of a single base U6, decreased IRP2 binding 20-fold, with only a small effect on IRP1 binding. Similarly, a C-bulge in the natural IREs (m-aconitase, erythroid ALAS (eALAS), and the transferrin receptor (TfR)), decreased IRP2 binding 10-fold, compared with the ferritin-IRE. Natural IRP1 and IRP2 in a cell extract produced results similar to those observed with recombinant IRPs. The results coincide with structural differences observed by NMR spectroscopy (11Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (171) Google Scholar, 12Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (77) Google Scholar) and Cu(phen)2 probing (6Wang Y.-H. Sczekan S.R. Theil E.C. Nucleic Acids Res. 1990; 18: 4463-4468Crossref PubMed Scopus (55) Google Scholar). 2Y. Ke and E. C. Theil, manuscript in preparation. The differential sensitivity of IRP1 and IRP2 binding to natural variations in IREs at the junction of the two helices (internal loop/bulge or C-bulge) suggests that the presence of two IRPs broadens the regulatory range of IREs and emphasizes the importance of the internal loop/bulge region in RNA-protein interactions. RNA, transcribed using T7 RNA polymerase and a chemically synthesized DNA template (9Sierzputowska-Gracz H. McKenzie R.A. Theil E.C. Nucleic Acids Res. 1995; 23: 145-152Crossref Scopus (48) Google Scholar, 29Milligan J.F. Uhlenbeck U.C. Methods Enzymol. 1989; 180: 51-61Crossref PubMed Scopus (1026) Google Scholar), was purified on 12% polyacrylamide/urea gels; concentrated by ethanol precipitation, resuspended in water and stored at −80 °C until use. 5′-32P labeling of RNA was carried out as described previously (6Wang Y.-H. Sczekan S.R. Theil E.C. Nucleic Acids Res. 1990; 18: 4463-4468Crossref PubMed Scopus (55) Google Scholar, 30Harrell C.M. McKenzie A.R. Patino M.M. Walden W.E. Theil E.C. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1-6Crossref PubMed Scopus (78) Google Scholar), with purification through NENSORBTMcolumns (DuPont). 5′-32P-Labeled RNAs were heated to 85 °C for 5 min in 100 mm KCl, 40 mm Hepes, pH 7.2 and annealed to 25 °C before each use. In competition experiments, unlabeled RNAs were heated and annealed as described for labeled RNA before adding to the binding reaction. If RNA was only heated to 65 °C, the percentage that bound IRP1 was greatly decreased (50–60%). Binding of recombinant IRPs was accomplished by incubation of RNA (0.9 fmol, ∼1.5 × 105 cpm) and protein at 10 °C for 30 min in 20 μl of 60 mm KCl, 24 mm Hepes·Na, pH = 7.2, 4 mm MgCl2, 5% glycerol, 2% 2-mercaptoethanol; protein:RNA was 15:1. Almost all (80–90%) of the ferritin-IRE was bound by IRP1, but only 30–45% of the RNA was bound by IRP2, suggesting that inactive IRP2 was present in preparations of IRP2; 2% 2-mercaptoethanol does not decrease binding by IRP1 or IRP2 (14Iwai K. Klausner R.D. Rouault T.A. EMBO J. 1995; 14: 5350-5357Crossref PubMed Scopus (193) Google Scholar). RNA-protein complexes were separated from RNA in 4% nondenaturing acrylamide gels (acrylamide:bis = 19:1) in Tris borate-EDTA buffer (90 mm Tris borate, 2 mmEDTA, pH 8.0), 8 volts/cm for 1 h at 10 °C. Binding of IRPs in rabbit reticulocyte lysates (∼30 μg/20 μl reaction mixture), prepared as before (31Shull G.E. Theil E.C. J. Biol. Chem. 1982; 257: 14187-14191Abstract Full Text PDF PubMed Google Scholar), used the same binding buffer, but with tRNA (50 μg/ml). The IRP2·IRE complex was identified with anti-IRP2 serum, generated against the 73-amino acid insertion in IRP2 (20Guo B., Yu, Y. Leibold E.A. J. Biol. Chem. 1994; 269: 24252-24260Abstract Full Text PDF PubMed Google Scholar); 2 μl of the serum were added after 20 min of incubation, followed by 10 min incubation, addition of heparin (7.5 mg/ml) (20Guo B., Yu, Y. Leibold E.A. J. Biol. Chem. 1994; 269: 24252-24260Abstract Full Text PDF PubMed Google Scholar, 32Leibold E.A. Munro H.N. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2171-2175Crossref PubMed Scopus (561) Google Scholar, 33Phillips J.D. Guo B., Yu, Y. Brown F.M. Leibold E.A. Biochemistry. 1996; 35: 15704-15714Crossref PubMed Scopus (42) Google Scholar) and electrophoresis in a 5% native acrylamide gel (acrylamide:bis = 19:1), 12 volts/cm at 4 °C. Order of antiserum addition had no significant effect on the results. Recombinant IRP1 was isolated from the cytosol of Saccharomyces cerevisiae BJ5465 (34Jones E.W. Methods Enzymol. 1991; 194: 428-453Crossref PubMed Scopus (367) Google Scholar) containing the rabbit liver IRP1 sequence (35Patino M.M. Walden W.E. J. Biol. Chem. 1992; 267: 19011-19016Abstract Full Text PDF PubMed Google Scholar) in plasmid pYES-His (Invitrogen, Inc.), grown in minimal medium without uracil, and with 3% glycerol, 2% galactose (36Rose M.D. Winston F.M. Heiter P. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990: 179-186Google Scholar). The IRP1 was purified as His-tagged protein with a nickel-chelate column (Amersham Pharmacia Biotech), followed by heparin-agarose chromatography in 20 mm Tris-Cl, pH 7.4, 50 mm KCl, 1 mm EDTA, 2 mm sodium citrate, 10% glycerol, 7 mm mercaptoethanol, and stored at −80 °C. Recombinant IRP2-His, which appears to be less stable than IRP1 (22Guo B. Phillips J.D., Yu, Y. Leibold E.A. J. Biol. Chem. 1995; 270: 21645-21651Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 37Iwai K. Drake S. Wehr N.B. Weissman A.M. LaVaute T. Minato N. Klausner R.D. Levine R.L. Rouault T.A. Proc. Natl. Acad. Sci. U. S. A. 1998; VOL: 4924-4928Crossref Scopus (265) Google Scholar), was prepared on nickel-chelate columns as described by Phillips et al. (33Phillips J.D. Guo B., Yu, Y. Brown F.M. Leibold E.A. Biochemistry. 1996; 35: 15704-15714Crossref PubMed Scopus (42) Google Scholar). Previous studies that compared IRP1 and IRP2 binding had shown that differential IRP binding occurred only with mutations in the hairpin loop (25Henderson B.R. Menotti E. Bonnard C. Kuhn L.C. J. Biol. Chem. 1994; 269: 17481-17489Abstract Full Text PDF PubMed Google Scholar, 26Henderson B.R. Menotti E. Kuhn L.C. J. Biol. Chem. 1996; 271: 4900-4908Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 27Menotti E. Henderson B.R. Kuhn L.C. J. Biol. Chem. 1998; 273: 1821-1824Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 28Butt J. Kim H.-Y. Basilion J.P. Cohen S. Iwai K. Philpott C.C. Altschul S. Klausner R.D. Rouault T.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4345-4349Crossref PubMed Scopus (129) Google Scholar), but not in natural IREs (20, 38-40). The hairpin loop is the most conserved part of the IREs; evolutionary divergence occurs in the stem and internal loop regions (1Theil E.C. Sigel A. Signel H. Metal Ions in Biological Systems. Marcel Dekker, New York1998: 403-434Google Scholar, 2Theil E.C. Metal Ions in Gene Regulation.in: Silver S. Walden W. International Thomson Publishing, New York1997Google Scholar, 3Rouault T.A. Klausner R.D. J. Biol. Inorg. Chem. 1996; 1: 494-499Crossref Scopus (40) Google Scholar, 4Hentze M.W. Kuhn L.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8175-8182Crossref PubMed Scopus (1141) Google Scholar, 5Leibold E.A. Guo B. Annu. Rev. Nutr. 1992; 12: 345-368Crossref PubMed Scopus (120) Google Scholar). Recent studies of IREs by NMR and other approaches, which showed significant structural differences in the internal loop/bulge and C-bulge IREs (11Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (171) Google Scholar, 12Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (77) Google Scholar, 41Ke Y. Theil E.C. FASEB J. 1998; 12: A1474Google Scholar), stimulated reexamination of whether IRP1 and IRP2 differentially bind to the internal loop/bulge and C-bulge IREs. To enhance RNA conformational homogeneity, we synthesized RNA of comparable size (28–30 nucleotides), purified the RNA using denaturing gel electrophoresis, heated the purified RNA to 85 °C, and annealed before each use (see “Experimental Procedures”). The influence of the internal loop/bulge characteristic of the ferritin IRE was investigated with recombinant IRPs, by examining the effect of the deletion of U6, which converted the internal loop/bulge into the C-bulge (Fig. 1, a and e). IRP2 recognizes the ferritin-IRE ΔU6 much more poorly than the ferritin-IRE (Fig. 2 Aand Table I) in contrast to IRP1. Mutated ferritin-IRE HL1, HL2, and C8A (Fig. 1, f–h and Fig. 2 A) were controls, to show that results under the conditions used were comparable to those previously observed (25Henderson B.R. Menotti E. Bonnard C. Kuhn L.C. J. Biol. Chem. 1994; 269: 17481-17489Abstract Full Text PDF PubMed Google Scholar, 26Henderson B.R. Menotti E. Kuhn L.C. J. Biol. Chem. 1996; 271: 4900-4908Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 28Butt J. Kim H.-Y. Basilion J.P. Cohen S. Iwai K. Philpott C.C. Altschul S. Klausner R.D. Rouault T.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4345-4349Crossref PubMed Scopus (129) Google Scholar, 42Leibold E.A. Laudano A. Yu Y. Nucleic Acids Res. 1990; 18: 1819-1824Crossref PubMed Scopus (86) Google Scholar).Figure 2Sensitivity of IRP1 and IRP2 binding to the structure of the IRE internal loop/bulge-recombinant proteins.5′-32P-IREs were incubated with or without purified, recombinant IRP1 or IRP2 at a ratio of 1:15, RNA:protein (A). In the competition experiments (B) 5′-32P-labeled ferritin-IRE was mixed with unlabeled RNA, at ratios of 1:10 or 1:50, before adding IRP. The results are from 3–5 experiments with at least two preparations of RNA and protein. Predicted structures of the RNAs are in Fig. 1. A,32P-RNA + IRP1 or IRP2; B, 32P-RNA + unlabeled competitor RNA + IRP2. ⇑ = IRP/RNA complex.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IQuantitation of recombinant IRP binding to the internal loop/bulge or the C bulge structures of natural and mutant IREIRE bindingIRP1 bindingIRP2 bindingMolar excess of unlabeled RNA required to prevent 32P-labeled RNAFer (IL/B)10010010xFer ΔU6 (B)87 ± 5<5>50xTfR (B)102 ± 310 ± 4>50xeALAS (B)100 ± 111 ± 2>50xm-Aconitase (B)80 ± 212 ± 4>50xFer C6A (IL/B)24 ± 7<2NDFer HL1 (IL/B)<1<1NDFer HL2 (IL/B)89 ± 4<5NDRecombinant IRP1 and IRP2 were incubated with 32P-labeled RNA, and the protein/RNA complexes were resolved by electrophoresis (see “Experimental Procedures”). The relative amount of the IRE bound by IRP was quantitated with a PhosphorImager (Molecular Dynamics) and Image Quant software. Data for IRE/IRP complexes were normalized to the Fer IRE·IRP complex. The data are the average of 2–4 experiments using 2 RNA preparations for each IRE; the error is presented as the S.D. Fer = ferritin.ND, = not determined. Open table in a new tab Recombinant IRP1 and IRP2 were incubated with 32P-labeled RNA, and the protein/RNA complexes were resolved by electrophoresis (see “Experimental Procedures”). The relative amount of the IRE bound by IRP was quantitated with a PhosphorImager (Molecular Dynamics) and Image Quant software. Data for IRE/IRP complexes were normalized to the Fer IRE·IRP complex. The data are the average of 2–4 experiments using 2 RNA preparations for each IRE; the error is presented as the S.D. Fer = ferritin. ND, = not determined. Natural IREs all have the same C-G base pair and the pentameric sequence, CAGUG in the terminal hexaloop, but vary in structure at the interhelix junction (internal loop/bulge or C-bulge). Conversion of the ferritin internal loop/bulge to a C-bulge by deletion (ΔU6) differentially altered IRP recognition (Fig. 2 A and Table I). Thus, IRP1 and IRP2 should also have different interactions with the natural C-bulge IREs (m-aconitase, TfR, and eALAS IREs) compared with the natural internal loop/bulge IRE (ferritin-IRE). The results proved our prediction. All natural C-bulge IREs bind IRP2 much more poorly than IRP1 (10–12%) when compared with ferritin-IRE binding (Fig. 2 A and Table I) and are similar to the ferritin-IRE ΔU6. The observation that the internal loop/bulge enhanced IRP2 binding was confirmed by competition experiments with unlabeled RNAs. A 10-fold molar excess of unlabeled ferritin-IRE (internal loop/bulge IRE) prevented binding of the labeled ferritin-IRE to IRP2, whereas >50-fold molar excess of unlabeled TfR, m-aconitase, or eALAS IREs (C-bulge IREs) were required (Fig. 2 B and Table I). To ensure that the differential binding of recombinant IRP2 to IREs with an internal loop/bulge or a C-bulge (Fig. 2 and Table I) was a property of IRPs, and independent of possible differences between natural and recombinant proteins, IRE-protein binding was examined with IRPs in rabbit reticulocyte lysates comparing of the ferritin-IRE to the TfR-IRE. IRPs in such cell extracts can regulate translation of IRE-containing mRNAs (43Dickey L.F. Wang Y.-H. Shull G.E. Wortman I.A. Theil E.C. J. Biol. Chem. 1988; 263: 3071-3074Abstract Full Text PDF PubMed Google Scholar, 44Walden W.E. Daniels-McQueen S. Brown P.H. Gaffield L. Russell D.A. Bielser D. Bailey L.C. Thach R.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9503-9507Crossref PubMed Scopus (81) Google Scholar, 45Dix D.J. Lin P.-N. Kimata Y. Theil E.C. Biochemistry. 1992; 31: 2818-2822Crossref PubMed Scopus (52) Google Scholar, 46Bhasker C.R. Burgiel G. Neupert B. Emery-Goodman A. Kuhn L.C. May B.K. J. Biol. Chem. 1993; 268: 12699-12705Abstract Full Text PDF PubMed Google Scholar). The ferritin-IRE formed two RNA-protein complexes in the red cell extracts, in relatively equal amounts (Fig. 3 A, lane 3), whereas the TfR-IRE formed only one RNA-protein complex (Fig. 3 A, lane 4). The complex in the lower band formed with the ferritin IRE was identified as an IRP2·RNA complex with IRP2 antibody (compare lanes 5, 6, and 3, 4 in Fig. 3 A). The binding of TfR-IRE to IRP1 is 0.52 ± 0.05 that of the ferritin-IRE (Fig. 3 A, lanes 5 and 6, upper bands). (Similar results were obtained with fresh preparations of purified natural IRP1, but with recombinant IRP1, ferritin-IRE and TfR-IRE binding was the same, either because of the His tag used or the absence of posttranslational modifications such as phosphorylation or both.) The binding of TfR-IRE to endogenous IRP2 in red cell extracts was 0.10 ± 0.03 that of the ferritin-IRE (Fig. 3 A), which was comparable with binding to recombinant IRP2 (Fig. 2 A). IRP isoforms IRP1 and IRP2 showed quantitative differences in binding to IREs from different mRNAs (Figs. 2 and 3 and Table I). The ferritin IRE is recognized best by both IRP1 and IRP2, compared with the m-aconitase, TfR, and eALAS IREs. Accordingly, a larger fraction of the ferritin IRE is likely to be complexed with IRPs than other IREs, which explains the observation that IRE-dependent regulation in vivo and in vitro has the greatest range for the ferritin IRE (38Kim H.-Y. LaVaute T. Iwai K. Klausner R.D. Rouault T.A. J. Biol. Chem. 1996; 271: 24226-24230Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 40Dandekar T. Stripecke R. Gray N.K. Goossen B. Constable A. Johansson H.E. Hentze M.W. EMBO J. 1991; 10: 1903-1909Crossref PubMed Scopus (281) Google Scholar,47Casey J.L. Hentze M.W. 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Eisenstein R.S. Proc. Natl. Acad. Sci U. S. A. 1997; 94: 10681-10686Crossref PubMed Scopus (58) Google Scholar). IRP2 is sensitive to engineered changes in the IRE hairpin loop and internal loop/bulge. Because the hairpin loop structure is conserved in all natural IREs, its contribution to IRP2 binding will be constant. However, the variation in structure of natural IREs, with C-bulge or the internal loop/bulge, will differentially influence IRP2 binding to natural IREs (Figs. 2 and 3 and Table I). The ferritin-IRE ΔU6 with the C-bulge was an even poorer competitor for IRP2 binding than natural IRE isoforms with a C-bulge (Fig. 2 B), suggesting context effects even within the group of IREs with a C-bulge. NMR studies suggest more conformational flexibility at the internal loop/bulge than at the C-bulge in IREs (11Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (171) Google Scholar, 12Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (77) Google Scholar). The IRE consensus sequence designed for NMR studies (11Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (171) Google Scholar), contained a C-bulge, ΔU6, and 3 G-C base pairs next to the bulge in the lower stem, creating an analogue of the ΔU6 ferritin-IRE, which likely behaves similarly in IRP2 binding. Note that in addition to the C-bulge or internal loop/bulge, effects of IRE flanking regions have been observed on the predicted structure (m-aconitase IRE) (52Schalinske K.L. Chen O.S. Eisenstein R.S. J. Biol. Chem. 1998; 273: 3740-3746Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and on both solution structure and translation regulation (ferritin-IRE) (53Dix D.J. Lin P.-N. McKenzie A.R. Walden W.E. Theil E.C. J. Mol. Biol. 1993; 231: 230-240Crossref PubMed Scopus (59) Google Scholar). RNA conformational flexibility which is matched to differences in the binding proteins, as recently emphasized for BIV-TAT/tar interactions (54Frankel A.D. Smith C.A. Cell. 1998; 92: 149-151Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), may also explain the differential binding of IRP1 and IRP2 to the IRE isoforms (Figs. 2 and 3 and Table I). For example, the appropriate conformation around the C residue required to bind IRP2 may be more readily achieved by an IRE with an internal loop/bulge, whereas IRP1 may be able to lock onto the C in any IRE. The C residue was disordered in both C-bulge and internal loop/bulge IREs (11Addess K.J. Basilion J.P. Klausner R.D. Rouault T.A. Pardi A. J. Mol. Biol. 1997; 274: 72-83Crossref PubMed Scopus (171) Google Scholar, 12Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (77) Google Scholar), but the internal loop/bulge forms a flexible pocket in the major groove near the conserved C residue (12Gdaniec Z. Sierzputowska-Gracz H. Theil E.C. Biochemistry. 1998; 37: 1505-1512Crossref PubMed Scopus (77) Google Scholar). IRP contacts the RNA surface on the minor groove (55Basilion J.P. Rouault T.A. Massinople C.M. Klausner R.D. Burgess W.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 574-578Crossref PubMed Scopus (110) Google Scholar), and the flexibility of the internal loop/bulge in the major groove may allow an “induced fit” needed for IRP2 binding on the minor groove surfaces. The question of IRE-dependent coordination of ferritin, m-aconitase, or TfR synthesis is raised by the tissue- and cell type-specific distribution of IRP1 and IRP2 and the differential binding of IRP2 to ferritin, m-aconitase, and TfR IREs in vitro (Figs. 2 and 3 and Table I). When IRP2 predominates in cells, IRE-dependent repression may be greater for ferritin than for other mRNAs, which can explain differential iron regulation of ferritin and m-aconitase mRNAs in rat liver and cultured cells (38Kim H.-Y. LaVaute T. Iwai K. Klausner R.D. Rouault T.A. J. Biol. Chem. 1996; 271: 24226-24230Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar,49Chen O.S. Schalinske K.L. Eisenstein R.S. J. Nutr. 1997; 127: 238-248Crossref PubMed Scopus (102) Google Scholar, 52Schalinske K.L. Chen O.S. Eisenstein R.S. J. Biol. Chem. 1998; 273: 3740-3746Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). On the other hand, the apparently equal regulation of TfR and ferritin by iron in B-lymphocyte cells lacking IRP1 (51Schalinske K.L. Blemings K.P. Steffen D.W. Chen O.S. Eisenstein R.S. Proc. Natl. Acad. Sci U. S. A. 1997; 94: 10681-10686Crossref PubMed Scopus (58) Google Scholar) could be attributed to other factors such as multiple IRE copies and/or to alternate structures (56Mullner E.W. Neupert B. Kuhn L.C. Cell. 1989; 58: 373-382Abstract Full Text PDF PubMed Scopus (404) Google Scholar, 57Theil E.C. Biochem. J. 1994; 304: 1-11Crossref PubMed Scopus (183) Google Scholar). Since both IRP2 and IRP1 can be phosphorylated by protein kinase C (24Schalinske K.L. Eisenstein R.S. J. Biol. Chem. 1996; 271: 7168-7175Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), phosphorylation of IRP2 may change IRE binding and could allow coordinate regulation of m-aconitase and TfR mRNAs with ferritin mRNA in IRP2-dominant cell types. Controlled coordination of IRE-dependent regulation through protein kinases would create a regulatory interface between the IRE-dependent regulatory pathways and other metabolic pathways. Differential binding among IRE isoforms, coupled with IRP responses to iron (17Kennedy M.C. Mende-Mueller L. Blondin G.A. Beinert H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11730-11734Crossref PubMed Scopus (301) Google Scholar, 18Haile D.J. Rouault R.A. Harford J.B. Kennedy M.C. Blondin G.A. Beinert H. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11735-11739Crossref PubMed Scopus (266) Google Scholar, 20Guo B., Yu, Y. Leibold E.A. J. Biol. Chem. 1994; 269: 24252-24260Abstract Full Text PDF PubMed Google Scholar), and the potential for regulated phosphorylation and modulation of RNA/protein recognition, indicate the potential for high precision and fine-tuning of IRE-dependent mRNA regulation.}, number={37}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Ke, YH and Wu, JY and Leibold, EA and Walden, WE and Theil, EC}, year={1998}, month={Sep}, pages={23637–23640} }
@misc{theil_1998, title={Methods of identifying transition metal complexes that selectively cleave regulatory elements of mRNA and uses thereof}, volume={5,834,199}, number={1998 Nov. 10}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Theil, E. C.}, year={1998} }
@book{principles of chemistry in biology: a teaching companion_1998, ISBN={0841235066}, publisher={Washington, DC: American Chemical Society; Distributed by Oxford University Press}, year={1998} }
@article{theil_1998, title={The iron responsive element (IRE) family of mRNA regulators: Regulation of iron transport and uptake compared in animals, plants, and microorganisms}, volume={35}, number={1998}, journal={Iron transport and storage in microorganisms, plants and animals}, author={Theil, E. C.}, year={1998}, pages={403–434} }
@article{ha_theil_allewell_1997, title={Preliminary analysis of amphibian red cell M ferritin in a novel tetragonal unit cell}, volume={53}, ISSN={["2059-7983"]}, DOI={10.1107/S0907444997003983}, abstractNote={The ferritins are a multigene family of proteins that concentrate and store iron in all prokaryotic and eukaryotic cells. 24 monomeric subunits which fold as four-helix bundles assemble to form a protein shell with 432 cubic symmetry and an external diameter of approximately 130 A. The iron is stored inside the protein shell as a mineralized core approximately 80 A in diameter. Recombinant amphibian red cell M ferritin crystallizes in approximately 2 M (NH(4))(2)SO(4) at pH 4.6 in a space group that has not been reported previously. Electron microscopy, precession photography, Patterson and Fourier maps of the native protein and a UO(2)(2+) derivative, and simulations were used to determine that the unit-cell dimensions are a = b = 169.6, c = 481.2 A, alpha = beta = gamma = 90 degrees and the space group is P4(1)2(1)2 or P4(3)2(1)2. A preliminary model of the structure was obtained by molecular replacement, with amphibian red cell L ferritin as the model. In contrast to previously determined ferritin crystal structures which have intermolecular contacts at the twofold and threefold molecular axes, M ferritin crystals have a novel intermolecular interaction mediated by interdigitation of the DE loops of two molecules at the fourfold molecular axes.}, journal={ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY}, author={Ha, Y and Theil, EC and Allewell, NM}, year={1997}, month={Sep}, pages={513–523} }
@article{pereira_tavares_lloyd_danger_edmondson_theil_huynh_1997, title={Rapid and parallel formation of Fe3+ multimers, including a trimer, during H-type subunit ferritin mineralization}, volume={36}, ISSN={["0006-2960"]}, DOI={10.1021/bi970348f}, abstractNote={Conversion of Fe ions in solution to the solid phase in ferritin concentrates iron required for cell function. The rate of the Fe phase transition in ferritin is tissue specific and reflects the differential expression of two classes of ferritin subunits (H and L). Early stages of mineralization were probed by rapid freeze-quench Mossbauer, at strong fields (up to 8 T), and EPR spectroscopy in an H-type subunit, recombinant frog ferritin; small numbers of Fe (36 moles/mol of protein) were used to increase Fe3+ in mineral precursor forms. At 25 ms, four Fe3+-oxy species (three Fe dimers and one Fe trimer) were identified. These Fe3+-oxy species were found to form at similar rates and decay subsequently to a distinctive superparamagentic species designated the "young core." The rate of oxidation of Fe2+ (1026 s(-1)) corresponded well to the formation constant for the Fe3+-tyrosinate complex (920 s(-1)) observed previously [Waldo, G. S., & Theil, E. C. (1993) Biochemistry 32, 13261] and, coupled with EPR data, indicates that several or possibly all of the Fe3+-oxy species involve tyrosine. The results, combined with previous Mossbauer studies of Y30F human H-type ferritin which showed decreases in several Fe3+ intermediates and stabilization of Fe2+ [Bauminger, E. R., et al. (1993) Biochem. J. 296, 709], emphasize the involvement of tyrosyl residues in the mineralization of H-type ferritins. The subsequent decay of these multiple Fe3+-oxy species to the superparamagnetic mineral suggests that Fe3+ species in different environments may be translocated as intact units from the protein shell into the ferritin cavity where the conversion to a solid mineral occurs.}, number={25}, journal={BIOCHEMISTRY}, author={Pereira, AS and Tavares, P and Lloyd, SG and Danger, D and Edmondson, DE and Theil, EC and Huynh, BH}, year={1997}, month={Jun}, pages={7917–7927} }
@article{theil_burton_beard_1997, title={Sustainable solution for dietary iron deficiency through plant biotechnology and breeding to increase seed ferritin control}, volume={51}, number={Suppl. 4}, journal={European Journal of Clinical Nutrition}, author={Theil, E. C. and Burton, J. W. and Beard, J. L.}, year={1997}, pages={S28–31} }
@article{fetter_cohen_danger_sanders_theil_1997, title={The influence of conserved tyrosine 30 and tissue-dependent differences in sequence on ferritin function: use of blue and purple Fe(III) species as reporters of ferroxidation}, volume={2}, DOI={10.1007/s007750050180}, abstractNote={Ferritins uniquely direct the vectorial transfer of hydrated Fe(II)/Fe(III) ions to a condensed ferric phase in the central cavity of the soluble protein. Secondary, tertiary and quaternary structure are conserved in ferritin, but only five amino acid residues are conserved among all known ferritins. The sensitivity of ferroxidation rates to small differences in primary sequence between ferritin subunits that are cell-specifically expressed or to the conservative replacement of the conserved tyrosine 30 residue was demonstrated by examining recombinant (frog) H-type (red blood cell predominant) and M-type subunit (liver predominant) proteins which are both fast ferritins; the proteins form two differently colored Fe(III)-protein complexes absorbing at 550 nm or 650 nm, respectively. The complexes are convenient reporters of Fe(III)-protein interaction because they are transient in contrast to the Fe(III)-oxy complexes measured in the past at 310–420 nm, which are stable because of contributions from the mineral itself. The A650-nm species formed 18-fold faster in the M-subunit protein than did the 550-nm species in H-subunit ferritin, even though all the ferroxidase residues are the same; the Vmax was fivefold faster but the Hill coefficents were identical (1.6), suggesting similar mechanisms. In H-subunit ferritin, substitution of phenylalanine for conserved tyrosine 30 (located in the core of the subunit four-helix bundle) slowed ferroxidation tenfold, whereas changing surface tyrosine 25 or tyrosine 28 had no effect. The Fe(III)-tyrosinate was fortunately not changed by the mutation, based on the resonance Raman spectrum, and remained a suitable reporter for Fe(III)-protein interactions. Thus, the A550/650 nm can also report on post-oxidation events such as transport through the protein. The impact of Y30F on rates of formation of Fe(III)-protein complexes in ferritin, combined with Mössbauer spectroscopic studies that showed the parallel formation of multiple Fe(III) postoxidation species (three dinuclear oxy and one trinuclear oxy species) (A. S. Periera et al., Biochemistry 36 : 7917–7927, 1997) and the loss of several of the multimeric Fe(III) post-oxidation species in a Y30F alteration of human recombinant H-ferritin (E. R. Bauminger et al., Biochem J. 296 : 709–719, 1993), indicate that at least one of the pathways for Fe oxidation/transfer in ferritin is through the center of the four-helix bundle and is influenced by structural features dependent on tyrosine 30.}, number={5}, journal={Journal of Biological Inorganic Chemistry}, author={Fetter, J. and Cohen, J. and Danger, D. P. and Sanders, Loehr J. and Theil, E. C.}, year={1997}, pages={652–661} }
@inbook{theil_1997, title={Translational regulation of bioiron}, DOI={10.1007/978-1-4615-5993-1_6}, booktitle={Metal ions in gene regulation}, publisher={New York: Chapman & Hall}, author={Theil, E. C.}, editor={S. Silver and Walden, W.Editors}, year={1997}, pages={131–156} }
@article{theil_1990, title={Regulation of ferritin and transferrin receptor messenger-rnas}, volume={265}, number={9}, journal={Journal of Biological Chemistry}, author={Theil, E. C.}, year={1990}, pages={4771–4774} }
@misc{theil_1987, title={FERRITIN - STRUCTURE, GENE-REGULATION, AND CELLULAR FUNCTION IN ANIMALS, PLANTS, AND MICROORGANISMS}, volume={56}, ISSN={["0066-4154"]}, DOI={10.1146/annurev.biochem.56.1.289}, abstractNote={The Hippo pathway was initially discovered in Drosophila melanogaster as a key regulator of tissue growth. It is an evolutionarily conserved signaling cascade regulating numerous biological processes, including cell growth and fate decision, organ size ...Read More}, journal={ANNUAL REVIEW OF BIOCHEMISTRY}, author={THEIL, EC}, year={1987}, pages={289–315} }