@article{buhrman_enriquez_dillard_baer_truong_grunden_rose_2021, title={Structure, Function, and Thermal Adaptation of the Biotin Carboxylase Domain Dimer from Hydrogenobacter thermophilus 2-Oxoglutarate Carboxylase}, volume={60}, ISSN={["0006-2960"]}, url={https://doi.org/10.1021/acs.biochem.0c00815}, DOI={10.1021/acs.biochem.0c00815}, abstractNote={2-Oxoglutarate carboxylase (OGC), a unique member of the biotin-dependent carboxylase family from the order Aquificales, captures dissolved CO2 via the reductive tricarboxylic acid (rTCA) cycle. Structure and function studies of OGC may facilitate adaptation of the rTCA cycle to increase the level of carbon fixation for biofuel production. Here we compare the biotin carboxylase (BC) domain of Hydrogenobacter thermophilus OGC with the well-studied mesophilic homologues to identify features that may contribute to thermal stability and activity. We report three OGC BC X-ray structures, each bound to bicarbonate, ADP, or ADP-Mg2+, and propose that substrate binding at high temperatures is facilitated by interactions that stabilize the flexible subdomain B in a partially closed conformation. Kinetic measurements with varying ATP and biotin concentrations distinguish two temperature-dependent steps, consistent with biotin's rate-limiting role in organizing the active site. Transition state thermodynamic values derived from the Eyring equation indicate a larger positive ΔH⧧ and a less negative ΔS⧧ compared to those of a previously reported mesophilic homologue. These thermodynamic values are explained by partially rate limiting product release. Phylogenetic analysis of BC domains suggests that OGC diverged prior to Aquificales evolution. The phylogenetic tree identifies mis-annotations of the Aquificales BC sequences, including the Aquifex aeolicus pyruvate carboxylase structure. Notably, our structural data reveal that the OGC BC dimer comprises a "wet" dimerization interface that is dominated by hydrophilic interactions and structural water molecules common to all BC domains and likely facilitates the conformational changes associated with the catalytic cycle. Mutations in the dimerization domain demonstrate that dimerization contributes to thermal stability.}, number={4}, journal={BIOCHEMISTRY}, publisher={American Chemical Society (ACS)}, author={Buhrman, Greg and Enriquez, Paul and Dillard, Lucas and Baer, Hayden and Truong, Vivian and Grunden, Amy M. and Rose, Robert B.}, year={2021}, month={Feb}, pages={324–345} }
 @article{fetics_guterres_kearney_buhrman_ma_nussinov_mattos_2015, title={Allosteric Effects of the Oncogenic RasQ61L Mutant on Raf-RBD}, volume={23}, ISSN={["1878-4186"]}, DOI={10.1016/j.str.2014.12.017}, abstractNote={The Ras/Raf/MEK/ERK signal transduction pathway is a major regulator of cell proliferation activated by Ras-guanosine triphosphate (GTP). The oncogenic mutant RasQ61L is not able to hydrolyze GTP in the presence of Raf and thus is a constitutive activator of this mitogenic pathway. The Ras/Raf interaction is essential for the activation of the Raf kinase domain through a currently unknown mechanism. We present the crystal structures of the Ras-GppNHp/Raf-RBD and RasQ61L-GppNHp/Raf-RBD complexes, which, in combination with MD simulations, reveal differences in allosteric interactions leading from the Ras/Raf interface to the Ras calcium-binding site and to the remote Raf-RBD loop L4. In the presence of Raf, the RasQ61L mutant has a rigid switch II relative to the wild-type and increased flexibility at the interface with switch I, which propagates across Raf-RBD. We show that in addition to local perturbations on Ras, RasQ61L has substantial long-range effects on the Ras allosteric lobe and on Raf-RBD.}, number={3}, journal={STRUCTURE}, author={Fetics, Susan K. and Guterres, Hugo and Kearney, Bradley M. and Buhrman, Greg and Ma, Buyong and Nussinov, Ruth and Mattos, Carla}, year={2015}, month={Mar}, pages={505–516} }
 @article{friedenberg_buhrman_chdid_olby_olivry_guillaumin_o’toole_goggs_kennedy_rose_et al._2015, title={Evaluation of a DLA-79 allele associated with multiple immune-mediated diseases in dogs}, volume={68}, ISSN={0093-7711 1432-1211}, url={http://dx.doi.org/10.1007/s00251-015-0894-6}, DOI={10.1007/s00251-015-0894-6}, abstractNote={Immune-mediated diseases are common and life-threatening disorders in dogs. Many canine immune-mediated diseases have strong breed predispositions and are believed to be inherited. However, the genetic mutations that cause these diseases are mostly unknown. As many immune-mediated diseases in humans share polymorphisms among a common set of genes, we conducted a candidate gene study of 15 of these genes across four immune-mediated diseases (immune-mediated hemolytic anemia, immune-mediated thrombocytopenia, immune-mediated polyarthritis (IMPA), and atopic dermatitis) in 195 affected and 206 unaffected dogs to assess whether causative or predictive polymorphisms might exist in similar genes in dogs. We demonstrate a strong association (Fisher’s exact p = 0.0004 for allelic association, p = 0.0035 for genotypic association) between two polymorphic positions (10 bp apart) in exon 2 of one allele in DLA-79, DLA-79*001:02, and multiple immune-mediated diseases. The frequency of this allele was significantly higher in dogs with immune-mediated disease than in control dogs (0.21 vs. 0.12) and ranged from 0.28 in dogs with IMPA to 0.15 in dogs with atopic dermatitis. This allele has two non-synonymous substitutions (compared with the reference allele, DLA-79*001:01), resulting in F33L and N37D amino acid changes. These mutations occur in the peptide-binding pocket of the protein, and based upon our computational modeling studies, are likely to affect critical interactions with the peptide N-terminus. Further studies are warranted to confirm these findings more broadly and to determine the specific mechanism by which the identified variants alter canine immune system function.}, number={3}, journal={Immunogenetics}, publisher={Springer Science and Business Media LLC}, author={Friedenberg, Steven G. and Buhrman, Greg and Chdid, Lhoucine and Olby, Natasha J. and Olivry, Thierry and Guillaumin, Julien and O’Toole, Theresa and Goggs, Robert and Kennedy, Lorna J. and Rose, Robert B. and et al.}, year={2015}, month={Dec}, pages={205–217} }
 @article{holzapfel_buhrman_mattos_2012, title={Shift in the Equilibrium between On and Off States of the Allosteric Switch in Ras-GppNHp Affected by Small Molecules and Bulk Solvent Composition}, volume={51}, ISSN={["0006-2960"]}, DOI={10.1021/bi300509j}, abstractNote={Ras GTPase cycles between its active GTP-bound form promoted by GEFs and its inactive GDP-bound form promoted by GAPs to affect the control of various cellular functions. It is becoming increasingly apparent that subtle regulation of the GTP-bound active state may occur through promotion of substates mediated by an allosteric switch mechanism that induces a disorder to order transition in switch II upon ligand binding at an allosteric site. We show with high-resolution structures that calcium acetate and either dithioerythritol (DTE) or dithiothreitol (DTT) soaked into H-Ras-GppNHp crystals in the presence of a moderate amount of poly(ethylene glycol) (PEG) can selectively shift the equilibrium to the "on" state, where the active site appears to be poised for catalysis (calcium acetate), or to what we call the "ordered off" state, which is associated with an anticatalytic conformation (DTE or DTT). We also show that the equilibrium is reversible in our crystals and dependent on the nature of the small molecule present. Calcium acetate binding in the allosteric site stabilizes the conformation observed in the H-Ras-GppNHp/NOR1A complex, and PEG, DTE, and DTT stabilize the anticatalytic conformation observed in the complex between the Ras homologue Ran and Importin-β. The small molecules are therefore selecting biologically relevant conformations in the crystal that are sampled by the disordered switch II in the uncomplexed GTP-bound form of H-Ras. In the presence of a large amount of PEG, the ordered off conformation predominates, whereas in solution, in the absence of PEG, switch regions appear to remain disordered in what we call the off state, unable to bind DTE.}, number={31}, journal={BIOCHEMISTRY}, author={Holzapfel, Genevieve and Buhrman, Greg and Mattos, Carla}, year={2012}, month={Aug}, pages={6114–6126} }
 @article{buhrman_kumar_cirit_haugh_mattos_2011, title={Allosteric Modulation of Ras-GTP Is Linked to Signal Transduction through RAF Kinase}, volume={286}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m110.193854}, abstractNote={Ras is a key signal transduction protein in the cell. Mutants of Gly12 and Gln61 impair GTPase activity and are found prominently in cancers. In wild type Ras-GTP, an allosteric switch promotes disorder to order transition in switch II, placing Gln61 in the active site. We show that the “on” and “off” conformations of the allosteric switch can also be attained in RasG12V and RasQ61L. Although both mutants have similarly impaired active sites in the on state, RasQ61L stabilizes an anti-catalytic conformation of switch II in the off state of the allosteric switch when bound to Raf. This translates into more potent activation of the MAPK pathway involving Ras, Raf kinase, MEK, and ERK (Ras/Raf/MEK/ERK) in cells transfected with RasQ61L relative to RasG12V. This differential is not observed in the Raf-independent pathway involving Ras, phosphoinositide 3-kinase (PI3K), and Akt (Ras/PI3K/Akt). Using a combination of structural analysis, hydrolysis rates, and experiments in NIH-3T3 cells, we link the allosteric switch to the control of signaling in the Ras/Raf/MEK/ERK pathway, supporting a GTPase-activating protein-independent model for duration of the Ras-Raf complex.}, number={5}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={Buhrman, Greg and Kumar, V. S. Senthil and Cirit, Murat and Haugh, Jason M. and Mattos, Carla}, year={2011}, month={Feb}, pages={3323–3331} }
 @article{biswas_buhrman_gagnon_mattos_brown_maxwell_2011, title={Comparative Analysis of the 15.5kD Box C/D snoRNP Core Protein in the Primitive Eukaryote Giardia lamblia Reveals Unique Structural and Functional Features}, volume={50}, ISSN={["0006-2960"]}, DOI={10.1021/bi1020474}, abstractNote={Box C/D ribonucleoproteins (RNP) guide the 2'-O-methylation of targeted nucleotides in archaeal and eukaryotic rRNAs. The archaeal L7Ae and eukaryotic 15.5kD box C/D RNP core protein homologues initiate RNP assembly by recognizing kink-turn (K-turn) motifs. The crystal structure of the 15.5kD core protein from the primitive eukaryote Giardia lamblia is described here to a resolution of 1.8 Å. The Giardia 15.5kD protein exhibits the typical α-β-α sandwich fold exhibited by both archaeal L7Ae and eukaryotic 15.5kD proteins. Characteristic of eukaryotic homologues, the Giardia 15.5kD protein binds the K-turn motif but not the variant K-loop motif. The highly conserved residues of loop 9, critical for RNA binding, also exhibit conformations similar to those of the human 15.5kD protein when bound to the K-turn motif. However, comparative sequence analysis indicated a distinct evolutionary position between Archaea and Eukarya. Indeed, assessment of the Giardia 15.5kD protein in denaturing experiments demonstrated an intermediate stability in protein structure when compared with that of the eukaryotic mouse 15.5kD and archaeal Methanocaldococcus jannaschii L7Ae proteins. Most notable was the ability of the Giardia 15.5kD protein to assemble in vitro a catalytically active chimeric box C/D RNP utilizing the archaeal M. jannaschii Nop56/58 and fibrillarin core proteins. In contrast, a catalytically competent chimeric RNP could not be assembled using the mouse 15.5kD protein. Collectively, these analyses suggest that the G. lamblia 15.5kD protein occupies a unique position in the evolution of this box C/D RNP core protein retaining structural and functional features characteristic of both archaeal L7Ae and higher eukaryotic 15.5kD homologues.}, number={14}, journal={BIOCHEMISTRY}, author={Biswas, Shyamasri and Buhrman, Greg and Gagnon, Keith and Mattos, Carla and Brown, Bernard A., II and Maxwell, E. Stuart}, year={2011}, month={Apr}, pages={2907–2918} }
 @article{nash_dallas_reyes_buhrman_ascencio-ibanez_hanley-bowdoin_2011, title={Functional Analysis of a Novel Motif Conserved across Geminivirus Rep Proteins}, volume={85}, ISSN={["1098-5514"]}, DOI={10.1128/jvi.02143-10}, abstractNote={ABSTRACT}, number={3}, journal={JOURNAL OF VIROLOGY}, author={Nash, Tara E. and Dallas, Mary B. and Reyes, Maria Ines and Buhrman, Gregory K. and Ascencio-Ibanez, J. Trinidad and Hanley-Bowdoin, Linda}, year={2011}, month={Feb}, pages={1182–1192} }
 @article{buhrman_holzapfel_fetics_mattos_2010, title={Allosteric modulation of Ras positions Q61 for a direct role in catalysis}, volume={107}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0912226107}, abstractNote={
            Ras and its effector Raf are key mediators of the Ras/Raf/MEK/ERK signal transduction pathway. Mutants of residue Q61 impair the GTPase activity of Ras and are found prominently in human cancers. Yet the mechanism through which Q61 contributes to catalysis has been elusive. It is thought to position the catalytic water molecule for nucleophilic attack on the γ-phosphate of GTP. However, we previously solved the structure of Ras from crystals with symmetry of the space group R32 in which switch II is disordered and found that the catalytic water molecule is present. Here we present a structure of wild-type Ras with calcium acetate from the crystallization mother liquor bound at a site remote from the active site and likely near the membrane. This results in a shift in helix 3/loop 7 and a network of H-bonding interactions that propagates across the molecule, culminating in the ordering of switch II and placement of Q61 in the active site in a previously unobserved conformation. This structure suggests a direct catalytic role for Q61 where it interacts with a water molecule that bridges one of the γ-phosphate oxygen atoms to the hydroxyl group of Y32 to stabilize the transition state of the hydrolysis reaction. We propose that Raf together with the binding of Ca
            2+
            and a negatively charged group mimicked in our structure by the acetate molecule induces the ordering of switch I and switch II to complete the active site of Ras.
          }, number={11}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Buhrman, Greg and Holzapfel, Genevieve and Fetics, Susan and Mattos, Carla}, year={2010}, month={Mar}, pages={4931–4936} }
 @article{buhrman_wink_mattos_2007, title={Transformation efficiency of RasQ61 mutants linked to structural features of the switch regions in the presence of Raf}, volume={15}, ISSN={["1878-4186"]}, DOI={10.1016/j.str.2007.10.011}, abstractNote={<h2>Summary</h2> Transformation efficiencies of Ras mutants at residue 61 range over three orders of magnitude, but the in vitro GTPase activity decreases 10-fold for all mutants. We show that Raf impairs the GTPase activity of RasQ61L, suggesting that the Ras/Raf complex differentially modulates transformation. Our crystal structures show that, in transforming mutants, switch II takes part in a network of hydrophobic interactions burying the nucleotide and precatalytic water molecule. Our results suggest that Y32 and a water molecule bridging it to the γ-phosphate in the wild-type structure play a role in GTP hydrolysis in lieu of the Arg finger in the absence of GAP. The bridging water molecule is absent in the transforming mutants, contributing to the burying of the nucleotide. We propose a mechanism for intrinsic hydrolysis in Raf-bound Ras and elucidate structural features in the Q61 mutants that correlate with their potency to transform cells.}, number={12}, journal={STRUCTURE}, author={Buhrman, Greg and Wink, Glenna and Mattos, Carla}, year={2007}, month={Dec}, pages={1618–1629} }
 @article{buhrman_parker_sohn_rudolph_mattos_2005, title={Structural mechanism of oxidative regulation of the phosphatase Cdc25B via an intramolecular disulfide bond}, volume={44}, ISSN={["0006-2960"]}, DOI={10.1021/bi047449f}, abstractNote={Cdc25B phosphatase, an important regulator of the cell cycle, forms an intramolecular disulfide bond in response to oxidation leading to reversible inactivation of phosphatase activity. We have obtained a crystallographic time course revealing the structural rearrangements that occur in the P-loop as the enzyme goes from its apo state, through the sulfenic (Cys-SO(-)) intermediate, to the stable disulfide. We have also obtained the structures of the irreversibly oxidized sulfinic (Cys-SO(2)(-)) and sulfonic (Cys-SO(3)(-)) Cdc25B. The active site P-loop is found in three conformations. In the apoenzyme, the P-loop is in the active conformation. In the sulfenic intermediate, the P-loop partially obstructs the active site cysteine, poised to undergo the conformational changes that accompany disulfide bond formation. In the disulfide form, the P-loop is closed over the active site cysteine, resulting in an enzyme that is unable to bind substrate. The structural changes that occur in the sulfenic intermediate of Cdc25B are distinctly different from those seen in protein tyrosine phosphatase 1B where a five-membered sulfenyl amide ring is generated as the stable end product. This work elucidates the mechanism by which chemistry and structure are coupled in the regulation of Cdc25B by reactive oxygen species.}, number={14}, journal={BIOCHEMISTRY}, author={Buhrman, G and Parker, B and Sohn, J and Rudolph, J and Mattos, C}, year={2005}, month={Apr}, pages={5307–5316} }
 @article{buhrman_serrano_mattos_2003, title={Organic solvents order the dynamic switch II in Ras crystals}, volume={11}, ISSN={["1878-4186"]}, DOI={10.1016/s0969-2126(03)00128-x}, abstractNote={<h2>Abstract</h2> Room temperature crystal structures of crosslinked H-Ras bound to GMPPNP were solved in 50% 2,2,2-trifluoroethanol, 60% 1,6-hexanediol, and 50% isopropanol. The disordered switch II region of Ras is ordered in the presence of 2,2,2-trifluoroethanol or 1,6-hexanediol. The overall backbone conformation of switch II in these organic solvents is the same as in the Ras-GMPPNP complexes with RalGDS, PI<sub>3</sub> kinase, and RasGAP, indicating a biologically relevant form. Key polar interactions that stabilize the ordered switch are enhanced in the presence of hydrophobic cosolvents. These results suggest that hydrophobic solvents can be used in general to order short biologically relevant segments of disordered regions in protein crystals by favoring H-bonding interactions between atoms that are highly solvated and mobile in aqueous solution.}, number={7}, journal={STRUCTURE}, author={Buhrman, G and Serrano, V and Mattos, C}, year={2003}, month={Jul}, pages={747–751} }