@article{gering_li_tang_swartz_chang_makris_2023, title={A Ferric-Superoxide Intermediate Initiates P450-Catalyzed Cyclic Dipeptide Dimerization}, volume={8}, ISSN={["1520-5126"]}, url={https://doi.org/10.1021/jacs.3c04542}, DOI={10.1021/jacs.3c04542}, abstractNote={The cytochrome P450 (CYP) AspB is involved in the biosynthesis of the diketopiperazine (DKP) aspergilazine A. Tryptophan-linked dimeric DKP alkaloids are a large family of natural products that are found in numerous species and exhibit broad and often potent bioactivity. The proposed mechanisms for C-N bond formation by AspB, and similar C-C bond formations by related CYPs, have invoked the use of a ferryl-intermediate as an oxidant to promote substrate dimerization. Here, the parallel application of steady-state and transient kinetic approaches reveals a very different mechanism that involves a ferric-superoxide species as a primary oxidant to initiate DKP-assembly. Single turnover kinetic isotope effects and a substrate analog suggest the probable nature and site for abstraction. The direct observation of CYP-superoxide reactivity rationalizes the atypical outcome of AspB and reveals a new reaction manifold in heme enzymes.}, journal={JOURNAL OF THE AMERICAN CHEMICAL SOCIETY}, author={Gering, Hannah E. and Li, Xiaojun and Tang, Haoyu and Swartz, Paul D. and Chang, Wei-Chen and Makris, Thomas M.}, year={2023}, month={Aug} }
 @article{schroder_william b. o'dell_swartz_meilleur_2021, title={Preliminary results of neutron and X-ray diffraction data collection on a lytic polysaccharide monooxygenase under reduced and acidic conditions}, volume={77}, ISSN={["2053-230X"]}, url={https://doi.org/10.1107/S2053230X21002399}, DOI={10.1107/S2053230X21002399}, abstractNote={Lytic polysaccharide monooxygenases (LPMOs) are copper-center enzymes that are involved in the oxidative cleavage of the glycosidic bond in crystalline cellulose and other polysaccharides. The LPMO reaction is initiated by the addition of a reductant and oxygen to ultimately form an unknown activated copper–oxygen species that is responsible for polysaccharide-substrate H-atom abstraction. Given the sensitivity of metalloproteins to radiation damage, neutron protein crystallography provides a nondestructive technique for structural characterization while also informing on the positions of H atoms. Neutron cryo-crystallography permits the trapping of catalytic intermediates, thereby providing insight into the protonation states and chemical nature of otherwise short-lived species in the reaction mechanism. To characterize the reaction-mechanism intermediates of LPMO9D from Neurospora crassa, a cryo-neutron diffraction data set was collected from an ascorbate-reduced crystal. A second neutron diffraction data set was collected at room temperature from an LPMO9D crystal exposed to low-pH conditions to probe the protonation states of ionizable groups involved in catalysis under acidic conditions.}, number={4}, journal={ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS}, publisher={International Union of Crystallography (IUCr)}, author={Schroder, Gabriela C. and William B. O'Dell and Swartz, Paul D. and Meilleur, Flora}, year={2021}, month={Apr}, pages={128–133} }
 @article{yao_swartz_hamilton_clark_2021, title={Remodeling hydrogen bond Interactions results in relaxed specificity of Caspase-3}, volume={41}, ISSN={["1573-4935"]}, DOI={10.1042/BSR20203495}, abstractNote={Abstract}, number={1}, journal={BIOSCIENCE REPORTS}, author={Yao, Liqi and Swartz, Paul and Hamilton, Paul T. and Clark, A. Clay}, year={2021}, month={Jan} }
 @article{shrestha_tung_grinshpon_swartz_hamilton_dimos_mydlarz_clark_2020, title={Caspases from scleractinian coral show unique regulatory features}, volume={295}, ISSN={["1083-351X"]}, url={http://dx.doi.org/10.1074/jbc.ra120.014345}, DOI={10.1074/jbc.RA120.014345}, abstractNote={Coral reefs are experiencing precipitous declines around the globe with coral diseases and temperature-induced bleaching being primary drivers of these declines. Regulation of apoptotic cell death is an important component in the coral stress response. Although cnidaria are known to contain complex apoptotic signaling pathways, similar to those in vertebrates, the mechanisms leading to cell death are largely unexplored. We identified and characterized two caspases each from Orbicella faveolata, a disease-sensitive reef-building coral, and Porites astreoides, a disease-resistant reef-building coral. The caspases are predicted homologs of the human executioner caspases-3 and -7, but OfCasp3a (Orbicella faveolata caspase-3a) and PaCasp7a (Porites astreoides caspase-7a), which we show to be DXXDases, contain an N-terminal caspase activation/recruitment domain (CARD) similar to human initiator/inflammatory caspases. OfCasp3b (Orbicella faveolata caspase-3b) and PaCasp3 (Porites astreoides caspase-3), which we show to be VXXDases, have short pro-domains, like human executioner caspases. Our biochemical analyses suggest a mechanism in coral which differs from that of humans, where the CARD-containing DXXDase is activated on death platforms but the protease does not directly activate the VXXDase. The first X-ray crystal structure of a coral caspase, of PaCasp7a determined at 1.57 Å resolution, reveals a conserved fold and an N-terminal peptide bound near the active site that may serve as a regulatory exosite. The binding pocket has been observed in initiator caspases of other species. These results suggest mechanisms for the evolution of substrate selection while maintaining common activation mechanisms of CARD-mediated dimerization.}, number={43}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, publisher={Elsevier BV}, author={Shrestha, Suman and Tung, Jessica and Grinshpon, Robert D. and Swartz, Paul and Hamilton, Paul T. and Dimos, Bradford and Mydlarz, Laura and Clark, A. Clay}, year={2020}, month={Oct}, pages={14578–14591} }
 @article{kearney_schwabe_marcus_roberts_dechene_swartz_mattos_2020, title={DRoP: Automated detection of conserved solvent-binding sites on proteins}, volume={88}, ISSN={["1097-0134"]}, DOI={10.1002/prot.25781}, abstractNote={Abstract}, number={1}, journal={PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS}, author={Kearney, Bradley M. and Schwabe, Michael and Marcus, Kendra C. and Roberts, Daniel M. and Dechene, Michelle and Swartz, Paul and Mattos, Carla}, year={2020}, month={Jan}, pages={152–165} }
 @article{agarwal_smith_de la rosa_verba_swartz_segura-totten_mattos_2020, title={Development of a structure-analysis pipeline using multiple-solvent crystal structures of barrier-to-autointegration factor}, volume={76}, ISSN={["2059-7983"]}, DOI={10.1107/S2059798320011341}, abstractNote={The multiple-solvent crystal structure (MSCS) approach uses high concentrations of organic solvents to characterize the interactions and effects of solvents on proteins. Here, the method has been further developed and an MSCS data-handling pipeline is presented that uses the Detection of Related Solvent Positions (DRoP) program to improve data quality. DRoP is used to selectively model conserved water molecules, so that an advanced stage of structural refinement is reached quickly. This allows the placement of organic molecules more accurately and convergence on high-quality maps and structures. This pipeline was applied to the chromatin-associated protein barrier-to-autointegration factor (BAF), resulting in structural models with better than average statistics. DRoP and Phenix Structure Comparison were used to characterize the data sets and to identify a binding site that overlaps with the interaction site of BAF with emerin. The conserved water-mediated networks identified by DRoP suggested a mechanism by which water molecules are used to drive the binding of DNA. Normalized and differential B-factor analysis is shown to be a valuable tool to characterize the effects of specific solvents on defined regions of BAF. Specific solvents are identified that cause stabilization of functionally important regions of the protein. This work presents tools and a standardized approach for the analysis and comprehension of MSCS data sets.}, journal={ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY}, author={Agarwal, Sorabh and Smith, Mychal and De La Rosa, Indhira and Verba, Kliment A. and Swartz, Paul and Segura-Totten, Miriam and Mattos, Carla}, year={2020}, month={Oct}, pages={1001–1014} }
 @article{thomas_grinshpon_swartz_clark_2018, title={Modifications to a common phosphorylation network provide individualized control in caspases}, volume={293}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.ra117.000728}, abstractNote={Caspase-3 activation and function have been well-defined during programmed cell death, but caspase activity, at low levels, is also required for developmental processes such as lymphoid proliferation and erythroid differentiation. Post-translational modification of caspase-3 is one method used by cells to fine-tune activity below the threshold required for apoptosis, but the allosteric mechanism that reduces activity is unknown. Phosphorylation of caspase-3 at a conserved allosteric site by p38-MAPK (mitogen-activated protein kinase) promotes survival in human neutrophils, and the modification of the loop is thought to be a key regulator in many developmental processes. We utilized phylogenetic, structural, and biophysical studies to define the interaction networks that facilitate the allosteric mechanism in caspase-3. We show that, within the modified loop, Ser150 evolved with the apoptotic caspases, whereas Thr152 is a more recent evolutionary event in mammalian caspase-3. Substitutions at Ser150 result in a pH-dependent decrease in dimer stability, and localized changes in the modified loop propagate to the active site of the same protomer through a connecting surface helix. Likewise, a cluster of hydrophobic amino acids connects the conserved loop to the active site of the second protomer. The presence of Thr152 in the conserved loop introduces a “kill switch” in mammalian caspase-3, whereas the more ancient Ser150 reduces without abolishing enzyme activity. These data reveal how evolutionary changes in a conserved allosteric site result in a common pathway for lowering activity during development or a more recent cluster-specific switch to abolish activity.}, number={15}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Thomas, Melvin E., III and Grinshpon, Robert and Swartz, Paul and Clark, A. Clay}, year={2018}, month={Apr}, pages={5447–5461} }
 @article{carey_kim_mccombs_swartz_kim_ghiladi_2018, title={Selective tuning of activity in a multifunctional enzyme as revealed in the F21W mutant of dehaloperoxidase B from Amphitrite ornata}, volume={23}, ISSN={0949-8257 1432-1327}, url={http://dx.doi.org/10.1007/s00775-017-1520-x}, DOI={10.1007/s00775-017-1520-x}, abstractNote={Possessing both peroxidase and peroxygenase activities with a broad substrate profile that includes phenols, indoles, and pyrroles, the enzyme dehaloperoxidase (DHP) from Amphitrite ornata is a multifunctional catalytic hemoglobin that challenges many of the assumptions behind the well-established structure-function paradigm in hemoproteins. While previous studies have demonstrated that the F21W variant leads to attenuated peroxidase activity in DHP, here we have studied the impact of this mutation on peroxygenase activity to determine if it is possible to selectively tune DHP to favor one function over another. Biochemical assays with DHP B (F21W) revealed minimal decreases in peroxygenase activity of 1.2-2.1-fold as measured by 4-nitrophenol or 5-Br-indole substrate conversion, whereas the peroxidase activity catalytic efficiency for 2,4,6-trichlorophenol (TCP) was more than sevenfold decreased. Binding studies showed a 20-fold weaker affinity for 5-bromoindole (K  d  = 2960 ± 940 μM) in DHP B (F21W) compared to WT DHP B. Stopped-flow UV/visible studies and isotope labeling experiments together suggest that the F21W mutation neither significantly changes the nature of the catalytic intermediates, nor alters the mechanisms that have been established for peroxidase and peroxygenase activities in DHP. The X-ray crystal structure (1.96 Å; PDB 5VLX) of DHP B (F21W) revealed that the tryptophan blocks one of the two identified TCP binding sites, specifically TCP interior , suggesting that the other site, TCP exterior , remains viable for binding peroxygenase substrates. Taken together, these studies demonstrate that blocking the TCP interior  binding site in DHP selectively favors peroxygenase activity at the expense of its peroxidase activity.}, number={2}, journal={JBIC Journal of Biological Inorganic Chemistry}, publisher={Springer Science and Business Media LLC}, author={Carey, Leiah M. and Kim, Kyung Beom and McCombs, Nikolette L. and Swartz, Paul and Kim, Cheal and Ghiladi, Reza A.}, year={2018}, month={Mar}, pages={209–219} }
 @article{william b. o'dell_swartz_weiss_meilleur_2017, title={Crystallization of a fungal lytic polysaccharide monooxygenase expressed from glycoengineered Pichia pastoris for X-ray and neutron diffraction}, volume={73}, ISSN={["2053-230X"]}, DOI={10.1107/s2053230x16020318}, abstractNote={Lytic polysaccharide monooxygenases (LPMOs) are carbohydrate-disrupting enzymes secreted by bacteria and fungi that break glycosidic bondsviaan oxidative mechanism. Fungal LPMOs typically act on cellulose and can enhance the efficiency of cellulose-hydrolyzing enzymes that release soluble sugars for bioethanol production or other industrial uses. The enzyme PMO-2 fromNeurospora crassa(NcPMO-2) was heterologously expressed inPichia pastoristo facilitate crystallographic studies of the fungal LPMO mechanism. Diffraction resolution and crystal morphology were improved by expressingNcPMO-2 from a glycoengineered strain ofP. pastorisand by the use of crystal seeding methods, respectively. These improvements resulted in high-resolution (1.20 Å) X-ray diffraction data collection at 100 K and the production of a largeNcPMO-2 crystal suitable for room-temperature neutron diffraction data collection to 2.12 Å resolution.}, number={2}, journal={ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS}, publisher={International Union of Crystallography (IUCr)}, author={William B. O'Dell and Swartz, Paul D. and Weiss, Kevin L. and Meilleur, Flora}, year={2017}, month={Feb}, pages={70–78} }
 @article{tucker_mackenzie_maciag_dirscherl ackerman_swartz_yoder_hamilton_clay clark_2016, title={Phage display and structural studies reveal plasticity in substrate specificity of caspase-3a from zebrafish}, volume={25}, ISSN={0961-8368}, url={http://dx.doi.org/10.1002/PRO.3032}, DOI={10.1002/PRO.3032}, abstractNote={Abstract}, number={11}, journal={Protein Science}, publisher={Wiley}, author={Tucker, Matthew B. and MacKenzie, Sarah H. and Maciag, Joseph J. and Dirscherl Ackerman, Hayley and Swartz, Paul and Yoder, Jeffrey A. and Hamilton, Paul T. and Clay Clark, A.}, year={2016}, month={Sep}, pages={2076–2088} }
 @article{maciag_mackenzie_tucker_schipper_swartz_clark_2016, title={Tunable allosteric library of caspase-3 identifies coupling between conserved water molecules and conformational selection}, volume={113}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1603549113}, abstractNote={Significance}, number={41}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Maciag, Joseph J. and Mackenzie, Sarah H. and Tucker, Matthew B. and Schipper, Joshua L. and Swartz, Paul and Clark, A. Clay}, year={2016}, month={Oct}, pages={E6080–E6088} }
 @article{bodenheimer_cuneo_swartz_he_o'neill_myles_evans_meilleur_section_2014, title={Crystallization and preliminary X-ray diffraction analysis of Hypocrea jecorina Cel7A in two new crystal forms}, volume={70}, ISSN={["2053-230X"]}, url={http://europepmc.org/abstract/med/24915091}, DOI={10.1107/s2053230x14008851}, abstractNote={Cel7A (previously known as cellobiohydrolase I) fromHypocrea jecorinawas crystallized in two crystalline forms, neither of which have been previously reported. Both forms co-crystallize under the same crystallization conditions. The first crystal form belonged to space groupC2, with unit-cell parametersa= 152.5,b= 44.9,c= 57.6 Å, β = 101.2°, and diffracted X-rays to 1.5 Å resolution. The second crystal form belonged to space groupP6322, with unit-cell parametersa=b≃ 155,c≃ 138 Å, and diffracted X-rays to 2.5 Å resolution. The crystals were obtained using full-length Cel7A, which consists of a large 434-residue N-terminal catalytic domain capable of cleaving cellulose, a 27-residue flexible linker and a small 36-residue C-terminal carbohydrate-binding module (CBM). However, a preliminary analysis of the electron-density maps suggests that the linker and CBM are disordered in both crystal forms. Complete refinement and structure analysis are currently in progress.}, number={Pt 6}, journal={ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS}, author={Bodenheimer, A.M. and Cuneo, M.J. and Swartz, P.D. and He, J. and O'Neill, H.M. and Myles, D.A. and Evans, B.R. and Meilleur, Flora and Section, F.}, year={2014}, month={Jun}, pages={773–776} }
 @article{kearney_johnson_roberts_swartz_mattos_2014, title={DRoP: A Water Analysis Program Identifies Ras-GTP-Specific Pathway of Communication between Membrane-Interacting Regions and the Active Site}, volume={426}, ISSN={["1089-8638"]}, DOI={10.1016/j.jmb.2013.10.036}, abstractNote={Ras GTPase mediates several cellular signal transduction pathways and is found mutated in a large number of cancers. It is active in the GTP-bound state, where it interacts with effector proteins, and at rest in the GDP-bound state. The catalytic domain is tethered to the membrane, with which it interacts in a nucleotide-dependent manner. Here we present the program Detection of Related Solvent Positions (DRoP) for crystallographic water analysis on protein surfaces and use it to study Ras. DRoP reads and superimposes multiple Protein Data Bank coordinates, transfers symmetry-related water molecules to the position closest to the protein surface, and ranks the waters according to how well conserved and tightly clustered they are in the set of structures. Coloring according to this rank allows visualization of the results. The effector-binding region of Ras is hydrated with highly conserved water molecules at the interface between the P-loop, switch I, and switch II, as well as at the Raf-RBD binding pocket. Furthermore, we discovered a new conserved water-mediated H-bonding network present in Ras-GTP, but not in Ras-GDP, that links the nucleotide sensor residues R161 and R164 on helix 5 to the active site. The double mutant RasN85A/N86A, where the final link between helix 5 and the nucleotide is not possible, is a severely impaired enzyme, while the single mutant RasN86A, with partial connection to the active site, has a wild-type hydrolysis rate. DRoP was instrumental in determining the water-mediated connectivity networks that link two lobes of the catalytic domain in Ras.}, number={3}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Kearney, Bradley Ni. and Johnson, Christian W. and Roberts, Daniel M. and Swartz, Paul and Mattos, Carla}, year={2014}, month={Feb}, pages={611–629} }
 @article{cade_swartz_mackenzie_clark_2014, title={Modifying Caspase-3 Activity by Altering Allosteric Networks}, volume={53}, ISSN={["0006-2960"]}, DOI={10.1021/bi500874k}, abstractNote={Caspases have several allosteric sites that bind small molecules or peptides. Allosteric regulators are known to affect caspase enzyme activity, in general, by facilitating large conformational changes that convert the active enzyme to a zymogen-like form in which the substrate-binding pocket is disordered. Mutations in presumed allosteric networks also decrease activity, although large structural changes are not observed. Mutation of the central V266 to histidine in the dimer interface of caspase-3 inactivates the enzyme by introducing steric clashes that may ultimately affect positioning of a helix on the protein surface. The helix is thought to connect several residues in the active site to the allosteric dimer interface. In contrast to the effects of small molecule allosteric regulators, the substrate-binding pocket is intact in the mutant, yet the enzyme is inactive. We have examined the putative allosteric network, in particular the role of helix 3, by mutating several residues in the network. We relieved steric clashes in the context of caspase-3(V266H), and we show that activity is restored, particularly when the restorative mutation is close to H266. We also mimicked the V266H mutant by introducing steric clashes elsewhere in the allosteric network, generating several mutants with reduced activity. Overall, the data show that the caspase-3 native ensemble includes the canonical active state as well as an inactive conformation characterized by an intact substrate-binding pocket, but with an altered helix 3. The enzyme activity reflects the relative population of each species in the native ensemble.}, number={48}, journal={BIOCHEMISTRY}, author={Cade, Christine and Swartz, Paul and MacKenzie, Sarah H. and Clark, A. Clay}, year={2014}, month={Dec}, pages={7582–7595} }
 @article{mackenzie_schipper_england_thomas_blackburn_swartz_clark_2013, title={Lengthening the Intersubunit Linker of Procaspase 3 Leads to Constitutive Activation}, volume={52}, ISSN={["0006-2960"]}, DOI={10.1021/bi400793s}, abstractNote={The conformational ensemble of procaspase 3, the primary executioner in apoptosis, contains two major forms, inactive and active, with the inactive state favored in the native ensemble. A region of the protein known as the intersubunit linker (IL) is cleaved during maturation, resulting in movement of the IL out of the dimer interface and subsequent active site formation (activation-by-cleavage mechanism). We examined two models for the role of the IL in maintaining the inactive conformer, an IL-extension model versus a hydrophobic cluster model, and we show that increasing the length of the IL by introducing 3-5 alanines results in constitutively active procaspases. Active site labeling and subsequent analyses by mass spectrometry show that the full-length zymogen is enzymatically active. We also show that minor populations of alternately cleaved procaspase result from processing at D169 when the normal cleavage site, D175, is unavailable. Importantly, the alternately cleaved proteins have little to no activity, but increased flexibility of the linker increases the exposure of D169. The data show that releasing the strain of the short IL, in and of itself, is not sufficient to populate the active conformer of the native ensemble. The IL must also allow for interactions that stabilize the active site, possibly from a combination of optimal length, flexibility in the IL, and specific contacts between the IL and interface. The results provide further evidence that substantial energy is required to shift the protein to the active conformer. As a result, the activation-by-cleavage mechanism dominates in the cell.}, number={36}, journal={BIOCHEMISTRY}, author={MacKenzie, Sarah H. and Schipper, Joshua L. and England, Erika J. and Thomas, Melvin E., III and Blackburn, Kevin and Swartz, Paul and Clark, A. Clay}, year={2013}, month={Sep}, pages={6219–6231} }
 @article{jiang_wright_swartz_franzen_2013, title={The role of T56 in controlling the flexibility of the distal histidine in dehaloperoxidase-hemoglobin from Amphitrite ornata}, volume={1834}, ISSN={["1878-1454"]}, DOI={10.1016/j.bbapap.2013.06.005}, abstractNote={The activation of dehaloperoxidase-hemoglobin (DHP) to form a ferryl intermediate requires the distal histidine, H55, to act as an acid base catalyst. The lack of ancillary amino acids in the distal pocket to assist in this process makes H55 even more important to the formation of active intermediates than in conventional peroxidases. Therefore, one can infer that the precise conformation H55 may greatly affect the enzymatic activity. Using site-direct mutagenesis at position T56, immediately adjacent to H55, we have confirmed that subtle changes in the conformation of H55 affect the catalytic efficiency of DHP. Mutating T56 to a smaller amino acid appears to permit H55 to rotate with relatively low barriers between conformations in the distal pocket, which may lead to an increase in catalytic activity. On the other hand, larger amino acids in the neighboring site appear to restrict the rotation of H55 due to the steric hindrance. In the case of T56V, which is an isosteric mutation, H55 appears less mobile, but forced to be closer to the heme iron than in wild type. Both proximity to the heme iron and flexibility of motion in some of the mutants can result in an increased catalytic rate, but can also lead to protein inactivation due to ligation of H55 to the heme iron, which is known as hemichrome formation. A balance of enzymatic rate and protein stability with respect to hemichrome formation appears to be optimum in wild type DHP (WT-DHP).}, number={10}, journal={BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS}, author={Jiang, Shu and Wright, Iain and Swartz, Paul and Franzen, Stefan}, year={2013}, month={Oct}, pages={2020–2029} }
 @article{walters_schipper_swartz_mattos_clark_2012, title={Allosteric modulation of caspase 3 through mutagenesis}, volume={32}, ISSN={["0144-8463"]}, DOI={10.1042/bsr20120037}, abstractNote={A mutation in the allosteric site of the caspase 3 dimer interface of Val266 to histidine abolishes activity of the enzyme, and models predict that the mutation mimics the action of small molecule allosteric inhibitors by preventing formation of the active site. Mutations were coupled to His266 at two sites in the interface, E124A and Y197C. We present results from X-ray crystallography, enzymatic activity and molecular dynamics simulations for seven proteins, consisting of single, double and triple mutants. The results demonstrate that considering allosteric inhibition of caspase 3 as a shift between discrete ‘off-state’ or ‘on-state’ conformations is insufficient. Although His266 is accommodated in the interface, the structural defects are propagated to the active site through a helix on the protein surface. A more comprehensive view of allosteric regulation of caspase 3 requires the representation of an ensemble of inactive states and shows that subtle structural changes lead to the population of the inactive ensemble.}, number={4}, journal={BIOSCIENCE REPORTS}, author={Walters, Jad and Schipper, Joshua L. and Swartz, Paul and Mattos, Carla and Clark, A. Clay}, year={2012}, month={Aug}, pages={401–411} }
 @article{walters_swartz_mattos_clark_2011, title={Thermodynamic, enzymatic and structural effects of removing a salt bridge at the base of loop 4 in (pro)caspase-3}, volume={508}, ISSN={["1096-0384"]}, DOI={10.1016/j.abb.2011.01.011}, abstractNote={Interactions between loops 2, 2' and 4, known as the loop bundle, stabilize the active site of caspase-3. Loop 4 (L4) is of particular interest due to its location between the active site and the dimer interface. We have disrupted a salt bridge between K242 and E246 at the base of L4 to determine its role in overall conformational stability and in maintaining the active site environment. Stability measurements show that only the K242A single mutant decreases stability of the dimer, whereas both single mutants and the double mutant demonstrate much lower activity compared to wild-type caspase-3. Structural studies of the caspase-3 variants show the involvement of K242 in hydrophobic interactions that stabilize helix 5, near the dimer interface, and the role of E246 appears to be to neutralize the positive charge of K242 within the hydrophobic cluster. Overall, the results suggest E246 and K242 are important in procaspase-3 for their interaction with neighboring residues, not with one another. Conversely, formation of the K242-E246 salt bridge in caspase-3 is needed for an accurate, stable conformation of loop L4 and proper active site formation in the mature enzyme.}, number={1}, journal={ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS}, author={Walters, Jad and Swartz, Paul and Mattos, Carla and Clark, A. Clay}, year={2011}, month={Apr}, pages={31–38} }
 @article{martin_guenther_sit_swartz_meilleur_lommel_rose_section_2010, title={Crystallization and preliminary X-ray diffraction analysis of red clover necrotic mosaic virus}, volume={66}, ISSN={["2053-230X"]}, url={http://europepmc.org/abstract/med/21045294}, DOI={10.1107/s1744309110032483}, abstractNote={Red clover necrotic mosaic virus (RCNMV) is a species that belongs to the Tombusviridae family of plant viruses with a T = 3 icosahedral capsid. RCNMV virions were purified and were crystallized for X-ray analysis using the hanging-drop vapor-diffusion method. Self-rotation functions and systematic absences identified the space group as I23, with two virions in the unit cell. The crystals diffracted to better than 4 Å resolution but were very radiation-sensitive, causing rapid decay of the high-resolution reflections. The data were processed to 6 Å in the analysis presented here.}, number={Pt 11}, journal={ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS}, author={Martin, S.L. and Guenther, R.H. and Sit, T.L. and Swartz, P.D. and Meilleur, Flora and Lommel, S.A. and Rose, Robert and Section, F.}, year={2010}, month={Nov}, pages={1458–1462} }
 @article{walters_pop_scott_drag_swartz_mattos_salvesen_clark_2009, title={A constitutively active and uninhibitable caspase-3 zymogen efficiently induces apoptosis}, volume={424}, ISSN={["1470-8728"]}, DOI={10.1042/bj20090825}, abstractNote={The caspase-3 zymogen has essentially zero activity until it is cleaved by initiator caspases during apoptosis. However, a mutation of V266E in the dimer interface activates the protease in the absence of chain cleavage. We show that low concentrations of the pseudo-activated procaspase-3 kill mammalian cells rapidly and, importantly, this protein is not cleaved nor is it inhibited efficiently by the endogenous regulator XIAP (X-linked inhibitor of apoptosis). The 1.63 Å (1 Å = 0.1 nm) structure of the variant demonstrates that the mutation is accommodated at the dimer interface to generate an enzyme with substantially the same activity and specificity as wild-type caspase-3. Structural modelling predicts that the interface mutation prevents the intersubunit linker from binding in the dimer interface, allowing the active sites to form in the procaspase in the absence of cleavage. The direct activation of procaspase-3 through a conformational switch rather than by chain cleavage may lead to novel therapeutic strategies for inducing cell death.}, journal={BIOCHEMICAL JOURNAL}, author={Walters, Jad and Pop, Cristina and Scott, Fiona L. and Drag, Marcin and Swartz, Paul and Mattos, Carla and Salvesen, Guy S. and Clark, A. Clay}, year={2009}, month={Dec}, pages={335–345} }
 @article{dechene_wink_smith_swartz_mattos_2009, title={Multiple solvent crystal structures of ribonuclease A: An assessment of the method}, volume={76}, ISSN={["1097-0134"]}, DOI={10.1002/prot.22393}, abstractNote={Abstract}, number={4}, journal={PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS}, author={Dechene, Michelle and Wink, Glenna and Smith, Mychal and Swartz, Paul and Mattos, Carla}, year={2009}, month={Sep}, pages={861–881} }
 @article{feeney_pop_swartz_mattos_clark_2006, title={Role of loop bundle hydrogen bonds in the maturation and activity of (pro) caspase-3}, volume={45}, ISSN={["0006-2960"]}, DOI={10.1021/bi0611964}, abstractNote={During maturation, procaspase-3 is cleaved at D175, which resides in a linker that connects the large and small subunits. The intersubunit linker also connects two active site loops that rearrange following cleavage and, in part, form the so-called loop bundle. As a result of chain cleavage, new hydrogen bonds and van der Waals contacts form among three active site loops. The new interactions are predicted to stabilize the active site. One unresolved issue is the extent to which the loop bundle residues also stabilize the procaspase active site. We examined the effects of replacing four loop bundle residues (E167, D169, E173, and Y203) on the biochemical and structural properties of the (pro)caspase. We show that replacing the residues affects the activity of the procaspase as well as the mature caspase, with D169A and E167A replacements having the largest effects. Replacement of D169 prevents caspase-3 autoactivation, and its cleavage at D175 no longer leads to an active enzyme. In addition, the E173A mutation, when coupled to a second mutation in the procaspase, D175A, may alter the substrate specificity of the procaspase. The mutations affected the active site environment as assessed by changes in fluorescence emission, accessibility to quencher, and cleavage by either trypsin or V8 proteases. High-resolution X-ray crystallographic structures of E167A, D173A, and Y203F caspases show that changes in the active site environment may be due to the increased flexibility of several residues in the N-terminus of the small subunit. Overall, the results show that these residues are important for stabilizing the procaspase active site as well as that of the mature caspase.}, number={44}, journal={BIOCHEMISTRY}, author={Feeney, Brett and Pop, Cristina and Swartz, Paul and Mattos, Carla and Clark, A. Clay}, year={2006}, month={Nov}, pages={13249–13263} }
 @article{kundu_richardson_granville_shaughnessy_hanley_swartz_richard_demarini_2004, title={Comparative mutagenicity of halomethanes and halonitromethanes in Salmonella TA100: structure–activity analysis and mutation spectra}, volume={554}, ISSN={0027-5107}, url={http://dx.doi.org/10.1016/j.mrfmmm.2004.05.015}, DOI={10.1016/j.mrfmmm.2004.05.015}, abstractNote={Halonitromethanes (HNMs) are a recently identified class of disinfection by-products (DPBs) in drinking water that are mutagenic in Salmonella and potent inducers of DNA strand breaks in mammalian cells. Here we compared the mutagenic potencies of the HNMs to those of their halomethane (HM) homologues by testing all nine HNMs and seven of the nine HMs (minus bromomethane and chloromethane) under the same conditions (the pre-incubation assay) in Salmonella TA100 ± S9. We also determined the mutation spectra for several DBPs. In the presence of S9, all nine HNMs, but only three HMs, dibromomethane (DBM), dichloromethane (DCM), and bromochloromethane (BCM), were mutagenic. Only two DBPs of each class were mutagenic in the absence of S9. The HNMs were generally more potent mutagens than their HM homologues, and the brominated forms of both classes of DBPs were more mutagenic and cytotoxic than their chlorinated homologues. The HNMs were at least 10 times more cytotoxic than the HMs, and the cytotoxicity rankings in the presence of S9 were similar for the HNMs and the HMs. The addition of a nitro-group to BCM did not change the mutation spectra significantly, with both homologues inducing primarily (55–58%) GC → AT transitions. The greater cytotoxic and mutagenic activities of the HNMs relative to the HMs are likely due to the greater intrinsic reactivity conferred by the nitro-group. Energy calculations predicted increased reactivity with increasing bromination and greater reactivity of the HNMs versus the HMs (Elumo values were ∼20 kcal/mol lower for the HNMs compared to their HM homologues). Given that the HNMs also are potent genotoxins in mammalian cells [Environ. Sci. Technol. 38 (2004) 62] and are more mutagenic and 10× more cytotoxic in Salmonella than the HMs, whose levels are regulated in drinking water, further study of their occurrence and potential health effects is warranted.}, number={1-2}, journal={Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis}, publisher={Elsevier BV}, author={Kundu, Bijit and Richardson, Susan D. and Granville, Courtney A. and Shaughnessy, Daniel T. and Hanley, Nancy M. and Swartz, Paul D. and Richard, Ann M. and DeMarini, David M.}, year={2004}, month={Oct}, pages={335–350} }
 @article{kundu_richardson_swartz_matthews_richard_demarini_2004, title={Mutagenicity in Salmonella of halonitromethanes: a recently recognized class of disinfection by-products in drinking water}, volume={562}, ISSN={1383-5718}, url={http://dx.doi.org/10.1016/j.mrgentox.2004.05.007}, DOI={10.1016/j.mrgentox.2004.05.007}, abstractNote={Halonitromethanes (HNMs) are a recently identified class of disinfection by-products (DBPs) in drinking water. They include chloronitromethane (CHN), dichloronitromethane (DCNM), trichloronitromethane (TCNM), bromonitromethane (BNM), dibromonitromethane (DBNM), tribromonitromethane (TBNM), bromochloronitromethane (BCNM),dibromochloronitromethane (DBCNM), and bromodichloronitromethane (BDCNM). Previous studies of TCNM, DCNM, CNM, and TBNM found that all four were mutagenic in bacteria, and a recent study showed that all nine induced DNA damage in CHO cells. Here, all nine HNMs were evaluated in the Salmonella plate-incorporation assay +/− S9 using strains TA98, TA100, TA104, TPT100, and the glutathione transferase theta (GSTT1-1)-expressing strain RSJ100. All were mutagenic, most with and without S9. In the absence of S9, six were mutagenic in TA98, six in TA100, and three in TA104; in the presence of S9, these numbers were five, seven, and three, respectively. Thus, the HNMs-induced base substitutions primarily at GC sites as well as frameshifts. Although five HNMs were activated to mutagens in RSJ100 −S9, they produced ≤2-fold increases in revertants and potencies <506 rev/μmol. The rank order of the HNMs by mutagenic potency in TA100 +S9 was (BCNM DBNM) > (TBNM CNM > BNM DCNM BDCNM) > (TCNM=DBCNM). The mean rev/μmol for the three groupings, respectively, were 1423, 498, and 0, which classifies the HNMs as weak mutagens in Salmonella. Reaction of the dihalo and monohalo HNMs with GSH, possibly GSTT1-1, is a possible mechanism for formation of ultimate mutagenic products. Because the HNMs are mutagenic in Salmonella (present study) and potent clastogens in mammalian cells [Environ. Sci. Technol. 38 (2004) 62], their presence in drinking water warrants further research on their potential health effects.}, number={1-2}, journal={Mutation Research/Genetic Toxicology and Environmental Mutagenesis}, publisher={Elsevier BV}, author={Kundu, Bijit and Richardson, Susan D and Swartz, Paul D and Matthews, Peggy P and Richard, Ann M and DeMarini, David M}, year={2004}, month={Aug}, pages={39–65} }