@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={Caspase (or cysteinyl-aspartate specific proteases) enzymes play important roles in apoptosis and inflammation, and the non-identical but overlapping specificity profiles (that is, cleavage recognition sequence) direct cells to different fates. Although all caspases prefer aspartate at the P1 position of the substrate, the caspase-6 subfamily shows preference for valine at the P4 position, while caspase-3 shows preference for aspartate. In comparison with human caspases, caspase-3a from zebrafish has relaxed specificity and demonstrates equal selection for either valine or aspartate at the P4 position. In the context of the caspase-3 conformational landscape, we show that changes in hydrogen bonding near the S3 subsite affect selection of the P4 amino acid. Swapping specificity with caspase-6 requires accessing new conformational space, where each landscape results in optimal binding of DxxD (caspase-3) or VxxD (caspase-6) substrate and simultaneously disfavors binding of the other substrate. Within the context of the caspase-3 conformational landscape, substitutions near the active site result in nearly equal activity against DxxD and VxxD by disrupting a hydrogen bonding network in the substrate binding pocket. The converse substitutions in zebrafish caspase-3a result in increased selection for P4 aspartate over valine. Overall, the data show that the shift in specificity that results in a dual function protease, as in zebrafish caspase-3a, requires fewer amino acid substitutions compared with those required to access new conformational space for swapping substrate specificity, such as between caspases-3 and -6.}, 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. 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. Apoptotic cell death is thought to be a unique characteristic of metazoans, although its evolutionary origins are unclear. Although caspases from human cells and model organisms such as Caenorhabditis elegans and Drosophila have been well-studied both biochemically and structurally (1Song Z. McCall K. Steller H. DCP-1, a Drosophila cell death protease essential for development.Science. 1997; 275 (8999799): 536-54010.1126/science.275.5299.536Crossref PubMed Scopus (251) Google Scholar, 2Dorstyn L. Mills K. Lazebnik Y. Kumar S. The two cytochrome c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells.J. Cell Biol. 2004; 167 (15533997): 405-41010.1083/jcb.200408054Crossref PubMed Scopus (99) Google Scholar, 3Ellis H.M. Horvitz H.R. Genetic control of programmed cell death in the nematode C. elegans.Cell. 1986; 44 (3955651): 817-82910.1016/0092-8674(86)90004-8Abstract Full Text PDF PubMed Scopus (1353) Google Scholar, 4Schwartz H.T. Horvitz H.R. The C. elegans protein CEH-30 protects male-specific neurons from apoptosis independently of the Bcl-2 homolog CED-9.Genes Dev. 2007; 21 (18056428): 3181-319410.1101/gad.1607007Crossref PubMed Scopus (58) Google Scholar, 5Shaham S. Horvitz H.R. Developing Caenorhabditis elegans neurons may contain both cell-death protective and killer activities.Genes Dev. 1996; 10 (8598288): 578-59110.1101/gad.10.5.578Crossref PubMed Scopus (203) Google Scholar, 6Yuan J. Horvitz H.R. The Caenorhabditis elegans genes ced-3 and ced-4 act cell autonomously to cause programmed cell death.Dev. Biol. 1990; 138 (2307287): 33-4110.1016/0012-1606(90)90174-HCrossref PubMed Scopus (444) Google Scholar), little is known about caspase activity and regulation in other basal species (7Ramirez M.L.G. 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Springer International Publishing, New York2016: 441-466Crossref Scopus (25) Google Scholar) were some of the first invertebrate caspases to be characterized, but they have proved to be poor models for studying the evolution of the vertebrate apoptotic network as their networks utilize fewer caspases and regulatory proteins compared with higher eukaryotes. C. elegans, for example, utilizes only one effector caspase (CED-3), which also bears a CARD-motif necessary for its activation (9Irmler M. Hofmann K. Vaux D. Tschopp J. Direct physical interaction between the Caenorhabditis elegans “death proteins” CED-3 and CED-4.FEBS Lett. 1997; 406 (9109415): 189-19010.1016/S0014-5793(97)00271-8Crossref PubMed Scopus (72) Google Scholar). Moreover, cytochrome c is not involved in the formation of the apoptosome in Drosophila, indicating that this organism lacks the intrinsic pathway found in humans (2Dorstyn L. Mills K. Lazebnik Y. Kumar S. The two cytochrome c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells.J. Cell Biol. 2004; 167 (15533997): 405-41010.1083/jcb.200408054Crossref PubMed Scopus (99) Google Scholar). In contrast, it now appears that vertebrates have retained many characteristics of the apoptotic machinery found in sponges, sea anemone, and coral (10Tchernov D. Kvitt H. Haramaty L. Bibby T.S. Gorbunov M.Y. Rosenfeld H. Falkowski P.G. Apoptosis and the selective survival of host animals following thermal bleaching in zooxanthellate corals.Proc. Natl. Acad. Sci. U. S. A. 2011; 108 (21636790): 9905-990910.1073/pnas.1106924108Crossref PubMed Scopus (126) Google Scholar, 11Wiens M. Krasko A. Perovic S. Müller W.E.G. Caspase-mediated apoptosis in sponges: Cloning and function of the phylogenetic oldest apoptotic proteases from Metazoa.Biochim. Biophys. Acta. 2003; 1593 (12581862): 179-18910.1016/s0167-4889(02)00388-9Crossref PubMed Scopus (58) Google Scholar, 12Furla P. Sabourault C. Zucchini N. Allemand D. Courtiade J. Richier S. Oxidative stress and apoptotic events during thermal stress in the symbiotic sea anemone, Anemonia viridis.FEBS J. 2006; 273 (16907933): 4186-419810.1111/j.1742-4658.2006.05414.xCrossref PubMed Scopus (93) Google Scholar). Genomic studies of cnidarians, the sister group to the bilateria, revealed many genes that were previously thought to have been vertebrate innovations, demonstrating that the extensive gene loss in C. elegans and in Drosophila resulted in apoptotic pathways that do not reflect the characteristics of ancestral metazoans (13Salvesen G.S. Walsh C.M. Functions of caspase 8: The identified and the mysterious.Semin. Immunol. 2014; 26 (24856110): 246-25210.1016/j.smim.2014.03.005Crossref PubMed Scopus (98) Google Scholar, 14Kortschak R.D. Samuel G. Saint R. Miller D.J. EST analysis of the cnidarian Acropora millepora reveals extensive gene loss and rapid sequence divergence in the model invertebrates.Curr. Biol. 2003; 13 (14680636): 2190-219510.1016/j.cub.2003.11.030Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). Basal metazoans, which appear to have a full complement of apoptotic signaling molecules, may therefore be more relevant to the evolutionary pathways of vertebrate apoptotic networks. Cnidarians including reef-building corals from the genus Scleractinia are ecologically important organisms that are on the decline (15Hughes T.P. Barnes M.L. Bellwood D.R. Cinner J.E. Cumming G.S. Jackson J.B.C. Kleypas J. Van De Leemput I.A. Lough J.M. Morrison T.H. Palumbi S.R. Van Nes E.H. Scheffer M. Coral reefs in the Anthropocene.Nature. 2017; 546 (28569801): 82-9010.1038/nature22901Crossref PubMed Scopus (942) Google Scholar) and cell death has been indicated to be important in these processes (16Dunn S.R. Schnitzler C.E. Weis V.M. Apoptosis and autophagy as mechanisms of dinoflagellate symbiont release during cnidarian bleaching: Every which way you lose.Proc. R. Soc. B Biol. Sci. 2007; 274 (17925275): 3079-308510.1098/rspb.2007.0711Crossref PubMed Scopus (128) Google Scholar). The two primary drivers of coral declines are marine diseases affecting reef-building corals (17Maynard J. Van Hooidonk R. Eakin C.M. Puotinen M. Garren M. Williams G. Heron S.F. Lamb J. Weil E. Willis B. Harvell C.D. Projections of climate conditions that increase coral disease susceptibility and pathogen abundance and virulence.Nature Clim. Change. 2015; 5: 688-69410.1038/nclimate2625Crossref Scopus (167) Google Scholar) as well as temperature-induced loss of the coral's symbiont known as bleaching (15Hughes T.P. Barnes M.L. Bellwood D.R. Cinner J.E. Cumming G.S. Jackson J.B.C. Kleypas J. Van De Leemput I.A. Lough J.M. Morrison T.H. Palumbi S.R. Van Nes E.H. Scheffer M. Coral reefs in the Anthropocene.Nature. 2017; 546 (28569801): 82-9010.1038/nature22901Crossref PubMed Scopus (942) Google Scholar). Coral possess an innate immune system that both defends the animals against pathogenic organisms and also serves as general stress responses (18Mansfield K.M. Gilmore T.D. Innate immunity and cnidarian-Symbiodiniaceae mutualism.Dev. Comp. Immunol. 2019; 90 (30268783): 199-20910.1016/j.dci.2018.09.020Crossref PubMed Scopus (32) Google Scholar). Therefore, the coral immune system is critical in the response of these organisms to both coral diseases and bleaching. Activation of the innate immune system activates apoptotic pathways (19Fuess L.E. Pinzón C J.H. Weil E. Grinshpon R.D. Mydlarz L.D. Life or death: Disease-tolerant coral species activate autophagy following immune challenge.Proc. R. Soc. Biol. Sci. 2017; 284 (28592676): 2017077110.1098/rspb.2017.0771Crossref PubMed Scopus (45) Google Scholar); however, to date very few functional studies have been performed to characterize caspase structure and subsequent function in corals (20Palmer C.V. Traylor-Knowles N. Towards an integrated network of coral immune mechanisms.Proc. R. Soc. B Biol. Sci. 2012; 279 (22896649): 4106-411410.1098/rspb.2012.1477Crossref PubMed Scopus (88) Google Scholar). There are several examples pointing to the importance of apoptotic pathways and caspases in coral survival to both disease and temperature stress. An increase in expression of apoptosis-related genes was detected in a diseased Caribbean soft coral resulting in a visible inflammatory response (black-melanized appearance) (21Fuess L.E. Mann W.T. Jinks L.R. Brinkhuis V. Mydlarz L.D. Transcriptional analyses provide new insight into the late-stage immune response of a diseased Caribbean coral.R. Soc. Open Sci. 2018; 5 (29892394): 17206210.1098/rsos.172062Crossref PubMed Scopus (21) Google Scholar). Also, executioner caspase genes were up-regulated in the branching coral Acropora infected with white band disease (22Libro S. Kaluziak S.T. Vollmer S.V. RNA-seq profiles of immune related genes in the staghorn coral Acropora cervicornis infected with white band disease.PLoS One. 2013; 8 (24278460): e8182110.1371/journal.pone.0081821Crossref PubMed Scopus (89) Google Scholar). Several studies have gleaned important insights into coral apoptosis post-temperature stress by demonstrating that corals activate cell death responses following expulsion of their algal symbiont (19Fuess L.E. Pinzón C J.H. Weil E. Grinshpon R.D. Mydlarz L.D. Life or death: Disease-tolerant coral species activate autophagy following immune challenge.Proc. R. Soc. Biol. Sci. 2017; 284 (28592676): 2017077110.1098/rspb.2017.0771Crossref PubMed Scopus (45) Google Scholar, 23Kaniewska P. Campbell P.R. Kline D.I. Rodriguez-Lanetty M. Miller D.J. Dove S. Hoegh-Guldberg O. Major cellular and physiological impacts of ocean acidification on a reef building coral.PLoS One. 2012; 7 (22509341): e3465910.1371/journal.pone.0034659Crossref PubMed Scopus (183) Google Scholar, 24Dunn S.R. Thomason J.C. Le Tissier M.D.A. Bythell J.C. Heat stress induces different forms of cell death in sea anemones and their endosymbiotic algae depending on temperature and duration.Cell Death Differ. 2004; 11 (15286684): 1213-122210.1038/sj.cdd.4401484Crossref PubMed Scopus (136) Google Scholar, 25Dunn S.R. Bythell J.C. Le Tissier M.D. Burnett W.J. Thomason J.C. Programmed cell death and cell necrosis activity during hyperthermic stress-induced bleaching of the symbiotic sea anemone Aiptasia sp.J. Exp. Mar. Biol. Ecol. 2002; 272: 29-5310.1016/S0022-0981(02)00036-9Crossref Scopus (129) Google Scholar). Specifically, the anti-apoptotic protein Bcl-2 in Acropora millepora is up-regulated during temperature stress (26Pernice M. Dunn S.R. Miard T. Dufour S. Dove S. Hoegh-Guldberg O. Regulation of apoptotic mediators reveals dynamic responses to thermal stress in the reef building coral Acropora millepora.PLoS One. 2011; 6 (21283671): e1609510.1371/journal.pone.0016095Crossref PubMed Scopus (64) Google Scholar), indicating that this species likely has an intrinsic apoptosis mechanism as well as mechanisms to regulate this process. Interestingly, it was shown that application of caspase inhibitors can prevent the death of bleached coral (10Tchernov D. Kvitt H. Haramaty L. Bibby T.S. Gorbunov M.Y. Rosenfeld H. Falkowski P.G. Apoptosis and the selective survival of host animals following thermal bleaching in zooxanthellate corals.Proc. Natl. Acad. Sci. U. S. A. 2011; 108 (21636790): 9905-990910.1073/pnas.1106924108Crossref PubMed Scopus (126) Google Scholar). Collectively, the data show the potential for complex apoptotic signaling pathways in coral but data on activation and control mechanisms, and how they compare with those in vertebrates, are lacking because of a dearth of biochemical characterization. To gain insight into caspase activity and regulation in coral, we expressed and characterized two caspases each from two species of Caribbean reef-building corals, Orbicella faveolata and Porites astreoides. The two coral species are found on opposite ends of the stress-tolerance spectrum where the disease-susceptible O. faveolata activates caspase-mediated apoptotic pathways upon immune challenge, whereas the disease-tolerant P. astreoides activates an adaptive autophagic response (19Fuess L.E. Pinzón C J.H. Weil E. Grinshpon R.D. Mydlarz L.D. Life or death: Disease-tolerant coral species activate autophagy following immune challenge.Proc. R. Soc. Biol. Sci. 2017; 284 (28592676): 2017077110.1098/rspb.2017.0771Crossref PubMed Scopus (45) Google Scholar). These findings indicate that understanding the apoptotic machinery in corals likely has significant implication in understanding species stress tolerance. In this investigation we describe the structural composition of each species' caspase repertoire, and we use these data to functionally characterize both initiator and effector caspases from both species. Two proteins referred to as PaCasp7a and OfCasp3a based on sequences similarity to human caspases contain CARD motifs at the N terminus, an unusual combination that has not been observed in caspases-3 or -7 enzymes from higher eukaryotes, and indeed these proteins function as initiator caspases. Additionally, two proteins PaCasp3 and OfCasp3b show canonical caspase-3/7 structural organization, with short pro-domains, and possess effector caspase function. We describe the first biochemical characterization of coral caspases and show that the PaCasp3 and OfCasp3b enzymes are not activated directly by the CARD-containing PaCasp7a and OfCasp3a, respectively. We also report the first X-ray crystal structure of a coral caspase, that of PaCasp7a determined at 1.57 Å resolution, which reveals an N-terminal peptide bound near the active site that may serve as a regulatory exosite. Overall, we find support for complex apoptotic mechanisms in these early metazoans, where the cellular machinery for both intrinsic and extrinsic apoptosis has ancient evolutionary origins. We examined seven caspase genes from O. faveolata based on sequences obtained from previous transcriptomic and genomic data (Fig. S1 and Table S1) (19Fuess L.E. Pinzón C J.H. Weil E. Grinshpon R.D. Mydlarz L.D. Life or death: Disease-tolerant coral species activate autophagy following immune challenge.Proc. R. Soc. Biol. Sci. 2017; 284 (28592676): 2017077110.1098/rspb.2017.0771Crossref PubMed Scopus (45) Google Scholar). The caspases were named based on the E-value from BLAST as well as the sequence similarity to the human orthologs. Results from examining the sequence homology and domain organization suggest that three of the caspases are apoptotic initiators and four are apoptotic effectors in O. faveolata (Fig. 1A). The sequence identities of the seven caspases compared with most human caspases are low, only ∼35% (Table 1), so it is difficult to determine the nature of each coral caspase based solely on sequence comparisons with human orthologs. In addition, two caspases from O. faveolata contain an N-terminal caspase activation and recruitment domain (CARD) motif, similar to those in HsCasp2 and HsCasp9, and one caspase contains tandem death effector domain (DED) motifs, similar to that found in HsCasp8 (Fig. 1A). The remaining four proteins show domain organization similar to the human effector caspases, with short pro-domains (Fig. 1A).Table 1Protein sequence identity/similarity (%) with human caspasesHsCasp3HsCasp7HsCasp6HsCasp2HsCasp8HsCasp10HsCasp9OfCasp737/5438/5232/4928/4334/5033/5334/50OfCasp3c36/5835/5635/5232/4837/5337/5433/49OfCasp3b35/6032/5733/5232/4937/5533/5334/50OfCasp3a47/6945/6538/5429/4839/5439/5528/46OfCasp237/5341/5535/4633/5234/5235/5232/48OfCasp8a39/5639/5334/4835/5332/5830/4934/51OfCasp8b33/5631/5031/4932/5235/5134/5232/46PaCasp336/5837/5936/5633/4937/5434/5435/50PaCasp7a43/6544/6036/5328/4637/5337/5328/44PaCasp7b38/5437/5234/4929/4631/4830/5033/48PaCasp239/5338/5233/4833/5035/5335/5232/49 Open table in a new tab In the case of P. astreoides, four caspase sequences consisted of two initiator-like caspases (called PaCasp7a and PaCasp2) and two effector-like caspases (called PaCasp7b and PaCasp3) (Fig. 1A and Fig. S1). Similar to the results for O. faveolata, the caspase sequences from P. astreoides also have only ∼35% identity with human caspases, regardless of comparisons to initiator or effector caspases (Table 1). The sequences from the two coral species displayed much higher identity to putative homologs in the other coral species. For example, PaCasp7a has a 77% sequence identity with OfCasp3a, whereas PaCasp3 has 71 and 73% sequence identity, respectively, with OfCasp3b and OfCasp3c. Likewise, PaCasp2 demonstrates 76% sequence identity with OfCasp2, and PaCasp7b shares 60% identity with OfCasp7 (Fig. 1B). A phylogenetic analysis of cnidarian and vertebrate caspases demonstrated that cnidarian caspases cluster in separate groups (Fig. 2A). All of the short pro-domain caspases, including PaCasp3 and OfCasp3b, cluster together between vertebrate effector (caspases-3/7) and initiator (caspases-8/10) caspases. Interestingly, the comparative genomics and phylogenetic analyses suggest that short cnidarian caspases, that is, those lacking a CARD or DED, share a common ancestor with vertebrate effector caspases-3 and -7 and with initiator caspases-8 and -10 (Fig. 2A). Homologs of caspase-8 in coral share the same clade with vertebrate caspases-8 and -10 and the CARD-containing OfCasp2 and PaCasp2 clustered with vertebrate caspase-2. With the exceptions of OfCasp2 and PaCasp2, the other CARD-containing coral caspases cluster with OfCasp3a and PaCasp7a and segregate into a different clade, although they share a common ancestor with vertebrate caspases-2 and -9. We analyzed the CARD motifs of cnidarian caspases independently of the protease domains and compared them to the CARD motifs of vertebrate caspases-2 and -9 as well as that of caspase-2 and RIPK1 domain containing adaptor with death domain (CRADD) motifs, which recruit caspase-2 to the PIDDosome (27Park H.H. Structural features of caspase-activating complexes.Int. J. Mol. Sci. 2012; 13 (22606010): 4807-481810.3390/ijms13044807Crossref PubMed Scopus (57) Google Scholar) (Fig. 2B). The CARD motifs of coral caspases-3 and -7 cluster together but are more closely related to the CARD of caspase-2 than those of caspase-9 or CRADD. Based on this analysis, there appear to be many CARD-containing caspase-3–like proteins in cnidaria. At present, it is not clear why CARD-containing caspase-3–like proteins provide an advantage for coral development and/or symbiosis because the animals also contain initiator caspases that presumably activate the short pro-domain effector caspases. CARD-containing caspase-3–like proteins are rarely observed in vertebrate effector caspases. Fish-specific caspases have been found, such as the CARD-containing caspase-8 for example (28Sakamaki K. Satou Y. Caspases: Evolutionary aspects of their functions in vertebrates.J. Fish Biol. 2009; 74 (20735596): 727-75310.1111/j.1095-8649.2009.02184.xCrossref PubMed Scopus (78) Google Scholar), but caspase-2 is, at present, the only characterized DXXDase with a CARD. We chose two caspases from each species to characterize further, based on the sequence comparisons with human effector caspases-3, -6, or -7. In the case of O. faveolata, we chose two caspase-3–like proteins that showed 47 and 35% sequence identity, respectively, with HsCasp3, and we named the two proteins OfCasp3a and OfCasp3b, respectively (Fig. 1A and Table 1). Interestingly, despite predicted similarity to HsCasp3, OfCasp3a also has an N-terminal CARD motif. One caspase from P. astreoides demonstrated the highest sequence identity with HsCasp7 (44%) and was named PaCasp7a, even though it also contains a CARD motif (Fig. 1A and Table 1). The second protein from P. astreoides showed similar sequence identity to human caspases-3, -6, -7, and -8 (36–37%) (Fig. 1A and Table 1), but the protein does not have a DED motif like caspase-8 and the domain organization is more similar to that of caspase-3. Consequently, we named the protein PaCasp3. Overall, the low sequence identity between the vertebrate and invertebrate caspases show that the classification is somewhat arbitrary without further biochemical characterizations of the proteins. Together, the phylogenetic analysis shows that the caspases from P. astreoides and O. faveolata have relatively low sequence identity (∼40%) to mammalian caspases as well as other vertebrate families, but the proteins had much higher sequence identities to caspases from other cnidarian species, such as Pocillopora damicornis, Stylophora pistillata, and Nematostella vectensis. An analysis of the coral caspase sequences shows that the proteins contain all of the conserved features that define a caspase. For example, each protein contains the catalytic dyad, histidine (CP-075) and cysteine (CP-117) (Fig. 3), where “CP” refers to the common position defined previously for caspases (29Grinshpon R.D. Williford A. Titus-McQuillan J. Clay Clark A. The CaspBase: A curated database for evolutionary biochemical studies of caspase functional divergence and ancestral sequence inference.Protein Sci. 2018; 27 (30076665): 1857-187010.1002/pro.3494Crossref PubMed Scopus (10) Google Scholar). The conserved sequence that contains the catalytic histidine (CP-115)-QACRG-(CP-119) is found in the four coral caspases, although PaCasp7a and OfCasp3a contain QACQG as in human caspase-8. One of the most highly variable regions, the intersubunit linker (IL) is the same length in OfCasp3b and PaCasp3 compared with that of HsCasp3, whereas those of PaCasp7a and OfCasp3a have one and two amino acids fewer than HsCasp3, respectively (Fig. 3). We examined the four coral caspases by size exclusion chromatography because CARD-containing human caspases are monomers or mixtures of weak protomer-dimer (30Clark A.C. Caspase allostery and conformational selection.Chem. Rev. 2016; 116 (26750439): 6666-670610.1021/acs.chemrev.5b00540Crossref PubMed Scopus (44) Google Scholar). Because the IL of the procaspase monomer is cleaved during activation, the protomer is defined as a single unit that contains a large and small subunit and a single active site. Thus, the dimer consists of two protomers, or is more formally considered a dimer of heterodimers. The data show that the CARD containing coral caspases, PaCasp7a and OfCasp3a, elute in a single peak with molecular mass of 42.6 and 44 kDa, respectively. The sizes are larger than that of a protomer but smaller than a dimer (Fig. S2 and Table S5), suggesting that the proteins form weak dimers similar to the human initiator caspases. In contrast, the short pro-domain containing caspases, PaCasp3 and OfCasp3b, are dimers similar to the human effector caspases, with molecular mass of 64.5 and 69.2 kDa, respectively (Fig. S3 and Table S5). We also determined the mass of the large and small subunits by MS. Caspase zymogens are cleaved in the IL, and the N-terminal CARD or pro-domain is removed during activation (30Clark A.C. Caspase allostery and conformational selection.Chem. Rev. 2016; 116 (26750439): 6666-670610.1021/acs.chemrev.5b00540Crossref PubMed Scopus (44) Google Scholar). The proteins also autoprocess during overexpression in Escherichia coli. The molecular size of the large and small subunits of each caspase, determined by MS, are shown in Table S5. When compared with the sequences for each protein (Fig. 3), the data show that OfCasp3a and PaCasp7a are cleaved in the intersubunit linker after (CP-127)-DVTD-(CP-130), whereas OfCasp3b and PaCasp3 are cleaved after (CP-127)-VESD-(CP-130). The actual amino acid positions, in addition to the common position number, are shown in Fig. 1A, and the cleavage sites are indicated by the arrow in Fig. 3. In addition, the first 20 or 31 amino acids, respectively, in the pro-domains of OfCasp3b and PaCasp3 are removed following cleavage after VIGD (Asp20) (OfCasp3b) or SSTD (Asp31) (PaCasp3). The CARD motifs of OfCasp3a and of PaCasp7a are removed following cleavage after DEAD (Asp123) and DQAD (Asp119), respectively (Figs. 1A and 3). We note that there are potentially other cleavage sites in the CARD motifs, but in our assays the CARD motif was completely removed. We characterized the substrate specificity for each of the four coral caspases using substrate-phage display assays, as described previously (31Tucker M.B. MacKenzie S.H. Maciag J.J. Dirscherl Ackerman H. Swartz P. Yoder J.A. Hamilton P.T. Clay Clark A. Phage display and structural studies reveal plasticity in substrate specificity of caspase-3a from zebrafish.Protein Sci. 2016; 25 (27577093): 2076-208810.1002/pro.3032Crossref PubMed Scopus (10) Google Scholar). In these assays, we utilize two substrate-phage libraries that determine the P5-P1′ substrate preferences, with either aspartate fixed at the P1 position (P5-XXXXDX-P1′) or random (called 6×), and the results were the same for both libraries. The data show that PaCasp7a and OfCasp3a have Group II specificity, with a preference for aspartate in the P4 position (DXXDase) (Fig. 4, A and B). In contrast, PaCasp3 and OfCasp3b prefer valine in the P4 position (VXXDase) (Fig. 4, C and D), which is defined as Group III specificity like HsCasp6. The activities of PaCasp7a and of OfCasp3a were also examined using DEVD-AFC and VEID-AFC substrates. In all cases, however, the activity against the tetrapeptide substrates was very low because of Km values >500 μm, so we could not reliably determine the steady-state catalytic parameters kcat or Km from the small peptide activity assays. In caspases, the Km is thought to correlate with substrate binding (KD), so the high Km suggests poor binding of the small peptide. Because of the low activity in small peptide assays, we tested the coral caspases for their ability to hydrolyze full-length human procaspases-3 and -6, which were made catalytically inactive}, 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={Water and ligand binding play critical roles in the structure and function of proteins, yet their binding sites and significance are difficult to predict a priori. Multiple solvent crystal structures (MSCS) is a method where several X‐ray crystal structures are solved, each in a unique solvent environment, with organic molecules that serve as probes of the protein surface for sites evolved to bind ligands, while the first hydration shell is essentially maintained. When superimposed, these structures contain a vast amount of information regarding hot spots of protein‐protein or protein‐ligand interactions, as well as conserved water‐binding sites retained with the change in solvent properties. Optimized mining of this information requires reliable structural data and a consistent, objective analysis tool. Detection of related solvent positions (DRoP) was developed to automatically organize and rank the water or small organic molecule binding sites within a given set of structures. It is a flexible tool that can also be used in conserved water analysis given multiple structures of any protein independent of the MSCS method. The DRoP output is an HTML format list of the solvent sites ordered by conservation rank in its population within the set of structures, along with renumbered and recolored PDB files for visualization and facile analysis. Here, we present a previously unpublished set of MSCS structures of bovine pancreatic ribonuclease A (RNase A) and use it together with published structures to illustrate the capabilities of DRoP.}, 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 ", {:sub=>"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", {:sub=>"interior"}, ", suggesting that the other site, TCP", {:sub=>"exterior"}, ", remains viable for binding peroxygenase substrates. Taken together, these studies demonstrate that blocking the TCP", {:sub=>"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 bonds via an 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 from Neurospora crassa (NcPMO-2) was heterologously expressed in Pichia pastoris to facilitate crystallographic studies of the fungal LPMO mechanism. Diffraction resolution and crystal morphology were improved by expressing NcPMO-2 from a glycoengineered strain of P. pastoris and 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 large NcPMO-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={The regulation of caspase‐3 enzyme activity is a vital process in cell fate decisions leading to cell differentiation and tissue development or to apoptosis. The zebrafish, Danio rerio, has become an increasingly popular animal model to study several human diseases because of their transparent embryos, short reproductive cycles, and ease of drug administration. While apoptosis is an evolutionarily conserved process in metazoans, little is known about caspases from zebrafish, particularly regarding substrate specificity and allosteric regulation compared to the human caspases. We cloned zebrafish caspase‐3a (casp3a) and examined substrate specificity of the recombinant protein, Casp3a, compared to human caspase‐3 (CASP3) by utilizing M13 bacteriophage substrate libraries that incorporated either random amino acids at P5‐P1′ or aspartate fixed at P1. The results show a preference for the tetrapeptide sequence DNLD for both enzymes, but the P4 position of zebrafish Casp3a also accommodates valine equally well. We determined the structure of zebrafish Casp3a to 2.28Å resolution by X‐ray crystallography, and when combined with molecular dynamics simulations, the results suggest that a limited number of amino acid substitutions near the active site result in plasticity of the S4 sub‐site by increasing flexibility of one active site loop and by affecting hydrogen‐bonding with substrate. The data show that zebrafish Casp3a exhibits a broader substrate portfolio, suggesting overlap with the functions of caspase‐6 in zebrafish development.}, 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={The native ensemble of caspases is described globally by a complex energy landscape where the binding of substrate selects for the active conformation, whereas targeting an allosteric site in the dimer interface selects an inactive conformation that contains disordered active-site loops. Mutations and posttranslational modifications stabilize high-energy inactive conformations, with mostly formed, but distorted, active sites. To examine the interconversion of active and inactive states in the ensemble, we used detection of related solvent positions to analyze 4,995 waters in 15 high-resolution (<2.0 Å) structures of wild-type caspase-3, resulting in 450 clusters with the most highly conserved set containing 145 water molecules. The data show that regions of the protein that contact the conserved waters also correspond to sites of posttranslational modifications, suggesting that the conserved waters are an integral part of allosteric mechanisms. To test this hypothesis, we created a library of 19 caspase-3 variants through saturation mutagenesis in a single position of the allosteric site of the dimer interface, and we show that the enzyme activity varies by more than four orders of magnitude. Altogether, our database consists of 37 high-resolution structures of caspase-3 variants, and we demonstrate that the decrease in activity correlates with a loss of conserved water molecules. The data show that the activity of caspase-3 can be fine-tuned through globally desolvating the active conformation within the native ensemble, providing a mechanism for cells to repartition the ensemble and thus fine-tune activity through conformational selection.}, 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) from Hypocrea jecorina was 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 group C2, with unit-cell parameters a=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 group P6₃22, with unit-cell parameters a=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={The multiple solvent crystal structures (MSCS) method uses organic solvents to map the surfaces of proteins. It identifies binding sites and allows for a more thorough examination of protein plasticity and hydration than could be achieved by a single structure. The crystal structures of bovine pancreatic ribonuclease A (RNAse A) soaked in the following organic solvents are presented: 50% dioxane, 50% dimethylformamide, 70% dimethylsulfoxide, 70% 1,6‐hexanediol, 70% isopropanol, 50% R,S,R‐bisfuran alcohol, 70% t‐butanol, 50% trifluoroethanol, or 1.0M trimethylamine‐N‐oxide. This set of structures is compared with four sets of crystal structures of RNAse A from the protein data bank (PDB) and with the solution NMR structure to assess the validity of previously untested assumptions associated with MSCS analysis. Plasticity from MSCS is the same as from PDB structures obtained in the same crystal form and deviates only at crystal contacts when compared to structures from a diverse set of crystal environments. Furthermore, there is a good correlation between plasticity as observed by MSCS and the dynamic regions seen by NMR. Conserved water binding sites are identified by MSCS to be those that are conserved in the sets of structures taken from the PDB. Comparison of the MSCS structures with inhibitor‐bound crystal structures of RNAse A reveals that the organic solvent molecules identify key interactions made by inhibitor molecules, highlighting ligand binding hot‐spots in the active site. The present work firmly establishes the relevance of information obtained by MSCS. Proteins 2009. © 2009 Wiley‐Liss, Inc.}, 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} }