@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. 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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. 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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. 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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{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
          The interconversion of states in the caspase-3 native ensemble is affected by binding of ligands that either stabilize or destabilize active-site loops. It is not clear how the ensemble is regulated in cells, aside from modulating levels of endogenous caspase inhibitors. We describe a library of caspase-3 variants with activities that vary by more than four orders of magnitude and show that removal of conserved water molecules may provide a strategy to design novel allosteric inhibitors that globally destabilize the active conformation within the ensemble. Our results suggest that posttranslational modifications fine-tune caspase activity by disrupting conserved water networks, and our database provides an approach to examine caspase signaling in cells by modifying caspase-3 activity while simultaneously maintaining endogenous enzyme levels.}, 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{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{ma_mackenzie_clark_2014, title={Redesigning the procaspase-8 dimer interface for improved dimerization}, volume={23}, number={4}, journal={Protein Science}, author={Ma, C. X. and MacKenzie, S. H. and Clark, A. C.}, year={2014}, pages={442–453} }
 @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{mackenzie_clark_2013, title={Slow folding and assembly of a procaspase-3 interface variant}, volume={52}, number={20}, journal={Biochemistry}, author={MacKenzie, S. H. and Clark, A. C.}, year={2013}, pages={3415–3427} }
 @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{schipper_mackenzie_sharma_clark_2011, title={A bifunctional allosteric site in the dimer interface of procaspase-3}, volume={159}, ISSN={["1873-4200"]}, DOI={10.1016/j.bpc.2011.05.013}, abstractNote={The dimer interface of caspase-3 contains a bifunctional allosteric site in which the enzyme can be activated or inactivated, depending on the context of the protein. In the mature caspase-3, the binding of allosteric inhibitors to the interface results in an order-to-disorder transition in the active site loops. In procaspase-3, by contrast, the binding of allosteric activators to the interface results in a disorder-to-order transition in the active site. We have utilized the allosteric site to identify a small molecule activator of procaspase and to characterize its binding to the protease. The data suggest that an efficient activator must stabilize the active conformer of the zymogen by expelling the intersubunit linker from the interface, and it must interact with active site residues found in the allosteric site. Small molecule activators that fulfill the two requirements should provide scaffolds for drug candidates as a therapeutic strategy for directly promoting procaspase-3 activation in cancer cells.}, number={1}, journal={BIOPHYSICAL CHEMISTRY}, author={Schipper, Joshua L. and MacKenzie, Sarah H. and Sharma, Anil and Clark, A. Clay}, year={2011}, month={Nov}, pages={100–109} }
 @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} }
 @misc{mackenzie_schipper_clark_2010, title={The potential for caspases in drug discovery}, volume={13}, number={5}, journal={Current Opinion in Drug Discovery & Development}, author={MacKenzie, S. H. and Schipper, J. L. and Clark, A. C.}, year={2010}, pages={568–576} }
 @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{milam_clark_2009, title={Folding and assembly kinetics of procaspase-3}, volume={18}, ISSN={["1469-896X"]}, DOI={10.1002/pro.259}, abstractNote={AbstractCaspases are vital to apoptosis and exist in the cell as inactive zymogens. Dimerization is central to procaspase activation because the active sites are comprised of loops from both monomers. Although initiator procaspases are stable monomers until activated on cell death scaffolds, the effector caspases, such as procaspase‐3, are stable dimers. The activation mechanisms are reasonably well understood in terms of polypeptide chain cleavage and subsequent active site rearrangements in the dimer, but the mechanisms that govern dimer assembly are not known. To further understand procaspase dimerization, we examined the folding and assembly of procaspase‐3 by fluorescence emission, circular dichroism, differential quenching by acrylamide, anisotropy, and enzyme activity assays. Single‐mixing stopped‐flow refolding studies showed a complex burst phase in which multiple monomeric species form rapidly. At longer times, the monomer folds through several intermediates, some of which appear to be off‐pathway or misfolded, before eventually forming a dimerization‐competent species. Enzyme activity studies demonstrated a slow rate of dimerization (∼70 M−1 s−1). In addition, single‐mixing stopped‐flow unfolding studies revealed a complex unfolding process with a slow rate of dimer dissociation. Interestingly, multiple dimeric species were observed in the burst phase for unfolding, suggesting that the native ensemble consists of at least two major conformations. Collectively, these results demonstrate complex folding and unfolding behavior for procaspase‐3 and suggest that slow dimerization results from the lack of stabilizing native contacts in the initial encounter complex.}, number={12}, journal={PROTEIN SCIENCE}, author={Milam, Sara L. and Clark, A. Clay}, year={2009}, month={Dec}, pages={2500–2517} }
 @misc{walters_milam_clark_2009, title={Practical approaches to protein folding and assembly: Spectroscopic strategies in thermodynamics and kinetics}, volume={455}, journal={Methods in enzymology: biothermodynamics,vol 455,  part a}, author={Walters, J. and Milam, S. L. and Clark, A. C.}, year={2009}, pages={1–39} }
 @misc{mattos_clark_2008, title={Minimizing frustration by folding in an aqueous environment}, volume={469}, number={1}, journal={Archives of Biochemistry and Biophysics}, author={Mattos, C. and Clark, A. C.}, year={2008}, pages={118–131} }
 @article{clark_2008, title={Protein folding: Are we there yet?}, volume={469}, number={1}, journal={Archives of Biochemistry and Biophysics}, author={Clark, A. C.}, year={2008}, pages={1–3} }
 @misc{mackenzie_clark_2008, title={Targeting cell death in tumors by activating Caspases}, volume={8}, number={2}, journal={Current Cancer Drug Targets}, author={MacKenzie, S. H. and Clark, A. C.}, year={2008}, pages={98–109} }
 @article{milam_nicely_feeney_mattos_clark_2007, title={Rapid folding and unfolding of Apaf-1 CARD}, volume={369}, ISSN={["0022-2836"]}, DOI={10.1016/j.jmb.2007.02.105}, abstractNote={Caspase recruitment domains (CARDs) are members of the death domain superfamily and contain six antiparallel helices in an α-helical Greek key topology. We have examined the equilibrium and kinetic folding of the CARD of Apaf-1 (apoptotic protease activating factor 1), which consists of 97 amino acid residues, at pH 6 and pH 8. The results showed that an apparent two state equilibrium mechanism is not adequate to describe the folding of Apaf-1 CARD at either pH, suggesting the presence of intermediates in equilibrium unfolding. Interestingly, the results showed that the secondary structure is less stable than the tertiary structure, based on the transition mid-points for unfolding. Single mixing and sequential mixing stopped-flow studies showed that Apaf-1 CARD folds and unfolds rapidly and suggest a folding mechanism that contains parallel channels with two unfolded conformations folding to the native conformation. Kinetic simulations show that a slow folding phase is described by a third conformation in the unfolded ensemble that interconverts with one or both unfolded species. Overall, the native ensemble is formed rapidly upon refolding. This is in contrast to other CARDs in which folding appears to be dominated by formation of kinetic traps.}, number={1}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Milam, Sara L. and Nicely, Nathan I. and Feeney, Brett and Mattos, Carla and Clark, A. Clay}, year={2007}, month={May}, pages={290–304} }
 @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{chen_clark_2006, title={Substitutions of prolines examine their role in kinetic trap formation of the caspase recruitment domain (CARD) of RICK}, volume={15}, ISSN={["1469-896X"]}, DOI={10.1110/ps.051943006}, abstractNote={AbstractCaspase recruitment domains (CARDs) are small helical protein domains that adopt the Greek key fold. For the two CARDs studied to date, RICK‐CARD and caspase‐1‐CARD (CP1‐CARD), the proteins unfold by an apparent two‐state process at equilibrium. However, the folding kinetics are complex for both proteins and may contain kinetically trapped species on the folding pathway. In the case of RICK‐CARD, the time constants of the slow refolding phases are consistent with proline isomerism. RICK‐CARD contains three prolines, P47 in turn 3, and P85 and P87. The latter two prolines constitute a nonconserved PxP motif in helix 6. To examine the role of the prolines in the complex folding kinetics of RICK‐CARD, we generated seven proline‐to‐alanine mutants, including three single mutants, three double mutants, and one triple mutant. We examined the spectroscopic properties, equilibrium folding, binding to CP1‐CARD, and folding kinetics. The results show that P85 is critical for maintaining the function of the protein and that all mutations decrease the stability. Results from single mixing and sequential mixing stopped‐flow studies strongly suggest the presence of parallel folding pathways consisting of at least two unfolded populations. The mutations affect the distribution of the two unfolded species, thereby affecting the population that folds through each channel. The two conformations also are present in the triple mutant, demonstrating that interconversion between them is not due to prolyl isomerism. Overall, the data show that the complex folding pathway, especially formation of kinetically trapped species, is not due to prolyl isomerism.}, number={3}, journal={PROTEIN SCIENCE}, author={Chen, YR and Clark, AC}, year={2006}, month={Mar}, pages={395–409} }
 @article{chen_rojanatavorn_clark_shih_2005, title={Characterization and enzymatic degradation of Sup35NM, a yeast prion-like protein}, volume={14}, ISSN={["1469-896X"]}, DOI={10.1110/ps.041234405}, abstractNote={AbstractTransmissible spongiform encephalopathies (TSEs) are believed to be caused by an unconventional infectious agent, the prion protein. The pathogenic and infectious form of prion protein, PrPSc, is able to aggregate and form amyloid fibrils, very stable and resistant to most disinfecting processes and common proteases. Under specific conditions, PrPSc in bovine spongiform encephalopathy (BSE) brain tissue was found degradable by a bacterial keratinase and some other proteases. Since this disease‐causing prion is infectious and dangerous to work with, a model or surrogate protein that is safe is needed for the in vitro degradation study. Here a nonpathogenic yeast prion‐like protein, Sup35NM, cloned and overexpressed in E. coli, was purified and characterized for this purpose. Aggregation and deaggregation of Sup35NM were examined by electron microscopy, gel electrophoresis, Congo red binding, fluorescence, and Western blotting. The degradation of Sup35NM aggregates by keratinase and proteinase K under various conditions was studied and compared. These results will be of value in understanding the mechanism and optimization of the degradation process.}, number={9}, journal={PROTEIN SCIENCE}, author={Chen, CY and Rojanatavorn, K and Clark, AC and Shih, JCH}, year={2005}, month={Sep}, pages={2228–2235} }
 @article{feeney_soderblom_goshe_clark_2006, title={Novel protein purification system utilizing an N-terminal fusion protein and a caspase-3 cleavable linker}, volume={47}, ISSN={1046-5928}, url={http://dx.doi.org/10.1016/j.pep.2005.10.005}, DOI={10.1016/j.pep.2005.10.005}, abstractNote={Coupled with over-expression in host organisms, fusion protein systems afford economical methods to obtain large quantities of target proteins in a fast and efficient manner. Some proteases used for these purposes cleave C-terminal to their recognition sequences and do not leave extra amino acids on the target. However, they are often inefficient and are frequently promiscuous, resulting in non-specific cleavages of the target protein. To address these issues, we created a fusion protein system that utilizes a highly efficient enzyme and leaves no residual amino acids on the target protein after removal of the affinity tag. We designed a glutathione S-transferase (GST)-fusion protein vector with a caspase-3 consensus cleavage sequence located between the N-terminal GST tag and a target protein. We show that the enzyme efficiently cleaves the fusion protein without leaving excess amino acids on the target protein. In addition, we used an engineered caspase-3 enzyme that is highly stable, has increased activity relative to the wild-type enzyme, and contains a poly-histidine tag that allows for efficient removal of the enzyme after cleavage of the fusion protein. Although we have developed this system using a GST tag, the system is amenable to any commercially available affinity tag.}, number={1}, journal={Protein Expression and Purification}, publisher={Elsevier BV}, author={Feeney, Brett and Soderblom, Erik J. and Goshe, Michael B. and Clark, A. Clay}, year={2006}, month={May}, pages={311–318} }
 @article{feeney_clark_2005, title={Reassembly of active caspase-3 is facilitated by the propeptide}, volume={280}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M505834200}, abstractNote={Changes in ionic homeostasis are early events leading up to the commitment to apoptosis. Although the direct effects of cations on caspase-3 activity have been examined, comparable studies on procaspase-3 are lacking. In addition, the effects of salts on caspase structure have not been examined. We have studied the effects of cations on the activities and conformations of caspase-3 and an uncleavable mutant of procaspase-3 that is enzymatically active. The results show that caspase-3 is more sensitive to changes in pH and ion concentrations than is the zymogen. This is due to the loss of both an intact intersubunit linker and the prodomain. The results show that, although the caspase-3 subunits reassemble to the heterotetramer, the activity return is low after the protein is incubated at or below pH 4.5 and then returned to pH 7.5. The data further show that the irreversible step in assembly results from heterotetramer rather than heterodimer dissociation and demonstrate that the active site does not form properly following reassembly. However, active-site formation is fully reversible when reassembly occurs in the presence of the prodomain, and this effect is specific for the propeptide of caspase-3. The data show that the prodomain facilitates both dimerization and active-site formation in addition to stabilizing the native structure. Overall, the results show that the prodomain acts as an intramolecular chaperone during assembly of the (pro)caspase subunits and increases the efficiency of formation of the native conformation.}, number={48}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Feeney, B and Clark, AC}, year={2005}, month={Dec}, pages={39772–39785} }
 @article{bose_clark_2005, title={pH effects on the stability and dimerization of procaspase-3}, volume={14}, number={1}, journal={Protein Science}, author={Bose, K. and Clark, A. C.}, year={2005}, pages={24–36} }
 @article{bobay_benson_naylor_feeney_clark_goshe_strauch_thompson_cavanagh_2004, title={Evaluation of the DNA Binding Tendencies of the Transition State Regulator AbrB†}, volume={43}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi048399h}, DOI={10.1021/bi048399h}, abstractNote={Global transition state regulator proteins represent one of the most diverse classes of prokaryotic transcription factors. One such transition state regulator, AbrB from Bacillus subtilis, is known to bind more than 60 gene targets yet displays specificity within this target set by binding each promoter with a different affinity. Microelectrospray ionization mass spectrometry (microESI-MS), circular dichroism, fluorescence, UV spectroscopy, and molecular modeling were used to elucidate differences among AbrB, DNA, and AbrB-DNA complexes. MicroESI-MS analysis of AbrB confirmed its stable macromolecular state as being tetrameric and verified the same stoichiometric state in complex with DNA targets. MicroESI-MS, circular dichroism, and fluorescence provided relative binding affinities for AbrB-DNA interactions in a qualitative manner. UV spectroscopy was used in a quantitative manner to determine solution phase dissociation constants for AbrB-DNA complexes. General DNA structural parameters for all known natural AbrB binding sequences were also studied and significant similarities in topological constraints (stretch, opening, and propeller twist) were observed. It is likely that these parameters contribute to the differential binding proclivities of AbrB. In addition to providing an improved understanding of transition state regulator-DNA binding properties and structural tendencies of target promoters, this comprehensive and corroborative spectroscopic study endorses the use of microESI-MS for rapidly ascertaining qualitative binding trends in noncovalent systems in a high-throughput manner.}, number={51}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Bobay, Benjamin G. and Benson, Linda and Naylor, Stephen and Feeney, Brett and Clark, A. Clay and Goshe, Michael B. and Strauch, Mark A. and Thompson, Richele and Cavanagh, John}, year={2004}, month={Dec}, pages={16106–16118} }
 @article{feeney_pop_tripathy_clark_2004, title={Ionic interactions near the loop L4 are important for maintaining the active-site environment and the dimer stability of (pro)caspase 3}, volume={384}, number={Dec 15 2004}, journal={Biochemical Journal (London, England : 1984)}, author={Feeney, B. and Pop, C. and Tripathy, A. and Clark, A. C.}, year={2004}, pages={515–525} }
 @article{chen_clark_2004, title={Kinetic traps in the folding/unfolding of procaspase-1 CARD domain}, volume={13}, ISSN={["1469-896X"]}, DOI={10.1110/ps.03521504}, abstractNote={AbstractWe have examined the folding and unfolding of the caspase recruitment domain of procaspase‐1 (CP1‐CARD), a member of the α‐helical Greek key protein family. The equilibrium folding/unfolding of CP1‐CARD is described by a two‐state mechanism, and the results show CP1‐CARD is marginally stable with a ΔG of 1.1 ± 0.2 kcal/mole and an m‐value of 0.65 ± 0.06 kcal/mole/M (10 mM Tris‐HCl at pH 8.0, 1 mM DTT, 25°C). Consistent with the equilibrium folding data, CP1‐CARD is a monomer in solution when examined by size exclusion chromatography. Single‐mixing stopped‐flow refolding and unfolding studies show that CP1‐CARD folds and unfolds rapidly, with no detectable slow phases, and the reactions appear to reach equilibrium within 10 msec. However, double jump kinetic experiments demonstrate the presence of an unfolded‐like intermediate during unfolding. The intermediate converts to the fully unfolded conformation with a half‐time of 10 sec. Interrupted refolding studies demonstrate the presence of one or more nativelike intermediates during refolding, which convert to the native conformation with a half‐time of about 60 sec. Overall, the data show that both unfolding and refolding processes are slow, and the pathways contain kinetically trapped species.}, number={8}, journal={PROTEIN SCIENCE}, author={Chen, YR and Clark, AC}, year={2004}, month={Aug}, pages={2196–2206} }
 @article{bose_pop_feeney_clark_2003, title={An uncleavable procaspase-3 mutant has a lower catalytic efficiency but an active site similar to that of mature caspase-3}, volume={42}, ISSN={["0006-2960"]}, DOI={10.1021/bi034998x}, abstractNote={We have examined the enzymatic activity of an uncleavable procaspase-3 mutant (D9A/D28A/D175A), which contains the wild-type catalytic residues in the active site. The results are compared to those for the mature caspase-3. Although at pH 7.5 and 25 degrees C the K(m) values are similar, the catalytic efficiency (k(cat)) is approximately 130-fold lower in the zymogen. The mature caspase-3 demonstrates a maximum activity at pH 7.4, whereas the maximum activity of procaspase-3 occurs at pH 8.3. The pK(a) values of both catalytic groups, H121 and C163, are shifted to higher pH for procaspase-3. We developed limited proteolysis assays using trypsin and V8 proteases, and we show that these assays allow the examination of amino acids in three of five active site loops. In addition, we examined the fluorescence emission of the two tryptophanyl residues in the active site over the pH range of 2.5-9 as well as the response to several quenching agents. Overall, the data suggest that the major conformational change that occurs upon maturation results in formation of the loop bundle among loops L4, L2, and L2'. The pK(a) values of both catalytic groups decrease as a result of the loop movements. However, loop L3, which comprises the bulk of the substrate binding pocket, does not appear to be unraveled and solvent-exposed, even at lower pH.}, number={42}, journal={BIOCHEMISTRY}, author={Bose, K and Pop, C and Feeney, B and Clark, AC}, year={2003}, month={Oct}, pages={12298–12310} }
 @article{chen_clark_2003, title={Equilibrium and kinetic folding of a alpha-helical Greek key protein domain: Caspase recruitment domain (CARD) of RICK}, volume={42}, ISSN={["0006-2960"]}, DOI={10.1021/bi0340752}, abstractNote={We have characterized the equilibrium and kinetic folding of a unique protein domain, caspase recruitment domain (CARD), of the RIP-like interacting CLARP kinase (RICK) (RICK-CARD), which adopts a α-helical Greek key fold. At equilibrium, the folding of RICK-CARD is well described by a two-state mechanism representing the native and unfolded ensembles. The protein is marginally stable, with a ΔGH2O of 3.0 ± 0.15 kcal/mol and an m-value of 1.27 ± 0.06 kcal mol-1 M-1 (30 mM Tris-HCl, pH 8, 1 mM DTT, 25 °C). While the m-value is constant, the protein stability decreases in the presence of moderate salt concentrations (below 200 mM) and then increases at higher salt concentrations. The results suggest that electrostatic interactions are stabilizing in the native protein, and the favorable Coulombic interactions are reduced at low ionic strength. Above 200 mM salt, the results are consistent with Hofmeister effects. The unfolding pathway of RICK-CARD is complex and contains at least three non-native conformati...}, number={20}, journal={BIOCHEMISTRY}, author={Chen, YR and Clark, AC}, year={2003}, month={May}, pages={6310–6320} }
 @article{pop_feeney_tripathy_clark_2003, title={Mutations in the procaspase-3 dimer interface affect the activity of the zymogen}, volume={42}, ISSN={["0006-2960"]}, DOI={10.1021/bi034999p}, abstractNote={The interface of the procaspase-3 dimer plays a critical role in zymogen maturation. We show that replacement of valine 266, the residue at the center of the procaspase-3 dimer interface, with glutamate resulted in an increase in enzyme activity of approximately 60-fold, representing a pseudoactivation of the procaspase. In contrast, substitution of V266 with histidine abolished the activity of the procaspase-3 as well as that of the mature caspase. While the mutations do not affect the dimeric properties of the procaspase, we show that the V266E mutation may affect the formation of a loop bundle that is important for stabilizing the active site. In contrast, the V266H mutation affects the positioning of loop L3, the loop that forms the bulk of the substrate binding pocket. In some cases, the amino acids affected by the mutations are >20 A from the interface. Overall, the results demonstrate that the integrity of the dimer interface is important for maintaining the proper active site conformation.}, number={42}, journal={BIOCHEMISTRY}, author={Pop, C and Feeney, B and Tripathy, A and Clark, AC}, year={2003}, month={Oct}, pages={12311–12320} }
 @article{shen_clark_huber_2003, title={The C-terminal tail of Arabidopsis 14-3-3 omega functions as an autoinhibitor and may contain a tenth alpha-helix}, volume={34}, ISSN={["1365-313X"]}, DOI={10.1046/j.1365-313X.2003.01739.x}, abstractNote={SummaryThe eukaryotic regulatory protein 14‐3‐3 is involved in many important plant cellular processes including regulation of nitrate assimilation through inhibition of phosphorylated nitrate reductase (pNR) in darkened leaves. Divalent metal cations (Me2+) and some polyamines interact with the loop 8 region of the 14‐3‐3 proteins and allow them to bind and inhibit pNR in vitro. The role of the highly variant C‐terminal regions of the 14‐3‐3 isoforms in regulation by polycations is not clear. In this study, we carried out structural analyses on the C‐terminal tail of the Arabidopsis 14‐3‐3ω isoform and evaluated its contributions to the inhibition of pNR. Nested C‐terminal truncations of the recombinant 14‐3‐3ω protein revealed that the removal of the C‐terminal tail renders the protein partially Mg2+‐independent in both pNR binding and inhibition of activity, suggesting that the C‐terminus functions as an autoinhibitor. The C‐terminus of 14‐3‐3ω appears to undergo a conformational change in the presence of polycations as demonstrated by its increased trypsin cleavage at Lys‐247. C‐terminal truncation of 14‐3‐3ω at Thr‐255 increased its interaction with antibodies to the C‐terminus of 14‐3‐3ω in non‐denaturing conditions, but not in denaturing conditions, suggesting that the C‐terminal tail contains ordered structures that might be disrupted by the truncation. Circular dichroism (CD) analysis of a C‐terminal peptide, from Trp‐234 to Lys‐249, revealed that the C‐terminal tail might contain a tenth α‐helix, in agreement with the in silico predictions. The function of the putative tenth α‐helix is not clear because substituting two prolyl residues within the predicted helix (E245P/I246P mutant), which prevented the corresponding peptide from adopting a helical conformation, did not affect the inhibition of pNR activity in the presence or absence of Mg2+. We propose that in the absence of polycations, access of target proteins to their binding groove in the 14‐3‐3 protein is restricted by the C‐terminus, which acts as part of a gate that opens with the binding of polycations to loop 8.}, number={4}, journal={PLANT JOURNAL}, author={Shen, W and Clark, AC and Huber, SC}, year={2003}, month={May}, pages={473–484} }
 @article{bose_clark_2001, title={Dimeric procaspase-3 unfolds via a four-state equilibrium process}, volume={40}, ISSN={["0006-2960"]}, DOI={10.1021/bi0110387}, abstractNote={We have examined the folding and assembly of a catalytically inactive mutant of procaspase-3, a homodimeric protein that belongs to the caspase family of proteases. The caspase family, and especially caspase-3, is integral to apoptosis. The equilibrium unfolding data demonstrate a plateau between 3 and 5 M urea, consistent with an apparent three-state unfolding process. However, the midpoint of the second transition as well as the amplitude of the plateau are dependent on the protein concentration. Overall, the data are well described by a four-state equilibrium model in which the native dimer undergoes an isomeration to a dimeric intermediate, and the dimeric intermediate dissociates to a monomeric intermediate, which then unfolds. By fitting the four-state model to the experimental data, we have determined the free energy change for the first step of unfolding to be 8.3 +/- 1.3 kcal/mol. The free energy change for the dissociation of the dimeric folding intermediate to two monomeric intermediates is 10.5 +/- 1 kcal/mol. The third step in the unfolding mechanism represents the complete unfolding of the monomeric intermediate, with a free energy change of 7.0 +/- 0.5 kcal/mol. These results show two important points. First, dimerization of procaspase-3 occurs as a result of the association of two monomeric folding intermediates, demonstrating that procaspase-3 dimerization is a folding event. Second, the stability of the dimer contributes significantly to the conformational free energy of the protein (18.8 of 25.8 kcal/mol).}, number={47}, journal={BIOCHEMISTRY}, author={Bose, K and Clark, AC}, year={2001}, month={Nov}, pages={14236–14242} }
 @article{pop_chen_smith_bose_bobay_tripathy_franzen_clark_2001, title={Removal of the pro-domain does not affect the conformation of the procaspase-3 dimer}, volume={40}, ISSN={["0006-2960"]}, DOI={10.1021/bi011037e}, abstractNote={We have investigated the oligomeric properties of procaspase-3 and a mutant that lacks the pro-domain (called pro-less variant). In addition, we have examined the interactions of the 28 amino acid pro-peptide when added in trans to the pro-less variant. By sedimentation equilibrium studies, we have found that procapase-3 is a stable dimer in solution at 25 degrees C and pH 7.2, and we estimate an upper limit for the equilibrium dissociation constant of approximately 50 nM. Considering the expression levels of caspase-3 in Jurkat cells, we predict that procaspase-3 exists as a dimer in vivo. The pro-less variant is also a dimer, with little apparent change in the equilibrium dissociation constant. Thus, in contrast with the long pro-domain caspases, the pro-peptide of caspase-3 does not appear to be involved in dimerization. Results from circular dichroism, fluorescence anisotropy, and FTIR studies demonstrate that the pro-domain interacts weakly with the pro-less variant. The data suggest that the pro-peptide adopts a beta-structure when in contact with the protein, but it is a random coil when free in solution. In addition, when added in trans, the pro-peptide does not inhibit the activity of the mature caspase-3 heterotetramer. On the other hand, the active caspase-3 does not efficiently hydrolyze the pro-domain at the NSVD(9) sequence as occurs when the pro-peptide is in cis to the protease domain. Based on these results, we propose a model for maturation of the procaspase-3 dimer.}, number={47}, journal={BIOCHEMISTRY}, author={Pop, C and Chen, YR and Smith, B and Bose, K and Bobay, B and Tripathy, A and Franzen, S and Clark, AC}, year={2001}, month={Nov}, pages={14224–14235} }
 @article{clark_noland_baldwin_2000, title={A rapid chromatographic method to separate the subunits of bacterial luciferase in urea-containing buffer}, volume={305}, journal={Bioluminescence and chemiluminescence, pt. C}, publisher={New York: Academic Press, 1978-}, author={Clark, A. C. and Noland, B. W. and Baldwin, T. O.}, year={2000}, pages={157–164} }