@article{yao_swartz_hamilton_clark_2021, title={Remodeling hydrogen bond Interactions results in relaxed specificity of Caspase-3}, volume={41}, ISSN={["1573-4935"]}, DOI={10.1042/BSR20203495}, abstractNote={Abstract
               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. 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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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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{resurrection of ancestral effector caspases identifies novel networks for evolution of substrate specificity._2019, url={https://doi.org/10.1042/BCJ20190625}, DOI={10.1042/bcj20190625}, abstractNote={Apoptotic caspases evolved with metazoans more than 950 million years ago (MYA), and a series of gene duplications resulted in two subfamilies consisting of initiator and effector caspases. The effector caspase genes (caspases-3, -6, and -7) were subsequently fixed into the Chordata phylum more than 650 MYA when the gene for a common ancestor (CA) duplicated, and the three effector caspases have persisted throughout mammalian evolution. All caspases prefer an aspartate residue at the P1 position of substrates, so each caspase evolved discrete cellular roles through changes in substrate recognition at the P4 position combined with allosteric regulation. We examined the evolution of substrate specificity in caspase-6, which prefers valine at the P4 residue, compared with caspases-3 and -7, which prefer aspartate, by reconstructing the CA of effector caspases (AncCP-Ef1) and the CA of caspase-6 (AncCP-6An). We show that AncCP-Ef1 is a promiscuous enzyme with little distinction between Asp, Val, or Leu at P4. The specificity of caspase-6 was defined early in its evolution, where AncCP-6An demonstrates a preference for Val over Asp at P4. Structures of AncCP-Ef1 and of AncCP-6An show a network of charged amino acids near the S4 pocket that, when combined with repositioning a flexible active site loop, resulted in a more hydrophobic binding pocket in AncCP-6An. The ancestral protein reconstructions show that the caspase-hemoglobinase fold has been conserved for over 650 million years and that only three substitutions in the scaffold are necessary to shift substrate selection toward Val over Asp.}, journal={The Biochemical journal}, year={2019}, month={Nov} }
 @article{cramer_hamilton_2017, title={An Internship May Not Be Enough: Enhancing Bioscience Industry Job Readiness through Practicum Experiences.}, volume={18}, url={http://europepmc.org/abstract/med/28512519}, DOI={10.1128/jmbe.v18i1.1248}, abstractNote={In contrast to the narrowing of options in academic careers, the bioscience industry offers robust employment opportunities for STEM-trained workers, especially those who display both scientific and business talent. Unfortunately, traditional science programs typically lack curricular features that develop this type of worker. The North Carolina State University Master of Microbial Biotechnology (MMB) program facilitates industry-specific experiential learning to fill this training gap. Similar programs often rely on a single industry internship to provide students relevant work experience, but completion of one internship might not suffice to position students for employment in a highly competitive job market. The MMB program requires students to complete an internship and three practicum projects in an industry setting, to promote development of key skills in a variety of areas, to build confidence in the ability to perform initial job duties, and to establish a more extensive work history in industry. In this Perspective we discuss an unmet need in undergraduate and graduate STEM education that can be filled by incorporating a similar set of industry-specific work experiences for students who desire to transition from academe into the life science industry.}, number={1}, journal={Journal of microbiology & biology education}, author={Cramer, JM and Hamilton, PT}, year={2017}, month={Apr} }
 @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={AbstractThe 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{carlson_hyde-deruyscher_hamilton_2015, title={High-Throughput and High-Content Screening Using Peptides}, DOI={10.1201/b18196-11}, journal={Phage Display In Biotechnology and Drug Discovery, Second Edition}, publisher={Informa UK Limited}, author={Carlson, Robert and Hyde-DeRuyscher, Robin and Hamilton, Paul}, year={2015}, month={Feb}, pages={249–274} }
 @book{carlson_hyde-deruyscher_hamilton_2015, title={High-throughput and high-content screening using peptides}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85053971671&partnerID=MN8TOARS}, DOI={10.1201/b18196}, abstractNote={Phage Display in Biotechnology and Drug Discovery, Second Edition provides a comprehensive view of the impact and promise of phage display in drug discovery and biotechnology. Building on the success of its previous edition, the book discusses current theories, principles, and methods in the field and demonstrates applications for peptide phage dis}, journal={Phage Display in Biotechnology and Drug Discovery, Second Edition}, author={Carlson, R.O. and Hyde-DeRuyscher, R. and Hamilton, P.T.}, year={2015}, pages={249–274} }
 @article{marks_hamilton_2014, title={Characterization of a thermophilic bacteriophage of Geobacillus kaustophilus}, volume={159}, ISSN={["1432-8798"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84907692380&partnerID=MN8TOARS}, DOI={10.1007/s00705-014-2101-8}, abstractNote={GBK2 is a bacteriophage, isolated from a backyard compost pile, that infects the thermophile Geobacillus kaustophilus. GBK2 has a circularly permuted genome of 39,078 bp with a G+C content of 43 %. Annotation of the genome reveals 62 putative open reading frames (ORFs), 25 of which (40.3 %) show homology to known proteins and 37 of which (59.7 %) are proteins with unknown functions. Twelve of the identified ORFs had the greatest homology to genes from the phage SPP1, a phage that infects the mesophile Bacillus subtilis. The overall genomic arrangement of GBK2 is similar to that of SPP1, with the majority of GBK2 SPP1-like genes coding for proteins involved in DNA replication and metabolism.}, number={10}, journal={ARCHIVES OF VIROLOGY}, publisher={Springer Science \mathplus Business Media}, author={Marks, Timothy J. and Hamilton, Paul T.}, year={2014}, month={Oct}, pages={2771–2775} }
 @article{luginbuhl_hamilton_2013, title={Cooperative Learning through Team-Based Projects in the Biotechnology Industry †}, volume={14}, DOI={10.1128/jmbe.v14i2.608}, abstractNote={We have developed a cooperative-learning, case studies project model that has teams of students working with biotechnology professionals on company-specific problems. These semester-long, team-based projects can be used effectively to provide students with valuable skills in an industry environment and experience addressing real issues faced by biotechnology companies. Using peer-evaluations, we have seen improvement in students’ professional skills such as time-management, quality of work, and level of contribution over multiple semesters. This model of team-based, industry-sponsored projects could be implemented in other college and university courses/programs to promote professional skills and expose students to an industry setting.}, number={2}, journal={Journal of Microbiology & Biology Education}, publisher={American Society for Microbiology}, author={Luginbuhl, Sarah C. and Hamilton, Paul T.}, year={2013}, pages={221–229} }
 @article{hamilton_jansen_ganesan_benson_hyde-deruyscher_beyer_gile_nair_hodges_grøn_et al._2013, title={Improved Bone Morphogenetic Protein-2 Retention in an Injectable Collagen Matrix Using Bifunctional Peptides}, volume={8}, ISSN={["1932-6203"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84881342868&partnerID=MN8TOARS}, DOI={10.1371/journal.pone.0070715}, abstractNote={To promote healing of many orthopedic injuries, tissue engineering approaches are being developed that combine growth factors such as Bone Morphogenetic Proteins (BMP) with biomaterial carriers. Although these technologies have shown great promise, they still face limitations. We describe a generalized approach to create target-specific modular peptides that bind growth factors to implantable biomaterials. These bifunctional peptide coatings provide a novel way to modulate biology on the surface of an implant. Using phage display techniques, we have identified peptides that bind with high affinity to BMP-2. The peptides that bind to BMP-2 fall into two different sequence clusters. The first cluster of peptide sequences contains the motif W-X-X-F-X-X-L (where X can be any amino acid) and the second cluster contains the motif F-P-L-K-G. We have synthesized bifunctional peptide linkers that contain BMP-2 and collagen-binding domains. Using a rat ectopic bone formation model, we have injected rhBMP-2 into a collagen matrix with or without a bifunctional BMP-2: collagen peptide (BC-1). The presence of BC-1 significantly increased osteogenic cellular activity, the area of bone formed, and bone maturity at the site of injection. Our results suggest that bifunctional peptides that can simultaneously bind to a growth factor and an implantable biomaterial can be used to control the delivery and release of growth factors at the site of implantation.}, number={8}, journal={PLOS ONE}, publisher={Public Library of Science (PLoS)}, author={Hamilton, Paul T. and Jansen, Michelle S. and Ganesan, Sathya and Benson, R. Edward and Hyde-DeRuyscher, Robin and Beyer, Wayne F. and Gile, Joseph C. and Nair, Shrikumar A. and Hodges, Jonathan A. and Grøn, Hanne and et al.}, editor={Gelain, FabrizioEditor}, year={2013}, month={Aug} }
 @article{hamilton_luginbuhl_hyman_2012, title={Preparing Science-Trained Professionals for the Biotechnology Industry: A Ten-Year Perspective on a Professional Science Master’s Program}, volume={13}, ISSN={1935-7877}, url={https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3577309/}, DOI={10.1128/jmbe.v13i1.375}, abstractNote={The biotechnology industry has a need for business-savvy scientists; however, this is not the way scientists are traditionally trained at universities and colleges. To address this need, universities have developed Professional Science Master’s (PSM) degree programs that offer advanced training in a technical field along with professional skills development through team-based projects and internships. Nearly ten years ago, the Department of Microbiology at NCSU started a PSM program in Microbial Biotechnology (MMB). This article provides an overview of the MMB program, and shares some of the lessons that we have learned.}, number={1}, journal={Journal of Microbiology & Biology Education : JMBE}, publisher={American Society for Microbiology}, author={Hamilton, Paul T. and Luginbuhl, Sarah C. and Hyman, Michael}, year={2012}, month={May}, pages={39–44} }
 @article{khoo_hamilton_o’toole_snyder_kenan_grinstaff_2009, title={Directed Assembly of PEGylated-Peptide Coatings for Infection-Resistant Titanium Metal}, volume={131}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-68249136655&partnerID=MN8TOARS}, DOI={10.1021/ja9020827}, abstractNote={Appropriate surface chemistry between a material and its surrounding biological environment is crucial to the eventual integration and performance of any implant, whether metal, plastic, or ceramic. A robust peptide-based coating technology capable of easily modifying the surface of titanium (Ti) metal through noncovalent binding is described. A short peptide possessing affinity for Ti was identified using a phage display screening process and subjected to an amino acid substitution exercise using solid-phase chemical synthesis. Through these studies, the HKH tripeptide motif was elucidated as an important contributor to Ti binding within the Ti-binding peptide. This peptide spontaneously and selectively adsorbs onto a Ti surface from dilute aqueous solution with submicromolar binding affinities as determined by ELISA and quartz crystal microbalance with dissipation monitoring (QCM-D), through a process largely dominated by electrostatic interactions. Atomic force microscopy (AFM) reveals a densely packed peptide adlayer with an average height of approximately 0.5 nm. Subsequently, a PEGylated analogue of the peptide was shown to rapidly coat Ti to afford a nonfouling surface that efficiently blocked the adsorption of fibronectin and significantly reduced the extent of Staphylococcus aureus attachment and biofilm formation in vitro. These PEGylated-peptide coatings show promise in terms of resolving two major hurdles common to implanted metals: (i) nonspecific protein adsorption and (ii) bacterial colonization. At the same time, the facile one-step modification process will facilitate the point-of-care application of these coatings in the surgical suite.}, number={31}, journal={J. Am. Chem. Soc.}, publisher={American Chemical Society (ACS)}, author={Khoo, Xiaojuan and Hamilton, Paul and O’Toole, George A. and Snyder, Brian D. and Kenan, Daniel J. and Grinstaff, Mark W.}, year={2009}, month={Aug}, pages={10992–10997} }
 @inproceedings{hamilton_solan_2008, title={A peptide coating for the endothelialization of intravascular devices}, volume={1}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84869012621&partnerID=MN8TOARS}, booktitle={8th World Biomaterials Congress 2008}, author={Hamilton, P.T. and Solan, A.}, year={2008}, pages={200} }
 @inproceedings{hamilton_buehrer_juzumiene_2008, title={Growth factor coated sutures for improved tendon repair}, volume={1}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84869010182&partnerID=MN8TOARS}, booktitle={8th World Biomaterials Congress 2008}, author={Hamilton, P.T. and Buehrer, B. and Juzumiene, D.}, year={2008}, pages={179} }
 @article{meyers_hamilton_walsh_kenan_grinstaff_2007, title={Endothelialization of Titanium Surfaces}, volume={19}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-34748856343&partnerID=MN8TOARS}, DOI={10.1002/adma.200700029}, abstractNote={In the extracellular matrix (ECM), chemical cues are present that control all aspects of cell biology. [1–3] These surfacebound and soluble factors provide the necessary adhesion and signaling for normal cellular activity, and without such matrix support, the cells will quickly apoptose. Implanted device surfaces lack the molecular features that provide guidance to the surrounding cells to afford optimal in vivo integration and function. One class of implanted materials are metal implants, which are commonly used in cardiovascular therapy (e.g., stents) and orthopedic procedures (e.g., hip replacement). While such materials are favored for their inertness and mechanical strength, negative consequences can arise from suboptimal tissue integration. For example, during a coronary angioplasty an occluded coronary artery is opened using a balloon catheter and then a metal stent is inserted to provide a permanent framework supporting vascular patency. If the artery re-occludes due to smooth muscle cell proliferation, in a process called restenosis, a second procedure is required to reestablish blood flow. Restenosis used to occur in about 25% of patients, but with the introduction of stents that elute a mitotic inhibitor, such as paclitaxel, this number has been reduced significantly. [4] However, there is new concern regarding the use of drug-eluting stents owing to an increased thrombolytic potential of two to three fold compared to bare metal stents. [5] An alternative approach to the use of drugs}, number={18}, journal={Adv. Mater.}, publisher={Wiley-Blackwell}, author={Meyers, S. R. and Hamilton, P. T. and Walsh, E. B. and Kenan, D. J. and Grinstaff, M. W.}, year={2007}, month={Sep}, pages={2492–2498} }
 @article{hamilton_carlson_hyde-deruyscher_2005, title={High Throughput and High Content Screening Using Peptides}, DOI={10.1201/9780849359125.ch9}, journal={Phage Display In Biotechnology and Drug Discovery}, publisher={Informa UK Limited}, author={Hamilton, Paul and Carlson, Robert and Hyde-Deruyscher, Robin}, year={2005}, month={Jul}, pages={347–384} }
 @article{ashraf_anderson_duke_hamilton_fredericks_2003, title={Identification and characterization of peptide probes directed against PKCα conformations}, volume={61}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0038025879&partnerID=MN8TOARS}, DOI={10.1034/j.1399-3011.2003.00056.x}, abstractNote={Abstract:  Phage display is a powerful technology that allows identification of high affinity peptides that bind specifically to a given molecular target. Using a highly complex peptide display library, we have identified separate classes of peptides that bind to protein kinase C alpha (PKCα) only under activation conditions. Furthermore, peptide binding was specific to PKCα and not to any of the other closely related PKC isoforms. The conformational and isoform specificity of the peptide binding was demonstrated using surface plasmon resonance as well as time‐resolved fluorescence assays. Kinase assays showed that these peptides were not direct substrates for PKC nor did they inhibit phosphorylation of PKC substrates. These peptides are most likely directed against protein–protein interaction sites on PKC. The data presented here offers another example of application of phage display technology to identify conformation‐dependent peptide probes against therapeutically important drug targets. These peptides are ideally suited to be used as surrogate ligands to identify compounds that bind specifically to PKCα, as well as conformational probes to detect activated forms of PKCα.}, number={5}, journal={The Journal of Peptide Research}, publisher={Wiley-Blackwell}, author={Ashraf, S. S. and Anderson, E. and Duke, K. and Hamilton, P.T. and Fredericks, Z.}, year={2003}, month={Mar}, pages={263–273} }
 @article{benson_gottlin_christensen_hamilton_2003, title={Intracellular Expression of Peptide Fusions for Demonstration of Protein Essentiality in Bacteria}, volume={47}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0041922662&partnerID=MN8TOARS}, DOI={10.1128/aac.47.9.2875-2881.2003}, abstractNote={ABSTRACT
          
            We describe a “protein knockout” technique that can be used to identify essential proteins in bacteria. This technique uses phage display to select peptides that bind specifically to purified target proteins. The peptides are expressed intracellularly and cause inhibition of growth when the protein is essential. In this study, peptides that each specifically bind to one of seven essential proteins were identified by phage display and then expressed as fusions to glutathione
            S
            -transferase in
            Escherichia coli.
            Expression of peptide fusions directed against
            E. coli
            DnaN, LpxA, RpoD, ProRS, SecA, GyrA, and Era each dramatically inhibited cell growth. Under the same conditions, a fusion with a randomized peptide sequence did not inhibit cell growth. In growth-inhibited cells, inhibition could be relieved by concurrent overexpression of the relevant target protein but not by coexpression of an irrelevant protein, indicating that growth inhibition was due to a specific interaction of the expressed peptide with its target. The protein knockout technique can be used to assess the essentiality of genes of unknown function emerging from the sequencing of microbial genomes. This technique can also be used to validate proteins as drug targets, and their corresponding peptides as screening tools, for discovery of new antimicrobial agents.
          }, number={9}, journal={Antimicrobial Agents and Chemotherapy}, publisher={American Society for Microbiology}, author={Benson, R. E. and Gottlin, E. B. and Christensen, D. J. and Hamilton, P. T.}, year={2003}, month={Aug}, pages={2875–2881} }
 @article{hamilton_christensen_2003, title={Surrogate Ligand-Based Assay Systems for Discovery of Antibacterial Agents for Genomic Targets}, DOI={10.1201/9780203911464.ch11}, journal={Microbial Genomics and Drug Discovery}, publisher={Informa UK Limited}, author={Hamilton, Paul and Christensen, Dale}, year={2003}, month={May}, pages={173–185} }
 @article{kay_hamilton_2001, title={Identification of Enzyme Inhibitors from Phage-Displayed Combinatorial Peptide Libraries}, volume={4}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0034786701&partnerID=MN8TOARS}, DOI={10.2174/1386207013330760}, abstractNote={In recent years, there have been a growing number of examples of the successful isolation of peptide ligands for enzymes from phage-displayed combinatorial peptide libraries. These peptides typically bind at or near the active site of the enzymes and can inhibit their activity. We review the literature on peptide ligands that have been isolated for enzymes other than proteases as well as present data on peptide ligands we have identified for E. coli dihydrofolate reductase (DHFR) which bind at, or near, the same site as the known inhibitors methotrexate or trimethoprim. Thus, while the peptide ligand isolated from phage-displayed libraries may not resemble the chemical structure of the normal substrate of the enzyme, the peptide can be used as an inhibitor to evaluate the function of the enzyme or for drug discovery efforts (i.e., as a lead compound for peptidomimetic design or as displaceable probe in high-throughput screens of libraries of small molecules).}, number={7}, journal={CCHTS}, publisher={Bentham Science Publishers Ltd.}, author={Kay, Brian and Hamilton, Paul}, year={2001}, month={Nov}, pages={535–543} }
 @article{christensen_gottlin_benson_hamilton_2001, title={Phage display for target-based antibacterial drug discovery}, volume={6}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0035395224&partnerID=MN8TOARS}, DOI={10.1016/s1359-6446(01)01853-0}, abstractNote={Increasing bacterial drug resistance and hard-to-eradicate opportunistic infections have created a need for new antibiotics. Sequencing of microbial genomes has yielded many new potential targets for antibacterial drug discovery. However, little is known about the biochemical activities of many of these targets, making it difficult to develop HTS assays for them. Peptides isolated by phage display can be used as 'surrogate ligands' in competition assays for screening of targets of unknown function with small-molecule libraries. These screening assays can be adapted into a variety of high-throughput formats, including those based on radioactive, luminescence or fluorescence detection.}, number={14}, journal={Drug Discovery Today}, publisher={Elsevier BV}, author={Christensen, Dale J and Gottlin, Elizabeth B and Benson, R.Edward and Hamilton, Paul T}, year={2001}, month={Jul}, pages={721–727} }
 @article{hyde-deruyscher_paige_christensen_hyde-deruyscher_lim_fredericks_kranz_gallant_zhang_rocklage_et al._2000, title={Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries}, volume={7}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0034092470&partnerID=MN8TOARS}, DOI={10.1016/s1074-5521(00)00062-4}, abstractNote={The rapidly expanding list of pharmacologically important targets has highlighted the need for ways to discover new inhibitors that are independent of functional assays. We have utilized peptides to detect inhibitors of protein function. We hypothesized that most peptide ligands identified by phage display would bind to regions of biological interaction in target proteins and that these peptides could be used as sensitive probes for detecting low molecular weight inhibitors that bind to these sites.We selected a broad range of enzymes as targets for phage display and isolated a series of peptides that bound specifically to each target. Peptide ligands for each target contained similar amino acid sequences and competition analysis indicated that they bound one or two sites per target. Of 17 peptides tested, 13 were found to be specific inhibitors of enzyme function. Finally, we used two peptides specific for Haemophilus influenzae tyrosyl-tRNA synthetase to show that a simple binding assay can be used to detect small-molecule inhibitors with potencies in the micromolar to nanomolar range.Peptidic surrogate ligands identified using phage display are preferentially targeted to a limited number of sites that inhibit enzyme function. These peptides can be utilized in a binding assay as a rapid and sensitive method to detect small-molecule inhibitors of target protein function. The binding assay can be used with a variety of detection systems and is readily adaptable to automation, making this platform ideal for high-throughput screening of compound libraries for drug discovery.}, number={1}, journal={Chemistry & Biology}, publisher={Elsevier BV}, author={Hyde-DeRuyscher, R and Paige, LA and Christensen, DJ and Hyde-DeRuyscher, N and Lim, A and Fredericks, ZL and Kranz, J and Gallant, P and Zhang, J and Rocklage, SM and et al.}, year={2000}, month={Jan}, pages={17–25} }
 @article{chang_norris_grøn_paige_hamilton_kenan_fowlkes_mcdonnell_1999, title={Dissection of the LXXLL Nuclear Receptor-Coactivator Interaction Motif Using Combinatorial Peptide Libraries: Discovery of Peptide Antagonists of Estrogen Receptors α and β}, volume={19}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0033513582&partnerID=MN8TOARS}, DOI={10.1128/mcb.19.12.8226}, abstractNote={ABSTRACT Recruitment of transcriptional coactivators following ligand activation is a critical step in nuclear receptor-mediated target gene expression. Upon binding an agonist, the receptor undergoes a conformational change which facilitates the formation of a specific coactivator binding pocket within the carboxyl terminus of the receptor. This permits the α-helical LXXLL motif within some coactivators to interact with the nuclear receptors. Until recently, the LXXLL motif was thought to function solely as a docking module; however, it now appears that sequences flanking the core motif may play a role in determining receptor selectivity. To address this issue, we used a combinatorial phage display approach to evaluate the role of flanking sequences in influencing these interactions. We sampled more than 108 variations of the core LXXLL motif with estradiol-activated estrogen receptor alpha (ERα) as a target and found three different classes of peptides. All of these peptides interacted with ERα in an agonist-dependent manner and disrupted ERα-mediated transcriptional activity when introduced into target cells. Using a series of ERα-mutants, we found that these three classes of peptides showed different interaction patterns from each other, suggesting that not all LXXLL motifs are the same and that receptor binding selectivity can be achieved by altering sequences flanking the LXXLL core motif. Most notable in this regard was the discovery of a peptide which, when overexpressed in cells, selectively disrupted ERβ- but not ERα-mediated reporter gene expression. This novel ERβ-specific antagonist may be useful in identifying and characterizing the ERβ-regulated process in estradiol-responsive cells. In conclusion, using a combinatorial approach to define cofactor-receptor interactions, we have clearly been able to demonstrate that not all LXXLL motifs are functionally equivalent, a finding which suggests that it may be possible to target receptor-LXXLL interactions to develop receptor-specific antagonists.}, number={12}, journal={Mol. Cell. Biol.}, publisher={American Society for Microbiology}, author={Chang, Ching-yi and Norris, John D. and Grøn, Hanne and Paige, Lisa A. and Hamilton, Paul T. and Kenan, Daniel J. and Fowlkes, Dana and McDonnell, Donald P.}, year={1999}, pages={8226–8239} }
 @article{paige_christensen_gron_norris_gottlin_padilla_chang_ballas_hamilton_mcdonnell_et al._1999, title={Estrogen receptor (ER) modulators each induce distinct conformational changes in ER   and ER  }, volume={96}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-2642544810&partnerID=MN8TOARS}, DOI={10.1073/pnas.96.7.3999}, abstractNote={Estrogen receptor (ER) modulators produce distinct tissue-specific biological effects, but within the confines of the established models of ER action it is difficult to understand why. Previous studies have suggested that there might be a relationship between ER structure and activity. Different ER modulators may induce conformational changes in the receptor that result in a specific biological activity. To investigate the possibility of modulator-specific conformational changes, we have applied affinity selection of peptides to identify binding surfaces that are exposed on the apo-ERs α and β and on each receptor complexed with estradiol or 4-OH tamoxifen. These peptides are sensitive probes of receptor conformation. We show here that ER ligands, known to produce distinct biological effects, induce distinct conformational changes in the receptors, providing a strong correlation between ER conformation and biological activity. Furthermore, the ability of some of the peptides to discriminate between different ER α and ER β ligand complexes suggests that the biological effects of ER agonists and antagonists acting through these receptors are likely to be different.}, number={7}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Paige, L. A. and Christensen, D. J. and Gron, H. and Norris, J. D. and Gottlin, E. B. and Padilla, K. M. and Chang, C.-y. and Ballas, L. M. and Hamilton, P. T. and McDonnell, D. P. and et al.}, year={1999}, month={Mar}, pages={3999–4004} }
 @article{norris_paige_christensen_chang_huacani_fan_hamilton_fowlkes_mcdonnell_1999, title={Peptide antagonists of the human estrogen receptor}, volume={285}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0033618510&partnerID=MN8TOARS}, DOI={10.1126/science.285.5428.744}, abstractNote={Estrogen receptor α transcriptional activity is regulated by distinct conformational states that are the result of ligand binding. Phage display was used to identify peptides that interact specifically with either estradiol- or tamoxifen-activated estrogen receptor α. When these peptides were coexpressed with estrogen receptor α in cells, they functioned as ligand-specific antagonists, indicating that estradiol-agonist and tamoxifen–partial agonist activities do not occur by the same mechanism. The ability to regulate estrogen receptor α transcriptional activity by targeting sites outside of the ligand-binding pocket has implications for the development of estrogen receptor α antagonists for the treatment of tamoxifen-refractory breast cancers.}, number={5428}, journal={Science}, author={Norris, J.D. and Paige, L.A. and Christensen, D.J. and Chang, C.-Y. and Huacani, M.R. and Fan, D. and Hamilton, P.T. and Fowlkes, D.M. and McDonnell, D.P.}, year={1999}, pages={744–746} }
 @article{hamilton_1995, title={Applying Genetic Engineering to the Structural Analysis of Proteins}, DOI={10.1007/978-1-4899-1079-0_9}, abstractNote={Developments in biochemistry and recombinant DNA technology make it pos-sible to genetically clone, isolate, characterize, and modify any protein of interest. Current techniques in protein purification permit N-terminal sequence analysis of picomolar quantities of proteins. From this amino acid sequence information, DNA probes can be designed and used to clone the gene sequence encoding that protein. The gene can be expressed at high levels in the gram-negative bacterium Escherichia coli using specialized plasmid vectors. The overexpressed protein can often be purified in sufficient quantities to allow biophysical characterization. Mutagenesis can be used to analyze the structure-function relationships of the protein and to generate new and novel proteins for therapeutic and diagnostic applications.}, journal={Physical Methods to Characterize Pharmaceutical Proteins}, publisher={Springer Science \mathplus Business Media}, author={Hamilton, Paul T.}, year={1995}, pages={329–350} }
 @article{hamilton_1995, title={Applying genetic engineering to the structural analysis of proteins.}, volume={7}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0029449448&partnerID=MN8TOARS}, journal={Pharmaceutical biotechnology}, author={Hamilton, P.T.}, year={1995}, pages={329–350} }
 @article{hamilton_martinson_malinowski_pearson_1993, title={Expression of a single-chain antibody as a maltose binding protein fusion}, volume={6}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84962999165&partnerID=MN8TOARS}, DOI={10.1093/protein/6.Supplement.88-b}, abstractNote={Journal Article Expression of a single-chain antibody as a maltose binding protein fusion Get access Paul Hamilton, Paul Hamilton Becton Dickinson Research Center, P.O. Box 12016, RTP, NC 27709 Search for other works by this author on: Oxford Academic PubMed Google Scholar Mary Martinson, Mary Martinson Becton Dickinson Research Center, P.O. Box 12016, RTP, NC 27709 Search for other works by this author on: Oxford Academic PubMed Google Scholar Doug Malinowski, Doug Malinowski Becton Dickinson Research Center, P.O. Box 12016, RTP, NC 27709 Search for other works by this author on: Oxford Academic PubMed Google Scholar Robert Pearson Robert Pearson Becton Dickinson Research Center, P.O. Box 12016, RTP, NC 27709 Search for other works by this author on: Oxford Academic PubMed Google Scholar Protein Engineering, Design and Selection, Volume 6, Issue Supplement, 1993, Page 88, https://doi.org/10.1093/protein/6.Supplement.88-b Published: 01 January 1993}, journal={Protein Engineering, Design and Selection}, author={Hamilton, P. and Martinson, M. and Malinowski, D. and Pearson, R.}, year={1993}, pages={88} }
 @article{reeve_beckler_cram_hamilton_brown_krzycki_kolodziej_alex_orme-johnson_walsh_1989, title={A hydrogenase-linked gene in Methanobacterium thermoautotrophicum strain delta H encodes a polyferredoxin.}, volume={86}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0024670961&partnerID=MN8TOARS}, DOI={10.1073/pnas.86.9.3031}, abstractNote={The genes mvhDGA, which encode the subunit polypeptides of the methyl viologen-reducing hydrogenase in Methanobacterium thermoautotrophicum strain delta H, have been cloned and sequenced. These genes, together with a fourth open reading frame designated mvhB, are tightly linked and appear to form an operon that is transcribed starting 42 base pairs upstream of mvhD. The organization and sequences of the mvhG and mvhA genes indicate a common evolutionary ancestry with genes encoding the small and large subunits of hydrogenases in eubacterial species. The product of the mvhB gene is predicted to contain six tandomly repeated bacterial-ferredoxin-like domains and, therefore, is predicted to be a polyferredoxin that could contain as many as 48 iron atoms in 12 Fe4S4 clusters.}, number={9}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Reeve, J. N. and Beckler, G. S. and Cram, D. S. and Hamilton, P. T. and Brown, J. W. and Krzycki, J. A. and Kolodziej, A. F. and Alex, L. and Orme-Johnson, W. H. and Walsh, C. T.}, year={1989}, month={May}, pages={3031–3035} }
 @article{hamilton_malinowski_1989, title={Nucleotide sequence of the major outer membrane protein gene from Chlamydia trachomatis serovar H}, volume={17}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0024978653&partnerID=MN8TOARS}, DOI={10.1093/nar/17.20.8366}, abstractNote={The gene encoding the major outer membrane protein (MOMP) from Chlamydia trachomatis, serovar H was isolated from a genomic library using the MOMP gene from serovar L2 as the probe (1) . The recombinant insert was subcloned into a plasmid vector (Bluescript), the gene localized by Southern blotting and the nucleotide sequence was determined by the dideoxy chain termination method. The H MOMP gene is 1193 bp long and encodes a polypeptide of 397 amino acids including a 22 amino acid leader sequence. This corresponds to a mature H MOMP of 40,672 daltons, in agreement with the molecular mass predicted by SDS-PAGE (2) . The H and L2 MOMP genes are 74% homologous at the nucleotide level and are 84% homologous in their protein sequences. The differences in the two sequences are clustered in four regions (435-483, 655-687, 906-952 and 1083-1194).}, number={20}, journal={Nucl Acids Res}, publisher={Oxford University Press (OUP)}, author={Hamilton, Paul T. and Malinowski, Douglas P.}, year={1989}, pages={8366–8366} }
 @article{reeve_beckler_cram_hamilton_brown_krzycki_kolodziej_alex_orme-johnson_walsh_1989, title={Polyferredoxin: A methanogen hydrogenase operon encodes a gene for a 12 × (4Fe4S) protein}, volume={36}, DOI={10.1016/0162-0134(89)84221-7}, number={3-4}, journal={Journal of Inorganic Biochemistry}, publisher={Elsevier BV}, author={Reeve, J. and Beckler, G. and Cram, D. and Hamilton, P. and Brown, J. and Krzycki, J. and Kolodziej, A. and Alex, L. and Orme-Johnson, W.H. and Walsh, C.T.}, year={1989}, month={Aug}, pages={221} }
 @article{reeve_beckler_brown_cram_haas_hamilton_morris_sherf_weil_1987, title={Divergence of Methanogens, Conservation of the His I Gene Sequence in all Three Biological Kingdoms and the Status of Methanobacterium Thermoautotrophicum}, DOI={10.1007/978-94-009-3539-6_31}, abstractNote={Methanogens are archaebacteria [1]. It was therefore somewhat surprising when it was shown that methanogen-derived genes could function in eubacterial species [2]. This has offered the opportunity of using gene cloning in Escherichia coli to investigate the basic structure of methanogen genes [3, 4] and to use E. coli to synthesize methanogen enzymes involved in methanogenesis [5] and nitrogen fixation [6].}, journal={Microbial Growth on C1 Compounds}, publisher={Springer Science \mathplus Business Media}, author={Reeve, John N. and Beckler, Gregory S. and Brown, James W. and Cram, David S. and Haas, Elizabeth S. and Hamilton, Paul T. and Morris, Christina J. and Sherf, Bruce A. and Weil, Clifford F.}, year={1987}, pages={255–260} }
 @article{reeve_hamilton_beckler_morris_clarke_1986, title={Structure of methanogen genes}, volume={7}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0022467785&partnerID=MN8TOARS}, DOI={10.1016/s0723-2020(86)80116-3}, abstractNote={An analysis of the structure and presumed regulatory signals in sequenced methanogen genes is presented. The evidence for polycistronic transcriptional units is extended by inclusion of a DNA sequence, cloned from Methanobrevibacter smithii, which precedes the M. smithii proC gene (Hamilton and Reeve, 1985a). Comparison of DNA sequences indicates that a consensus core ribosome binding sequence for methanogens would be 5'AGGTGA and that many of the ribosome binding sequences are, in fact, longer than the core sequence containing 7 bases with the potential to hybridize to 16SrRNA. The majority, but not all, of the sequenced methanogen genes conform to the rule in which RNY codons (R=purine, Y=pyrimidine, N=purine or pyrimidine) occur most frequently in the translated reading frame (Shepherd 1981,1983). Codon usages by different methanogens reflect the need to accommodate genomes with very different overall %mol G+C contents. Codons such as AUA, AGA, and AGG, rarely used by E. coli are frequently used by methanogens. The dinucleotide, CG, which occurs very infrequently in eucaryotic DNAs (Subak-Sharpe et al., 1967; Lennon and Faser, 1983; Nussinov, 1984) is also rarely found in methanogen genes. Methylation and demethylation of the cytosine residue in CG sequences has been implicated in regulation of eucaryotic gene expression (Weisbrod, 1982) and this role used as an argument for the infrequent occurrence of CG-containing sequences. The rarity of CG in the DNA sequences so far obtained from methanogens may indicate a similar regulatory usage of CG-containing sequences in archaebacterial methanogens.}, number={1}, journal={Systematic and Applied Microbiology}, publisher={Elsevier BV}, author={Reeve, John N. and Hamilton, Paul T. and Beckler, Gregory S. and Morris, Christina J. and Clarke, Colin H.}, year={1986}, month={Mar}, pages={5–12} }
 @article{hamilton_reeve_1985, title={Sequence divergence of an archaebacterial gene cloned from a mesophilic and a thermophilic methanogen}, volume={22}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0022321706&partnerID=MN8TOARS}, DOI={10.1007/bf02115691}, abstractNote={A 1.6-kb fragment of DNA from the thermophilic, methane-producing, anaerobic archaebacterium Methanobacterium thermoautotrophicum delta H has been cloned and sequenced. This DNA complements mutations in both the purE1 and purE2 loci of Escherichia coli. The sequence of the M. thermoautotrophicum DNA predicts that complementation in E. coli results from the synthesis of a polypeptide with a molecular weight of 36,249. A polypeptide apparently of this molecular weight is synthesized in E. coli minicells containing recombinant plasmids that carry the cloned fragment of methanogen DNA. We have previously cloned and sequenced a purE-complementing gene from the mesophilic methanogen Methanobrevibacter smithii. The two methanogen-derived purE-complementing genes are 53% homologous and encode polypeptides that are 45% homologous in their amino acid sequences but would be 74% homologous if conservative amino acid substitutions were considered as maintaining sequence homology. The genome of M. thermoautotrophicum has a molar G + C content of 49.7%, whereas the genome of M. smithii is 30.6% G + C. Conservation of encoded amino acids while accommodating the very different G + C contents is accomplished by use of different codons that encode the same amino acid. The majority of base changes occur at the third codon position. The intergenic regions of the cloned M. thermoautotrophicum DNA contain sequences previously identified as ribosome binding sites and as putative methanogen promoters. Although the two purE-complementing genes are apparently derived from a common ancestor, only the gene from M. smithii maintains a codon usage that conforms to the RNY rule.}, number={4}, journal={J Mol Evol}, publisher={Springer Science \mathplus Business Media}, author={Hamilton, Paul T. and Reeve, John N.}, year={1985}, pages={351–360} }
 @article{hamilton_reeve_1985, title={Structure of genes and an insertion element in the methane producing archaebacterium Methanobrevibacter smithii}, volume={200}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0021871395&partnerID=MN8TOARS}, DOI={10.1007/bf00383311}, abstractNote={DNA fragments cloned from the methanogenic archaebacterium Methanobrevibacter smithii which complement mutations in the purE and proC genes of E. coli have been sequenced. Sequence analyses, transposon mutagenesis and expression in E. coli minicells indicate that purE and proC complementations result from the synthesis of M. smithii polypeptides with molecular weights of 36,697 and 27,836 respectively. The encoding genes appear to be located in operons. The M. smithii genome contains 69% A/T basepairs (bp) which is reflected in unusual codon usages and intergenic regions containing approximately 85% A/T bp. An insertion element, designated ISM1, was found within the cloned M. smithii DNA located adjacent to the proC complementing region. ISM1 is 1381 bp in length, has 29 bp terminal inverted repeat sequences and contains one major ORF encoded in 87% of the ISM1 sequence. ISM1 is mobile, present in approximately 10 copies per genome and integration duplicates 8 bp at the site of insertion. The duplicated sequences show homology with sequences within the 29 bp terminal repeat sequence of ISM1. Comparison of our data with sequences from halophilic archaebacteria suggests that 5'GAANTTTCA and 5'TTTTAATATAAA may be consensus promoter sequences for archaebacteria. These sequences closely resemble the consensus sequences which precede Drosophila heat-shock genes (Pelham 1982; Davidson et al. 1983). Methanogens appear to employ the eubacterial system of mRNA: 16SrRNA hybridization to ensure initiation of translation; the consensus ribosome binding sequence is 5'AGGTGA.}, number={1}, journal={Molec. Gen. Genet.}, publisher={Springer Science \mathplus Business Media}, author={Hamilton, Paul T. and Reeve, John N.}, year={1985}, pages={47–59} }
 @article{reeve_trun_hamilton_1982, title={Beginning genetics with methanogens.}, volume={19}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0020009847&partnerID=MN8TOARS}, journal={Basic life sciences}, author={Reeve, J.N. and Trun, N.J. and Hamilton, P.T.}, year={1982}, pages={233–244} }