Crystal Structure of an Invertebrate Caspase
2004; Elsevier BV; Volume: 279; Issue: 8 Linguagem: Inglês
10.1074/jbc.m312472200
ISSN1083-351X
AutoresC.M. Forsyth, Donna Lemongello, Douglas LaCount, Paul D. Friesen, Andrew J. Fisher,
Tópico(s)RNA Interference and Gene Delivery
ResumoCaspases play an essential role in the execution of apoptosis. These cysteine proteases are highly conserved among metazoans and are translated as inactive zymogens, which are activated by proteolytic cleavages to generate the large and small subunits and remove the N-terminal prodomain. The 2.3 Å resolution crystal structure of active Sf-caspase-1, the principal effector caspase of the insect Spodoptera frugiperda, is presented here. The structure represents the first nonhuman caspase to be resolved. The structure of the cleaved and active protease was determined with the tetrapeptide inhibitor N-acetyl-Asp-Glu-Val-Asp-chloromethylketone covalently bonded to the active site cysteine. As expected, the overall fold of Sf-caspase-1 is exceedingly similar to that of the five active caspases from humans solved to date. The overall structure and active site arrangement of Sf-caspase-1 is most comparable with that of the human effector caspases, with which it shares highest sequence homology. The most prominent structural difference with Sf-caspase-1 is the position of the N-terminal region of the large subunit. Unlike the N terminus of human caspases, the N terminus of Sf-caspase-1 originates from the active site side where it interacts with active site loop L2 and then extends to the backside of the heterodimer. This unusual structural arrangement raises the possibility that the N-terminal prodomain plays a regulatory role during effector caspase activation or enzyme activity in insects. Caspases play an essential role in the execution of apoptosis. These cysteine proteases are highly conserved among metazoans and are translated as inactive zymogens, which are activated by proteolytic cleavages to generate the large and small subunits and remove the N-terminal prodomain. The 2.3 Å resolution crystal structure of active Sf-caspase-1, the principal effector caspase of the insect Spodoptera frugiperda, is presented here. The structure represents the first nonhuman caspase to be resolved. The structure of the cleaved and active protease was determined with the tetrapeptide inhibitor N-acetyl-Asp-Glu-Val-Asp-chloromethylketone covalently bonded to the active site cysteine. As expected, the overall fold of Sf-caspase-1 is exceedingly similar to that of the five active caspases from humans solved to date. The overall structure and active site arrangement of Sf-caspase-1 is most comparable with that of the human effector caspases, with which it shares highest sequence homology. The most prominent structural difference with Sf-caspase-1 is the position of the N-terminal region of the large subunit. Unlike the N terminus of human caspases, the N terminus of Sf-caspase-1 originates from the active site side where it interacts with active site loop L2 and then extends to the backside of the heterodimer. This unusual structural arrangement raises the possibility that the N-terminal prodomain plays a regulatory role during effector caspase activation or enzyme activity in insects. Apoptosis is a cellular pathway that eliminates damaged, potentially dangerous, superfluous, or unwanted cells in metazoan organisms. This programmed cell death pathway is a naturally occurring physiological process that is vital to normal organismal development and tissue homeostasis. Abnormalities in the regulation of apoptosis can trigger many diseases including cancer, neurodegenerative disorders, autoimmune disorders, and ischemic injury. The apoptotic pathway is highly conserved in the Metazoa kingdom and can be triggered by both intracellular and extracellular stimuli. Apoptosis is executed through the activity of the caspases that are aspartyl-specific proteases (1Alnemri E.S. Livingston D.J. Nicholson D.W. Salvesen G. Thornberry N.A. Wong W.W. Yuan J. Cell. 1996; 87: 171Abstract Full Text Full Text PDF PubMed Scopus (2129) Google Scholar, 2Nicholson D.W. Thornberry N.A. Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2175) Google Scholar, 3Grütter M.G. Curr. Opin. Struc. Biol. 2000; 10: 649-655Crossref PubMed Scopus (439) Google Scholar, 4Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4104) Google Scholar, 5Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6133) Google Scholar, 6Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2428) Google Scholar). Because of their central role in apoptosis, these cysteine proteases are attractive targets for therapeutic intervention.Synthesized as dormant single-chain zymogens, the caspases are activated by a hierarchical series of proteolytic cleavages. The apical initiator caspases typically contain a large N-terminal prodomain that interacts with cellular factors that initiate apoptosis. These initiator caspases oligomerize through their prodomains that also associate with other proteins and promote enzyme activation in the absence of proteolytic processing (7Boatright K.M. Renatus M. Scott F.L. Sperandio S. Shin H. Pedersen I.M. Ricci J.E. Edris W.A. Sutherlin D.P. Green D.R. Salvesen G.S. Mol. Cell. 2003; 11: 529-541Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar). The activated initiator caspases subsequently cleave the proform of downstream effector caspases, which usually possess a short N-terminal prodomain. 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These structures reveal that the heterodimer of large and small subunits associates with another heterodimer to form a tetramer (or dimer-of-heterodimers). Both the large and small subunits contribute residues that form the active site pocket. Residues from the small subunit shape the binding pocket for the substrate residues P2-P4. Both subunits contribute to the selective recognition of aspartate at the P1 position. The carboxylate group of the substrate P1 aspartic acid salt-links with two conserved arginines, one from the large subunit and one from the small subunit, respectively. The P4 subsite in human caspase-1 is a large hydrophobic pocket that accommodates the preferred tryptophan side chain of the substrate. The corresponding pocket in caspase-3 is narrower and smaller, which thereby accommodates the smaller aspartate side chain. The conserved catalytic cysteine residue, which attacks the carbonyl carbon of the scissile bond, is situated at the end of β-strand 4. The imidazole side chain of conserved active site histidine is adjacent to cysteine and likely plays a role in catalysis (14Wilson K.P. Black J.-A.F. Thomson J.A. Kim E.E. Griffith J.P. Navia M.A. Murcko M.A. Chambers S.P. Aldape R.A. Raybuck S.A. Livingston D.J. Nature. 1994; 370: 270-275Crossref PubMed Scopus (752) Google Scholar, 15Walker N.P.C. Talanian R.V. Brady K.D. Dang L.C. Bump N.J. Ferenz C.R. Franklin S. Ghayur T. Hackett M.C. Hammill L.D. Herzong L. Hugunin M. Houy W. Mankovich J.A. McGuiness L. Orlewicz E. Paskind M. Pratt C.A. Reis P. Summani A. Terranova M. Welch J.P. Xiong L. Möller A. Tracey D.E. Kamen R. Wong W.W. Cell. 1994; 78: 343-352Abstract Full Text PDF PubMed Scopus (526) Google Scholar).Since discovery of the Caenorhabditis elegans caspase CED-3 (23Yuan J. Shaham S. Ledoux S. Ellis H.M. Horvitz H.R. Cell. 1993; 75: 641-652Abstract Full Text PDF PubMed Scopus (2234) Google Scholar), numerous caspases have been identified in diverse metazoan organisms, including invertebrates (24Song Z. McCall K. Steller H. Science. 1997; 275: 536-540Crossref PubMed Scopus (249) Google Scholar, 25Inohara N. Koseki T. Hu Y. Chen S. Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10717-10722Crossref PubMed Scopus (278) Google Scholar, 26Ahmad M. Srinivasula S.M. Wang L. Litwack G. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 1997; 272: 1421-1424Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 27Bayascas J.R. Yuste V.J. Benito E. Garcia-Fernandez J. Comella J.X. Cell Death Differ. 2002; 9: 1078-1089Crossref PubMed Scopus (35) Google Scholar, 28Fraser A.G. Evan G.I. EMBO J. 1997; 16: 2805-2813Crossref PubMed Scopus (170) Google Scholar, 29Dorstyn L. Colussi P.A. Quinn L.M. Richardson H. Kumar S. Proc. Natl. Acad. Sci. U. S. 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Chem. 1997; 272: 1421-1424Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), a pancaspase inhibitor with a novel mechanism of stoichiometric inhibition (32Bump N.J. Hackett M. Hugunin M. Seshagiri S. Brady K. Chen P. Ferenz C. Ferenz S. Franklin S. Ghayur T. Li P. Licari P. Mankovich J. Shi L. Greenberg A.H. Miller L.K. Wong W.W. Science. 1995; 269: 1885-1888Crossref PubMed Scopus (600) Google Scholar, 33Xu G. Cirilli M. Huang Y. Rich R.L. Myszka D.G. Wu H. Nature. 2001; 410: 494-497Crossref PubMed Scopus (157) Google Scholar, 34Fisher A.J. delaCruz W. Zoog S.J. Schneider C.L. Friesen P.D. EMBO J. 1999; 18: 2031-2039Crossref PubMed Scopus (99) Google Scholar, 35dela Cruz W.P. Friesen P.D. Fisher A.J. J. Biol. Chem. 2001; 276: 32933-32939Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Sf-caspase-1 displays 41 and 39% protein sequence identity to human effector caspases-7 and -3, respectively (26Ahmad M. Srinivasula S.M. Wang L. Litwack G. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 1997; 272: 1421-1424Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). It is most closely related to DrICE and DCP-1 (65 and 61% identical, respectively), which are short prodomain effector caspases of Drosophila melanogaster. Like its mammalian orthologs, Sf-caspase-1 is activated by two sequential caspase-mediated cleavages that take place in the cytosol (13LaCount D.J. Hanson S.F. Schneider C.L. Friesen P.D. J. Biol. Chem. 2000; 275: 15657-15664Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). First, a P35-resistant apical caspase cleaves pro-Sf-caspase-1 at Asp195 to separate the large and small subunits (p25 and p12, respectively). Subsequent cleavage by a separate caspase (probably Sf-caspase-1 itself) at Asp28 removes the N-terminal prodomain from the p25 fragment to generate the mature large subunit p19. It is expected that the activated subunits are assembled as a dimer-of-heterodimers that is common to all effector caspases (3Grütter M.G. Curr. Opin. Struc. Biol. 2000; 10: 649-655Crossref PubMed Scopus (439) Google Scholar).Because caspase activity and activation are decisive phases in executing apoptosis, a better understanding of the conserved features in caspase structure and function is necessary. Here we report the three-dimensional x-ray crystal structure of Sf-caspase-1. We compare the structural details of this invertebrate effector caspase with those of human effector and initiator caspases. The observed similarities and differences of these critical enzymes provide new insight into conserved features of the caspases that are expected to contribute to the design of therapeutic strategies for treatment of apoptosis-associated disorders.EXPERIMENTAL PROCEDURESProtein Expression, Purification, and Crystallization—The Sf-caspase-1 gene was cloned into the pET22b(+) expression vector (Novagen, Madison, WI) for production of Sf-caspase-1 with a C-terminal hexahistidine extension in Escherichia coli. Purified recombinant His-tagged Sf-caspase-1 was fully functional and stoichiometrically inhibited by baculovirus P35 as described previously (13LaCount D.J. Hanson S.F. Schneider C.L. Friesen P.D. J. Biol. Chem. 2000; 275: 15657-15664Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). For purification, bacterial pellets were resuspended in binding buffer (50 mm NaH2PO4, pH 8.0, 300 mm NaCl, 10 mm imidazole), lysed, and passed over a metal (Ni2+)-chelate column. N-Acetyl-Asp-Glu-Val-Asp-chloromethylketone inhibitor (Ac-DEVD-cmk) 1The abbreviations used are: Ac, acetyl; cmk, chloromethylketone; DEVD, Asp-Glu-Val-Asp tetrapeptide; rms, root mean square. (Calbiochem, San Diego, CA) was added, then dialyzed against binding buffer, and purified over a second high pressure metal-chelate column. The purified Sf-caspase-1, with acetyl-DEVD-methylketone inhibitor bound to the active site, was dialyzed against 25 mm Tris, pH 9.0, and concentrated to 10 mg/ml before crystallization trials.Sf-caspase-1 with bound inhibitor was crystallized by sitting drop vapor diffusion using 2-μl drops of the protein mixed with an equal volume of reservoir buffer (1.6 mm ammonium sulfate, 2% polyethylene glycol 1000, 0.1 m HEPES, pH 7.5). The crystals grew at room temperature to the size of 0.7 × 0.15 × 0.15 mm by 4 weeks. The crystals were transferred to 1.8 mm ammonium sulfate, 2% polyethylene glycol 1000, 0.1 m HEPES, pH 7.5, 30% ethylene glycol and frozen to -175 °C for data collection. X-ray diffraction data were collected to 2.3 Å resolution at Beamline 9-2 of the Stanford Synchrotron Radiation Laboratory, were processed with DENZO, and were scaled with SCALEPACK (Table I) (36Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38361) Google Scholar). The crystals belong to the trigonal space group P3221 with unit cell parameters of a = 151.7 Å, c = 79.5 Å.Table IData collection and refinement statisticsX-ray sourceSSRL BL 9-2Wavelength (Å)0.980Space groupP3221Cell parameters (Å)a = b = 151.7, c = 79.5Resolution (Å)30.0−2.30 (2.35−2.30)No. of reflections100,942No. of unique reflections45,879Completeness (%)97.6 (97.0)I/σ12.2 (2.98)Rmerge (%)6.4 (26.5)RefinementResolution (Å)30.0−2.30R factor (%) (43,689 reflections)18.3Rfree (%) (2,190 reflections)23.2r.m.s for bond distances (Å)0.017r.m.s for bond angles (deg)1.80No. of non-hydrogen protein atoms6,083No. of water molecules318No. of ethylene glycol atoms (8 molecules)32 Open table in a new tab Phase Determination, Model Building, and Refinement—The structure was solved by the molecular replacement method (37Rossmann M.G. The Molecular Replacement Method: A Collection of Papers on the Use of Non-Crystallographic Symmetry. Gordon and Breach, New York1972Google Scholar). The p17-p12 subunits of human caspase-3 (16Rotonda J. Nicholson D.W. Fazil K.M. Gallant M. Gareau Y. Labelle M. Peterson E.P. Rasper D.M. Ruel R. Vaillancourt J.P. Thornberry N.A. Becker J.W. Nat. Struc. Biol. 1996; 3: 619-625Crossref PubMed Scopus (400) Google Scholar), in which all residues were truncated to alanine, were used as a search model in the program AMoRe (38Navaza J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1993; 49: 588-591Crossref PubMed Google Scholar) in the CCP4 package (39Collaborative Computational Project Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19704) Google Scholar). Because it was originally thought that two complete dimer-of-heterodimers were in the crystallographic asymmetric unit (corresponding to a VM of 2.1 Å3/Da), the human caspase-3 dimer-of-heterodimers was used to search for two complete copies in the Sf-caspase-1 crystal asymmetric unit. The rotation function of AMoRe found many peaks, which were subjected to the first round of a translation search to identify the position of the first dimer-of-heterodimers. The first translation search found a peak corresponding to a correlation coefficient and R factor of 25.9 and 51.6%, respectively, with the next highest peak giving values of 20.1 and 53.8% and many other peaks with values of 12.1 and 55.8%. (Searching the enantiomorphic space group, P3121, resulted in no peaks higher than 12 and 57% for the correlation coefficient and R factor, respectively.) The search model was held fixed at this position, and a second translation search was conducted using the same caspase-3 dimer-of-heterodimers model to locate the second protomer in the asymmetric unit. This search resulted in one peak that stood out among the others (correlation coefficient and R factor of 34.8 and 49.3%, respectively, with all other peaks at ∼20 and 53%, respectively). Upon packing inspection of models in the trigonal unit cell, it was identified that the second translation search result was positioned on a crystallographic 2-fold axis, which was coincident with the dimer-of-heterodimers 2-fold axis, generating essentially two molecules superimposed on each other. This signified that there were 1.5 dimer-of-heterodimers in the crystallographic asymmetric unit, with one p19-p12 heterodimer situated at the crystallographic 2-fold axis to generate the functional dimer-of-heterodimers. This corresponded to a more reasonable Matthews coefficient of 2.8 Å3/Da (56% solvent) assuming three p19-p12 heterodimers/asymmetric unit (40Matthews B.W. J. Mol. Biol. 1968; 33: 491-497Crossref PubMed Scopus (7899) Google Scholar).Each of the three p19-p12 heterodimers was subjected to rigid body refinement in the CNS program, which lowered the R factor to 45.1% for all data to 3 Å resolution. The molecular replacement phases were refined by 3-fold molecular averaging and solvent flattening using DM in the CCP4 package (39Collaborative Computational Project Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19704) Google Scholar, 41Cowtan K. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography. 1994; 31: 34-38Google Scholar). One thousand rounds of averaging and phase extension starting at 8 Å resolution resulted in an overall final figure of merit of 0.86 at 3 Å resolution. The 3-fold averaged electron density map was readily interpretable, and a single p19-p12 heterodimer was built into the density with aid of the molecular graphics program O (42Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar). After initial model building, the complete asymmetric unit was generated by rotating and translating the p19-p12 heterodimer built in the averaged map. The model was then subjected to subsequent rounds of simulated annealing with the program CNS (43Brünger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16929) Google Scholar) applying the maximum likelihood as a refinement target. Initially, high restraints were placed on the noncrystallographic symmetry between the three p19-p12 heterodimers in the asymmetric unit. During stages of refinement the weight placed on the noncrystallographic symmetry restraint was re-evaluated to give the lowest Rfree value. As the model improved, the noncrystallographic weight that gave the lowest Rfree decreased until the noncrystallographic symmetry restraints were released completely, resulting in lower Rfree values. The final conventional R factor is 18.3%, and the Rfree value is 23.2% for 95 and 5% of all recorded data to 2.3 Å resolution, respectively (44Brünger A.T. Nature. 1992; 355: 472-474Crossref PubMed Scopus (3849) Google Scholar). The data collection and refinement statistics are given in Table I. 91.5 and 8.5% of the 759 residues in the asymmetric unit plot to the "most favored" and "additionally allowed" regions of the Ramachandran plot respectively as defined by the program PROCHECK (45Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). None of the 759 residues plot in the "generously allowed" or "disallowed" region of the Ramachandran plot.RESULTSOverall Structure—The asymmetric unit contains three large subunit (p19)-small subunit (p12) heterodimers. The p19-p12 heterodimer designated A-chain combines with the B-chain heterodimer to form one biologically functional dimer-of-heterodimers. The designated C-chain p19-p12 heterodimer is positioned next to a crystallographic 2-fold axis, which places a symmetry related C-chain p19-p12 heterodimer from the neighboring unit cell adjacent to it, thus generating the functional dimer-of-heterodimers. The final model consists of residues 40-191 (p19) and 201-295 (p12) for the A chain; residues 41-191 and 200-296 for the B-chain; and residues 40-191 and 201-300 for the C-chain. Sf-caspase-1 expression and purification in E. coli can produce a mixture of large subunits, in which some of the p19 subunits are cleaved after Asp184, generating a composite of p19/p18 large subunits with the p12 small subunit (13LaCount D.J. Hanson S.F. Schneider C.L. Friesen P.D. J. Biol. Chem. 2000; 275: 15657-15664Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 26Ahmad M. Srinivasula S.M. Wang L. Litwack G. Fernandes-Alnemri T. Alnemri E.S. J. Biol. Chem. 1997; 272: 1421-1424Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). However, our preparations were homogenous with respect to the large subunit as indicated by SDS-PAGE (not shown). In addition, clear electron density was observed in the crystal structure for residues Asp184 to Arg191 in all three p19 subunits in the crystallographic asymmetric unit. The Ac-DEVD-cmk inhibitor is clearly defined in all three active sites of the asymmetric unit. However, the chlorine atom is displaced by the active site Cys178, in which Sγ is covalently bonded to the methyl group. The average temperature factor (B) for all atoms is 31.3, 29.8, and 26.1 Å2 for the A-, B-, and C-chains respectively. The C-chain likely has an overall lower average B value because it makes more lattice contacts than either the A- or B-chain. Because the C-chain heterodimer residues were the most ordered, and the overall average temperature value is lower than the A- or B-chain, most of the following discussion will concentrate on the C-chain heterodimer.In Sf-caspase-1, two p19-p12 heterodimers assemble to form the biologically active dimer-of-heterodimers producing a central 12-stranded β-sheet. A total of ∼4,900 Å2 of surface area is buried between the two p19-p12 heterodimers. The two active sites in the dimer-of-heterodimers are separated by ∼35 Å. The overall topology of Sf-caspase-1 (Fig. 1) is very similar to that of the human caspases. The overall shape of the dimer-of-heterodimers is roughly a compact cylinder (∼60 Å long × 40 Å wide) consisting of a twisted 12-stranded β-sheet running down the axis of the cylinder surrounded by 10 α-helices. Each p19-p12 heterodimer folds into a single-domain α/β motif with a central six-stranded β-sheet (β2, β1, β3, β4, β7, and β8 from outside to inside) sandwiched by five α-helices, two on one side of the β-sheet (αC and αD) and three on the other (αB, αE, and αF) (Fig. 1). The secondary structure designations follow that of the human caspase-1 structure (14Wilson K.P. Black J.-A.F. Thomson J.A. Kim E.E. Griffith J.P. Navia M.A. Murcko M.A. Chambers S.P. Aldape R.A. Raybuck S.A. Livingston D.J. Nature. 1994; 370: 270-275Crossref PubMed Scopus (752) Google Scholar), and the active site loop designations (L1-L4) correspond to that previously reported (46Chai J. Wu Q. Shiozaki E. Srinivasula S.M. Alnemri E.S. Shi Y. Cell. 2001; 107: 399-407Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). The first α-helix in human caspase-1, αA, is missing because the structural element is not conserved in Sf-caspase-1, as well as the human effector caspases. The p19 large subunit of Sf-caspase-1 contributes the outer four parallel β-strands and three helices, whereas the p12 small subunit adds two α-helices and two antiparallel β-strands adjacent to the 2-fold axis, which forms the central four antiparallel β-strands in the dimer-of-heterodimers (Fig. 1). Following strand β3, three short β-strands (β3a, β3b, and β3c) form a β-meander motif before the short helix αD (Fig. 1). The β7-αE loop (L3, Fig. 1) contains a short β-strand (β7a) that hydrogen bonds to the main chain of the tetrapeptide inhibitor. The αF-β8 loop (L4) adopts a hairpin loop conformation containing two antiparallel β-strands (β7b and β7c), which forms part of the active site pocket. The first ordered N-terminal residues of the p19 subunit (residues 40-44) forms a β-strand (βa), which together with β6 clamps β5 from the 2-fold related p19-p12 heterodimer (Fig. 1). The location of these N-terminal residues of the p19 in Sf-caspase-1 differs significantly from the human caspases (see below). The active site Cys178 is located at the end of β4, and the active site His136 is situated at the start of β3a. The active site pocket, which binds the tetrapeptide substrate recognition sequence, is formed by loops L1-L4 from both the p19 and p12 subunits.Structural Comparison with Human Caspases—The overall structure and topology of Sf-caspase-1 is very similar to that of the human caspases. A stereo superposition (Fig. 2) demonstrates the similarities of the Sf-caspase-1 p19-p12 heterodimer (in red) with the heterodimers of activated human caspases-1, -3, -7, -8, and -9. Over an average of 220 equivalent α-carbons, the root mean square (rms) deviation from Sf-caspase-1 is 1.22, 0.61, 0.59, 0.87, and 1.05 Å, respectively, for each of these human caspases. The rms deviation parallels the sequence identity with the higher identity correlating with lower rms deviation.Fig. 2Structural comparison of insect Sf-caspase-1 to all known activated h
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