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• W2034891969 abstract "Apoptosis is a highly regulated multistep process for programmed cellular destruction. It is centered on the activation of a group of intracellular cysteine proteases known as caspases. The baculoviral p35 protein effectively blocks apoptosis through its broad spectrum caspase inhibition. It harbors a caspase recognition sequence within a highly protruding reactive site loop (RSL), which gets cleaved by a target caspase before the formation of a tight complex. The crystal structure of the post-cleavage complex between p35 and caspase-8 shows that p35 forms a thioester bond with the active site cysteine of the caspase. The covalent bond is prevented from hydrolysis by the N terminus of p35, which repositions into the active site of the caspase to eliminate solvent accessibility of the catalytic residues. Here, we report mutational analyses of the pre-cleavage and post-cleavage p35/caspase interactions using surface plasmon resonance biosensor measurements, pull-down assays and kinetic inhibition experiments. The experiments identify important structural elements for caspase inhibition by p35, including the strict requirement for a Cys at the N terminus of p35 and the rigidity of the RSL. A bowstring kinetic model for p35 function is derived in which the tension generated in the bowstring system during the pre-cleavage interaction is crucial for the fast post-cleavage conformational changes required for inhibition. Apoptosis is a highly regulated multistep process for programmed cellular destruction. It is centered on the activation of a group of intracellular cysteine proteases known as caspases. The baculoviral p35 protein effectively blocks apoptosis through its broad spectrum caspase inhibition. It harbors a caspase recognition sequence within a highly protruding reactive site loop (RSL), which gets cleaved by a target caspase before the formation of a tight complex. The crystal structure of the post-cleavage complex between p35 and caspase-8 shows that p35 forms a thioester bond with the active site cysteine of the caspase. The covalent bond is prevented from hydrolysis by the N terminus of p35, which repositions into the active site of the caspase to eliminate solvent accessibility of the catalytic residues. Here, we report mutational analyses of the pre-cleavage and post-cleavage p35/caspase interactions using surface plasmon resonance biosensor measurements, pull-down assays and kinetic inhibition experiments. The experiments identify important structural elements for caspase inhibition by p35, including the strict requirement for a Cys at the N terminus of p35 and the rigidity of the RSL. A bowstring kinetic model for p35 function is derived in which the tension generated in the bowstring system during the pre-cleavage interaction is crucial for the fast post-cleavage conformational changes required for inhibition. The development and homeostasis of multicellular organisms depend on a delicate balance of cell proliferation and programmed cell death or apoptosis. Failure to control either of these processes can lead to serious diseases that threaten the existence of the organism (1Thompson C.B. Science. 1995; 267: 1456-1461Crossref PubMed Scopus (6170) Google Scholar, 2Johnstone R.W. Ruefli A.A. Lowe S.W. Cell. 2002; 108: 153-164Abstract Full Text Full Text PDF PubMed Scopus (1976) Google Scholar). For example, the down-regulation of apoptosis is often associated with cancer, autoimmune disorders, and persistent viral infections. The up-regulation of apoptosis is observed in many forms of degenerative disorders such as Alzheimer's disease, ischemic injury from stroke, and post-menopausal osteoporosis.The central effectors of apoptotic cell death are caspases, a group of cysteine proteases specific for aspartate residues (3Nicholson D.W. Cell Death Differ. 1999; 6: 1028-1042Crossref PubMed Scopus (1294) Google Scholar). Caspases are highly regulated at several different levels. First, they are synthesized as inactive single-chain zymogens. Second, caspase activation is achieved through controlled proteolytic cascades, with upstream caspases (Group III, such as caspase-8 and caspase-9) activated by signal-mediated oligomerization and autoprocessing and downstream caspases (Group II, such as caspase-3 and casapse-7) activated by upstream caspases. While caspases have a dominant requirement for Asp at the P1 position, neighboring sequences at P5-P1′ (in particular P4-P2) influence the substrate specificity of each group of caspases (4Stennicke H.R. Renatus M. Meldal M. Salvesen G.S. Biochem. J. 2000; 350: 563-568Crossref PubMed Scopus (262) Google Scholar).Active caspases are in addition subject to inhibition by specific viral and cellular caspase inhibitors (5Ekert P.G. Silke J. Vaux D.L. Cell Death Differ. 1999; 6: 1081-1086Crossref PubMed Scopus (389) Google Scholar, 6Stennicke H.R. Ryan C.A. Salvesen G.S. Trends Biochem. Sci. 2002; 27: 94-101Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Most notably, the p35 protein from baculoviruses is an effective and the only wide-spectrum caspase inhibitor. It blocks apoptosis induced by numerous stimuli and in diverse organisms (7Bump N.J. Hackett M. Hugunin M. Seshagiri S. Brady K. Chen P. Ferenz C. Franklin S. Ghayur T. Li P. Mankovich J. Shi L.F. Greenburg A.H. Miller L.K. Wong W.W. Science. 1995; 269: 1885-1888Crossref PubMed Scopus (600) Google Scholar, 8Xue D. Horvitz H.R. Nature. 1995; 377: 248-251Crossref PubMed Scopus (436) Google Scholar, 9Zhou Q. Krebs J.F. Snipas S.J. Price A. Alnemri E.S. Tomaselli K.J. Salvesen G.S. Biochemistry. 1998; 37: 10757-10765Crossref PubMed Scopus (194) Google Scholar). Transgenic expression of p35 shows immense promise in controlling apoptosis and degenerative diseases (10Araki T. Shibata M. Takano R. Hisahara S. Imamura S. Fukuuchi Y. Saruta T. Okano H. Miura M. Cell Death Differ. 2000; 7: 485-492Crossref PubMed Scopus (17) Google Scholar, 11Beidler D.R. Tewari M. Friesen P.D. Poirier G. Dixit V.M. J. Biol. Chem. 1995; 270: 16526-16528Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 12Clem R.J. Fechheimer M. Miller L.K. Science. 1991; 254: 1388-1390Crossref PubMed Scopus (703) Google Scholar, 13Datta R. Kojima H. Banach D. Bump N.J. Talanian R.V. Alnemri E.S. Weichselbaum R.R. Wong W.W. Kufe D.W. J. Biol. Chem. 1997; 272: 1965-1969Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 14Hay B.A. Wolff T. Rubin G.M. Development (Camb.). 1994; 120: 2121-2129PubMed Google Scholar, 15Hisahara S. Araki T. Sugiyama F. Yagami K. Suzuki M. Abe K. Yamamura K. Miyazaki J. Momoi T. Saruta T. Bernard C.C. Okano H. Miura M. EMBO J. 2000; 19: 341-348Crossref PubMed Scopus (96) Google Scholar, 16Martinou I. Fernandez P.A. Missotten M. White E. Allet B. Sadoul R. Martinou J.C. J. Cell Biol. 1995; 128: 201-208Crossref PubMed Scopus (131) Google Scholar, 17Morishima N. Okano K. Shibata T. Maeda S. FEBS Lett. 1998; 427: 144-148Crossref PubMed Scopus (25) Google Scholar, 18Rabizadeh S. LaCount D.J. Friesen P.D. Bredesen D.E. J. Neurochem. 1993; 61: 2318-2321Crossref PubMed Scopus (175) Google Scholar, 19Robertson N.M. Zangrilli J. Fernandes-Alnemri T. Friesen P.D. Litwack G. Alnemri E.S. Cancer Res. 1997; 57: 43-47PubMed Google Scholar, 20Sugimoto A. Friesen P.D. Rothman J.H. EMBO J. 1994; 13: 2023-2028Crossref PubMed Scopus (186) Google Scholar). Previous biochemical and structural studies showed that caspase inhibition by p35 requires the cleavage of a caspase recognition sequence (DQMD87) within a solvent exposed and highly protruding reactive site loop (RSL) 1The abbreviations used are: RSL, reactive site loop; Ac-DEVD-AFC, acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 1The abbreviations used are: RSL, reactive site loop; Ac-DEVD-AFC, acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (21Fisher A.J. Cruz W. Zoog S.J. Schneider C.L. Friesen P.D. EMBO J. 1999; 18: 2031-2039Crossref PubMed Scopus (99) Google Scholar), followed by the formation of a tight post-cleavage complex with the caspase.Previously, we reported the crystal structure of the post-cleavage complex between p35 and caspase-8, a group III initiator caspase involved in Fas-mediated apoptosis (22Xu G. Cirilli M. Huang Y. Rich R.L. Myszka D.G. Wu H. Nature. 2001; 410: 494-497Crossref PubMed Scopus (157) Google Scholar). The structure revealed that the caspase is inhibited via a covalent thioester linkage between the active site Cys360 of caspase-8 (Cys285of caspase-1 numbering) and the cleavage residue Asp87 of p35. During normal substrate cleavage, the thioester intermediate is quickly hydrolyzed by an enzyme bound water molecule. In the p35-caspase-8 complex, the thioester bond is preserved by the N terminus of p35, which interacts with the caspase active site and excludes solvent from the catalytic His317 of caspase-8 (His237 of caspase-1 numbering). The interaction between the p35 N terminus and the caspase is realized through a series of dramatic post-cleavage conformational changes (22Xu G. Cirilli M. Huang Y. Rich R.L. Myszka D.G. Wu H. Nature. 2001; 410: 494-497Crossref PubMed Scopus (157) Google Scholar).In the structure of the p35-caspase-8 post-cleavage complex, three regions of p35 directly contact the caspase, the N terminus, the KL region (including the KL loop and the K and L strands) and the substrate sequence of p35 (Fig. 1). To further understand the structural determinant of caspase inhibition by p35, we performed structure-based mutagenesis at these three regions of p35. The mutants were extensively analyzed for their effects on the pre-cleavage association, as assessed by surface plasmon resonance biosensor measurements, and on the post-cleavage inhibition, as determined by both qualitative pull-down assays and quantitative kinetic inhibition experiments. These experiments not only identified functionally important structural elements of p35 in caspase inhibition, but also led to a novel bowstring kinetic model of caspase inhibition by p35. In this model, the p35 may be considered as a bowstring system with the RSL being the string. The tension produced in the string during the pre-cleavage association with a target caspase appears to control the efficiency of caspase inhibition by facilitating fast post-cleavage conformational changes.RESULTS AND DISCUSSIONCaspase inhibition by p35 presumably proceeds through two distinct steps: a pre-cleavage association, which is the reversible mutual recognition between p35 and the caspase, followed by the cleavage, and the formation of an irreversible post-cleavage complex (Fig. 1 A). To obtain a thorough understanding on the mechanism of caspase inhibition by p35, we mutated a series of p35 residues in contact with caspase-8 in the post-cleavage complex, including the p35 N terminus, the KL region and the RSL (Fig. 1 B). These mutants were characterized to derive an integral understanding of the inhibitory process.Kinetic Characterization of the Pre-cleavage AssociationTo trap the pre-cleavage interaction, we used an active site mutant of caspase-3 (C163A) that was processed to its active conformation through trans-activation by its upstream caspase, caspase-8. Measurement by surface plasmon resonance for the interaction between p35 and caspase-3 (C163A) gave rise to an association rate of 6.7 × 105m−1 s−1, similar to many diffusion-controlled rigid body macromolecular associations (25Park Y.C. Ye H. Hsia C. Segal D. Rich R.L. Liou H.-L. Myszka D.G. Wu H. Cell. 2000; 101: 777-787Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 26Cunningham B.C. Wells J.A. J. Mol. Biol. 1993; 234: 554-563Crossref PubMed Scopus (485) Google Scholar), and a dissociation rate of 0.091s−1 (Table I). This fast rate of association suggests that the interaction between p35 and a caspase does not involve complex conformational changes, which would otherwise slow down the interaction. The calculated equilibrium dissociation constant is 0.13 μm, which is ∼200-fold stronger than theK m between caspase-3 and a peptide substrate (9Zhou Q. Krebs J.F. Snipas S.J. Price A. Alnemri E.S. Tomaselli K.J. Salvesen G.S. Biochemistry. 1998; 37: 10757-10765Crossref PubMed Scopus (194) Google Scholar). Because the direct contact between p35 and a caspase during pre-cleavage interaction is likely not much more extensive than substrate recognition by a caspase (since the KL loop does not appear to be energetically indispensable, see below), the stronger interaction between p35 and a caspase is likely due to the decrease in entropic loss often associated with the recognition of flexible peptide substrates. These kinetic and thermodynamic observations are consistent with the apparent rigidity of the highly solvent accessible RSL in the free p35 structure (21Fisher A.J. Cruz W. Zoog S.J. Schneider C.L. Friesen P.D. EMBO J. 1999; 18: 2031-2039Crossref PubMed Scopus (99) Google Scholar) (Fig. 1 C).Table IBiochemical characterization of structure-based p35 mutantsPost-cleavage interactionPre-cleavage interactionCleavagePull-downK ik on, caspase-3k off, caspase-3K d, caspase-3Aggregation1-aShown by gel filtration.Caspase-8Caspase-3Caspase-8Caspase-3Caspase-8Caspase-3p35WT−++++0.15 nm, relative 1.00.87 nm relative 1.06.7 ± 0.6 × 105m−1s−1, relative 1.00.091 ± 0.012 s−1, relative 1.00.13 ± 0.1 μm, relative 1.0N-terminal regionC2S−++−−1.01.30.91C2A−++−−1.31.10.83C2G1-bPreviously published mutants (22).−++−−1.21.31.2Δ2–51-bPreviously published mutants (22).+++−−NF1-cNF, the Biosensor data could not be fit with single bimolecular interaction.NFNFV3G1-bPreviously published mutants (22).+++−−NFNFNFV3A+++−−NFNFNFV3I−++++2.11.11.11.00.94I4A+++−−NFNFNFF5A+++−−P6A+++−−D10A−++++4.12.31.11.21.0Q13G−++++4.71.41.11.31.2ΔD10+++−−Δ (10–11)+++−−D10∇G−++++15140.830.911.1D10∇GG+++−−NFNFNFKL-loop regionS253A−++++3.91.80.941.01.1W254A−++++122.50.572.03.4K256A−++++3.41.30.812.03.4Y260A−++++194.10.761.41.8Δ252–258−++−−0.874.14.6Δ253–257−++++131.51.10.820.73ΔW254−++++131.61.11.21.1253/254/260/AAA−++++362.11.21.31.1Substrate regionY82A1-aShown by gel filtration.−++−−S83A−++++1.92.81.32.61.9D84A−++++58930.0518.3158D84N−++++47610.141392Q85A−++++552.80.973.23.3Q85K−++++4005.90.766.38.1Q85E−++++31.50.850.921.1Q85N−++++1491.30.888.39.2M86A−++++373.10.971.61.7M86Q−++++217.20.737.210M86V−++++7.31.10.490.0470.092M86I−++++134.90.510.230.4388–91/AAAA−++++a Shown by gel filtration.b Previously published mutants (22Xu G. Cirilli M. Huang Y. Rich R.L. Myszka D.G. Wu H. Nature. 2001; 410: 494-497Crossref PubMed Scopus (157) Google Scholar).c NF, the Biosensor data could not be fit with single bimolecular interaction. Open table in a new tab Strict Requirement of the Cys2 Residue and Importance of the Structural Integrity of the N-terminal Segment in Free p35In the crystal structure of the p35/caspase-8 complex, the N terminus of p35 repositions into the active site of caspase-8 to block thioester bond hydrolysis (22Xu G. Cirilli M. Huang Y. Rich R.L. Myszka D.G. Wu H. Nature. 2001; 410: 494-497Crossref PubMed Scopus (157) Google Scholar). Two p35 N-terminal residues, Cys2 and Val3, directly contact the caspase and both the C2G and V3G mutations have been shown to render p35 defective in caspase inhibition (22Xu G. Cirilli M. Huang Y. Rich R.L. Myszka D.G. Wu H. Nature. 2001; 410: 494-497Crossref PubMed Scopus (157) Google Scholar). To further understand the structural requirement of these two contacting residues, we performed both alanine mutations and conservative substitutions on these residues (Table I, Fig. 2, A andB).Figure 2Biochemical characterization of caspase inhibition by p35. A, selected examples of the kinetic analysis of the pre-cleavage interaction between p35 and active site mutant of caspase-3 (C163A) using Biacore. Black linesrepresent the responses obtained for 0, 0.033, 0.1, and 0.3 μm p35 mutant M86V (left) and 0, 0.016, 0.031, 0.063, 0.13, 0.25, and 0.50 μm p35 mutant C2S (right), flowed across caspase-3 (C163A) immobilized on the biosensor surface. Red lines indicate the fit of the data to a simple bimolecular interaction model. B, pull-down of caspase-3 by wild-type and mutant His-tagged p35. The large subunit of caspase-3 often ran as a close doublet and the p10 fragment of cleaved p35 ran closely to the small subunit of caspase-3. C, selected examples of progress curve analyses showing substrate hydrolysis (represented by relative fluorescence units (RFU)) with time for caspase-8 inhibition by different concentrations of the p35 mutant Y260A (left) and for an overlay of caspase-8 inhibition by wild-type and several p35 mutants at 0.1 μm concentration (right).View Large Image Figure ViewerDownload (PPT)Similar to the phenotype of the C2G mutation, both C2A and the isosteric C2S mutants failed to pull-down with either caspase-3 or caspase-8, although both were cleaved efficiently by the caspases. As these mutants exhibited identical solution behavior with wild-type p35, as shown by gel-filtration profiles, the mutational effects are likely due to the direct deletion of interactions with the caspases. The drastic phenotype of the C2S mutant suggests that the interaction between residue Cys2 of p35 and a target caspase exhibits a strict specificity. This specificity may be explained by the structural observation that the side chain thiol of Cys2 may form a hydrogen bond with the imidazole ring of His317 in caspase-8 (22Xu G. Cirilli M. Huang Y. Rich R.L. Myszka D.G. Wu H. Nature. 2001; 410: 494-497Crossref PubMed Scopus (157) Google Scholar). None of these mutations affected the pre-cleavage association.Conservative substitution V3I did not significantly change its inhibitory activity against caspases. In contrast, the V3A mutant failed to form a stable complex with either caspase-3 or caspase-8, although it could be readily cleaved by these caspases. Since Val3 is buried in the free p35 structure, this mutant phenotype may be explained by either a direct effect on a p35-caspase complex or an indirect effect through perturbation of the free p35 structure. Although it is not possible to distinguish these effects, the fact that the V3A mutant tends to aggregate in solution supports that structural perturbation may be at play in this mutant.To determine whether residues at the N-terminal segment that do not directly contact the caspase (residues 4–13) can influence the ability of p35 to inhibit caspases through structural perturbation, we selectively mutated a few residues at the N terminus. Most N-terminal residues such as Ile4, Phe5, and Pro6 are buried in the free p35 structure, with the exception of Asp10 and Gln13. Interestingly, the I4A, F5A, and P6A mutations in p35 were detrimental to the inhibition, while the D10A and Q13G mutants behaved similarly as the wild-type p35 in their ability to inhibit either caspase-3 or caspase-8. The I4A, F5A, and P6A mutants showed aggregation in their gel-filtration profiles and therefore may have possible local structural perturbations. Accordingly, biosensor measurements showed that the pre-cleavage association of these p35 mutants to caspase-3 (C163A) exhibited complex behavior, indicating the presence of a mixture of stoichiometries in the interactions. In contrast, both the D10A and Q13A mutants behaved as the wild-type p35 in solution. These results support that structural perturbation of the buried N-terminal arm in the free p35 structure can abrogate the inhibitory activity of p35.The KL Region Makes Modest and Variable Energetic Contribution to Caspase Interaction but Supports a Crucial Contact with the RSLIn the structure of the p35-caspase-8 complex, residues in the KL region make direct contact with residues 414–427 in the small subunit of caspase-8 at the L4 loop and the α4 helix. However, these residues in caspase-8 only show limited sequence conservation among different caspases. Since there are significant conformational changes in the KL loop region between the bound and the free p35, we had earlier proposed that this flexibility of the KL loop might be important for its ability to interact with different caspases (22Xu G. Cirilli M. Huang Y. Rich R.L. Myszka D.G. Wu H. Nature. 2001; 410: 494-497Crossref PubMed Scopus (157) Google Scholar).We created a series of point mutations and deletions to determine the functional role of the KL region (Table I and Fig. 2, B andC). We generated single-site mutations on residues in direct contact with caspase-8, S253A, W254A, K256A, and Y260A. We also created triple alanine mutations on S253, W254, and Y260, the three residues that show most extensive surface area burial in the complex. In addition, we created deletion mutations that remove one (ΔTrp254), five (Δ253–257) and seven (Δ252–258) residues from the KL loop and the adjoining β strands.Surprisingly, the mutational data suggest that the KL loop does not seem to play an indispensable role as would have been predicted from the structure. The W254A, Y260A, ΔTrp254, and Δ253–257 mutants exhibited only modest, but significant, effects against caspase-8 and behaved essentially as wild-type against caspase-3, an effector caspase. In addition, the pre-cleavage interactions between the p35 mutants and caspase-3 (C163A) are also very similar to the wild-type interaction. These results show that the KL region is not crucial for caspase inhibition and suggest that there may be significant differences in the role of the KL region against different caspases. Interestingly, a low resolution structure of the complex between p35 and an insect effector caspase showed that there is an orientational difference between the p35 in complex with an initiator caspase, caspase-8, and with the effector caspase (27Eddins M.J. Lemongello D. Friesen P.D. Fisher A.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 299-302Crossref PubMed Scopus (18) Google Scholar). Although the KL region appears to contact the target caspase in both complexes, their energetic roles may be different.The relative unimportance of the KL region also explains the ability of p35 to inhibit gingipain-K, a caspase-like bacterial cysteine protease that cleaves a different site on the RSL of p35 (28Snipas S.J. Stennicke H.R. Riedl S. Potempa J. Travis J. Barrett A.J. Salvesen G.S. Biochem. J. 2001; 357: 575-580Crossref PubMed Scopus (31) Google Scholar) (Fig. 1 C). Because gingipain-K cleaves further downstream (91DSIK94) from the caspase recognition site (84DQMD87), it is unlikely that gingipain-K would be close enough to the KL loop for a direct interaction.Interestingly, the KL loop deletion mutant Δ252–258 completely abolished the inhibitory activity of p35 against both caspase-3 and caspase-8, without drastically affecting the formation of the pre-cleavage complex. We have previously shown that residue Tyr82 near the beginning of the RSL is essential for the ability of p35 to inhibit caspases (22Xu G. Cirilli M. Huang Y. Rich R.L. Myszka D.G. Wu H. Nature. 2001; 410: 494-497Crossref PubMed Scopus (157) Google Scholar). We had postulated that Tyr82 helps to glue the RSL with the neighboring KL strands, in addition to its role in direct caspase contact. Because mutant Δ253–257 does not exhibit a drastic decrease in caspase inhibition, and the two additional residues deleted in Δ252–258 do not directly contact the caspase, the defective phenotype of the Δ252–258 mutant may be best explained by the perturbation of the local conformation of the K and L strands. Among other interactions, the K and L strands harbor two large aromatic residues Phe248 and Trp262 that interact with Tyr82 of the RSL. As the Cα distance between residues 251 and 259 is 5.2 Å, longer than a typical Cα distance between two adjacent residues, the deletion mutant Δ252–258 has to undergo conformational changes to join these two residues, leading to structural perturbations. Therefore, a crucial role of the KL region appears to provide a proper “glue” patch for the RSL.Substrate Region Residues; Pre-cleavage Association Directly Affects Inhibition, but May Not Be Rate-limitingThe non-covalent interaction between p35 and caspase-8 centers around the tetrapeptide caspase recognition sequence (P4-D84QMD87-P1) in the reactive site loop of p35. It has been shown previously that the P1 Asp residue is essential for p35 function (21Fisher A.J. Cruz W. Zoog S.J. Schneider C.L. Friesen P.D. EMBO J. 1999; 18: 2031-2039Crossref PubMed Scopus (99) Google Scholar, 29Bertin J. Mendrysa S.M. LaCount D.J. Gaur S. Krebs J.F. Armstrong R.C. Tomaselli K.J. Friesen P.D. J. Virol. 1996; 70: 6251-6259Crossref PubMed Google Scholar), consistent with the absolute specificity of caspases to cleave after Asp residues. To elucidate the specific role of these residues in the function of p35, we generated a series of point mutations at the P2-P4 positions of the caspase recognition sequence.None of the mutations on the P2-P4 positions completely abolished the caspase inhibitory activity of p35, but produced a range of different effects against caspase-3 and caspase-8 (Table I and Fig. 2) that are largely consistent with the substrate specificity of these caspases (30Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1838) Google Scholar). For example, mutations on the P2 residue are relatively mild against both caspases, consistent with P2 being the most tolerable. In contrast, P4 mutations are more drastic, especially for caspase-3, which is consistent with the known preference of Asp in this position for the group II apoptotic effector caspases such as caspase-3. The most surprising is the mutational phenotype on the P3 residue, which generated drastic effects on caspase-8, but did not seem to harm its inhibition on caspase-3, even though both caspases appear to prefer Glu at this position based on combinatorial substrate analyses (3Nicholson D.W. Cell Death Differ. 1999; 6: 1028-1042Crossref PubMed Scopus (1294) Google Scholar, 30Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1838) Google Scholar).How does the strength of pre-cleavage association affect the post-cleavage caspase inhibition? It appears that an efficient pre-cleavage interaction is required for inhibition because most of the p35 mutants that have a decreased pre-cleavage interaction are also less effective in caspase inhibition. However, substrate recognition or pre-cleavage interaction seems not to be the rate-limiting step in caspase inhibition by wild-type p35, as exemplified by the M86V mutant, which exhibits significantly stronger pre-cleavage association, but is wild-type in inhibition.Bowstring Kinetic Model of p35 InhibitionThe molecular event following p35 cleavage suggests the existence of two opposing forces in caspase inhibition by p35. As the post-cleavage strand of p35 departs the active site of the caspase, a series of cooperative conformational changes occur in p35 that allows the release of its N terminus from the core of p35 for interacting with the caspase active site. This has to occur before the caspase is able to hydrolyze the thioester intermediate formed between the caspase and p35. The existence of such a race between the catalytic power of the caspase and the rate of post-cleavage conformational changes may be exemplified by the observed leakage in caspase inhibition by p35, which often requires higher than stoichiometric quantity of p35 to completely inhibit a target caspase (9Zhou Q. Krebs J.F. Snipas S.J. Price A. Alnemri E.S. Tomaselli K.J. Salvesen G.S. Biochemistry. 1998; 37: 10757-10765Crossref PubMed Scopus (194) Google Scholar). In addition, since stronger pre-cleavage association does not necessarily translate into stronger inhibition, the rate-limiting step of the inhibition could rest on the rate of these post-cleavage conformational changes.So what controls the rate of conformational changes and therefore the efficiency of caspase inhibition? A most conspicuous feature of the free p35 structure is the conformational rigidity of the entirely solvent exposed RSL, as shown by the visibility of the loop in the electron density map (21Fisher A.J. Cruz W. Zoog S.J. Schneider C.L. Friesen P.D. EMBO J. 1999; 18: 2031-2039Crossref PubMed Scopus (99) Google Scholar). In addition, our measurements on the association rate and the strength of the pre-cleavage association also suggest a nearly rigid-body interaction between p35 and the caspase.During the pre-cleavage association in which the caspase recognition sequence in RSL interacts with the active site of the caspase, this rigidity is likely to transform into tension in the RSL, because RSL residues after the cleavage site would have to be stretched by 4 Å due to the pinching of the caspase recognition sequence (Fig. 3 A). The Cα distance between the cleavage residue Asp87 and Lys97 is 26.7 Å in the free p35 structure. Assuming that residue Lys97 does not move significantly in the pre-cleavage complex, this distance may be increased to 30.6 Å, which would very likely create tension in this already rigid RSL.Figure 3Bowstring kinetic model of caspase inhibition by p35. A, mapping of known “tension” mutations on free p35 (cyan). The conformation of the bound/cleaved p35 RSL is superimposed (yellow). Y82A and Δ252–258 may compromise the interaction between the" @default.
• W2034891969 created "2016-06-24" @default.
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• W2034891969 date "2003-02-01" @default.
• W2034891969 modified "2023-10-12" @default.
• W2034891969 title "Mutational Analyses of the p35-Caspase Interaction" @default.
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