title: Substrate and inhibitor-induced dimerization and cooperativity in caspase-1 but not caspase-3.
abstract: Caspases are intracellular cysteine-class proteases with aspartate specificity that is critical for driving processes as diverse as the innate immune response and apoptosis, exemplified by caspase-1 and caspase-3, respectively. Interestingly, caspase-1 cleaves far fewer cellular substrates than caspase-3 and also shows strong positive cooperativity between the two active sites of the homodimer, unlike caspase-3. Biophysical and kinetic studies here present a molecular basis for this difference. Analytical ultracentrifugation experiments show that mature caspase-1 exists predominantly as a monomer under physiological concentrations that undergoes dimerization in the presence of substrate; specifically, substrate binding shifts the KD for dimerization by 20-fold. We have created a hemi-active site-labeled dimer of caspase-1, where one site is blocked with the covalent active site inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. This hemi-labeled enzyme is about 9-fold more active than the apo-dimer of caspase-1. These studies suggest that substrate not only drives dimerization but also, once bound to one site in the dimer, promotes an active conformation in the other monomer. Steady-state kinetic analysis and modeling independently support this model, where binding of one substrate molecule not only increases substrate binding in preformed dimers but also drives the formation of heterodimers. Thus, the cooperativity in caspase-1 is driven both by substrate-induced dimerization as well as substrate-induced activation. Substrate-induced dimerization and activation seen in caspase-1 and not in caspase-3 may reflect their biological roles. Whereas caspase-1 cleaves a dramatically smaller number of cellular substrates that need to be concentrated near inflammasomes, caspase-3 is a constitutively active dimer that cleaves many more substrates located diffusely throughout the cell.
authors: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143.
authors: Debajyoti
authors: D
authors: Datta
authors: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143.
authors: Christopher L
authors: CL
authors: McClendon
authors: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143.
authors: Matthew P
authors: MP
authors: Jacobson
authors: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143. Electronic address: Jim.Wells@ucsf.edu.
authors: James A
authors: JA
authors: Wells
mesh_terms: D000495:Allosteric Site; D000590:Amino Acid Chloromethyl Ketones / Q000737:chemistry; D019540:Area Under Curve; D001703:Biophysics / Q000379:methods; D020170:Caspase 1 / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D020134:Catalytic Domain; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007249:Inflammation; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D014461:Ultracentrifugation
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D004791:Enzyme Inhibitors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D053148:Caspase 3; D020169:Caspases; D020170:Caspase 1
references: J Biol Chem. 1989 Apr 5;264(10):5323-6
references: 2784432
references: Methods Enzymol. 1994;244:615-31
references: 7845238
references: J Mol Biol. 2011 Feb 11;406(1):75-91
references: 21111746
references: Cell Mol Life Sci. 2011 Mar;68(5):765-83
references: 21072676
references: Proc Natl Acad Sci U S A. 2004 Jun 29;101(26):9745-50
references: 15192144
references: J Biol Chem. 2009 Aug 14;284(33):21777-21781
references: 19473994
references: Protein Expr Purif. 2005 May;41(1):148-53
references: 15802232
references: Nature. 1994 Jul 28;370(6487):270-5
references: 8035875
references: Structure. 2004 Aug;12(8):1361-71
references: 15296730
references: Immunity. 2001 Nov;15(5):825-35
references: 11728343
references: J Biol Chem. 1967 Jul 25;242(14):3301-7
references: 6029440
references: Nature. 1992 Apr 30;356(6372):768-74
references: 1574116
references: Nature. 2011 Apr 21;472(7343):366-9
references: 21441910
references: Biophys J. 2000 Mar;78(3):1606-19
references: 10692345
references: Nature. 1999 May 20;399(6733):263-7
references: 10353249
references: Cell. 2008 Sep 5;134(5):866-76
references: 18722006
references: Science. 1992 Apr 3;256(5053):97-100
references: 1373520
references: Methods. 1999 Apr;17(4):313-9
references: 10196102
references: J Immunol. 2006 Apr 1;176(7):3877-83
references: 16547218
references: J Biol Chem. 1997 Apr 11;272(15):9677-82
references: 9092497
references: Mol Cell. 2003 Feb;11(2):543-9
references: 12620240
references: Chembiochem. 2008 Dec 15;9(18):2920-8
references: 19021141
references: Structure. 2009 Aug 12;17(8):1042-50
references: 19679084
references: J Biol Chem. 1997 Jul 18;272(29):17907-11
references: 9218414
references: Nature. 2007 Dec 13;450(7172):983-90
references: 18075577
references: Mol Cell Proteomics. 2010 May;9(5):880-93
references: 20173201
references: J Exp Med. 1997 Mar 3;185(5):933-40
references: 9120399
references: Mol Cell. 2006 Aug;23(4):523-33
references: 16916640
references: Cell. 2001 Nov 2;107(3):399-407
references: 11701129
references: Mol Cell. 2003 Feb;11(2):519-27
references: 12620238
references: J Biol Chem. 1996 Sep 6;271(36):21853-8
references: 8702986
references: Proc Natl Acad Sci U S A. 2009 Mar 3;106(9):3071-6
references: 19208804
references: J Mol Biol. 2008 Sep 19;381(5):1157-67
references: 18590738
references: Science. 2005 Jun 3;308(5727):1424-8
references: 15933191
references: Biochem J. 2009 Oct 12;423(3):429-39
references: 19694615
references: Anal Biochem. 2004 Mar 15;326(2):234-56
references: 15003564
references: Protein Sci. 2005 Jan;14(1):24-36
references: 15576551
references: Proc Natl Acad Sci U S A. 1989 Jul;86(14):5227-31
references: 2787508
references: Proc Natl Acad Sci U S A. 2006 May 16;103(20):7595-600
references: 16682620
references: Chem Biol. 1997 Feb;4(2):149-55
references: 9190289