title: Detecting the active conformation of calpain with calpastatin-based reagents.
abstract: The specific, calcium-dependent, high affinity interaction between calpain and its endogenous inhibitor calpastatin was exploited to selectively detect the calcium-bound, catalytically competent, conformation of calpain in vitro. Modification of calpastatin domain-1 (Val(114)-Ser(270)) or its N-terminal fragment (Val(114)-Pro(202)), at selected unique cysteine residues with maleimide-AlexaFluor546 did not compromise calpastatin function (inhibition of calpain) or its binding with calpain. Ca(2+)-dependent binding between catalytically dead calpain-2 (Cys(105)Ala) fused with eGFP and these fluorigenic calpastatin peptides generates fluorescent resonance energy transfer (FRET). The FRET signal documents proximity of calpain-2, C-terminally linked fluorophore to specific sites within calpastatin when the proteins form a complex. These results provide important insights into the calcium-dependent interaction between calpain and calpastatin and for holo-calpain-2 in solution experimentally validate some key features of their predicted interactions. These data also provide proof of concept that the calpastatin-based reagents may be useful to selectively detect the active conformation of calpain.
authors.affiliation: 5735 Hitchner Hall, Department of Biochemistry, Microbiology and Molecular Biology, University of Maine, Orono, Maine 04469-5375, USA. dorothy.croall@umit.maine.edu
authors.forename: Dorothy E
authors.initials: DE
authors.lastname: Croall
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Vanhooser
authors.forename: Robert E
authors.initials: RE
authors.lastname: Cashon
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; C033668:calpastatin; D002154:Calpain; D002118:Calcium
references.citation: Genome Biol. 2007;8(6):218
references.pmid: 17608959
references.citation: Biochem J. 2000 May 15;348 Pt 1:37-43
references.pmid: 10794711
references.citation: Mol Cell Biol. 2006 Jul;26(14):5481-96
references.pmid: 16809781
references.citation: J Biochem. 2001 May;129(5):653-64
references.pmid: 11328585
references.citation: FEBS Lett. 1986 Nov 24;208(2):199-202
references.pmid: 3780962
references.citation: Nat Rev Mol Cell Biol. 2002 Dec;3(12):906-18
references.pmid: 12461557
references.citation: Methods Mol Biol. 2000;144:47-54
references.pmid: 10818747
references.citation: J Biol Chem. 2002 Mar 15;277(11):9022-6
references.pmid: 11809743
references.citation: J Biol Chem. 2003 Mar 7;278(10):7800-9
references.pmid: 12500971
references.citation: J Cell Sci. 2005 Sep 1;118(Pt 17):3829-38
references.pmid: 16129881
references.citation: Biochemistry. 1994 Nov 15;33(45):13223-30
references.pmid: 7947729
references.citation: Biol Chem. 2004 Jun;385(6):465-72
references.pmid: 15255177
references.citation: Anal Biochem. 1999 Aug 15;273(1):73-80
references.pmid: 10452801
references.citation: Nat Chem Biol. 2005 Sep;1(4):203-9
references.pmid: 16408036
references.citation: Biosens Bioelectron. 2005 Sep 15;21(3):438-44
references.pmid: 16076433
references.citation: Protein Sci. 2003 Oct;12(10):2327-36
references.pmid: 14500891
references.citation: Structure. 2003 Dec;11(12):1521-6
references.pmid: 14656436
references.citation: Proc Natl Acad Sci U S A. 2000 Jan 18;97(2):588-92
references.pmid: 10639123
references.citation: J Biol Chem. 1993 Nov 5;268(31):23593-600
references.pmid: 8226886
references.citation: FEBS Lett. 2008 Jun 25;582(15):2149-54
references.pmid: 18519038
references.citation: Physiol Rev. 2003 Jul;83(3):731-801
references.pmid: 12843408
references.citation: BMC Dev Biol. 2006 Jan 24;6:3
references.pmid: 16433929
references.citation: J Biol Chem. 2003 May 2;278(18):16336-46
references.pmid: 12591934
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: Nat Struct Biol. 1997 Jul;4(7):532-8
references.pmid: 9228945
references.citation: Mol Cell. 2006 Aug;23(4):509-21
references.pmid: 16916639
references.citation: J Biol Chem. 1994 Jul 22;269(29):18977-84
references.pmid: 8034655
references.citation: Mol Cell Biol. 2004 Jan;24(1):84-95
references.pmid: 14673145
references.citation: J Biol Chem. 1994 Sep 30;269(39):24430-6
references.pmid: 7929105
references.citation: Eur J Biochem. 2003 Feb;270(3):404-14
references.pmid: 12542690
references.citation: IUBMB Life. 2000 Jul;50(1):63-8
references.pmid: 11087123
references.citation: Biochemistry. 2005 Mar 15;44(10):3955-64
references.pmid: 15751971
references.citation: Proc Natl Acad Sci U S A. 2004 Jul 6;101(27):9955-9
references.pmid: 15226507
references.citation: Nat Chem Biol. 2005 Jun;1(1):33-8
references.pmid: 16407991
references.citation: EMBO J. 1999 Dec 15;18(24):6880-9
references.pmid: 10601010
references.citation: Physiol Rev. 1991 Jul;71(3):813-47
references.pmid: 2057527
references.citation: Biochemistry. 1988 Mar 22;27(6):1964-72
references.pmid: 2837276
references.citation: J Biol Chem. 2004 Jan 23;279(4):2761-71
references.pmid: 14594950
references.citation: Mol Cell Biol. 2000 Jun;20(12):4474-81
references.pmid: 10825211
references.citation: Exp Cell Res. 2004 Sep 10;299(1):179-87
references.pmid: 15302585
references.citation: J Mol Biol. 2004 Nov 5;343(5):1313-26
references.pmid: 15491615
references.citation: Anal Biochem. 2000 Jun 15;282(1):161-4
references.pmid: 10860517
references.citation: Bioorg Med Chem Lett. 2004 Jun 7;14(11):2725-8
references.pmid: 15125922
references.citation: Proc Natl Acad Sci U S A. 2000 Feb 29;97(5):2253-8
references.pmid: 10688895
references.citation: Anal Biochem. 1987 Nov 1;166(2):368-79
references.pmid: 2449095
references.citation: Methods Mol Biol. 2000;144:33-40
references.pmid: 10818745
references.citation: J Mol Biol. 2003 Apr 18;328(1):131-46
references.pmid: 12684003
references.citation: FEBS Lett. 2007 Jun 26;581(16):2894-8
references.pmid: 17543955
references.citation: Arch Biochem Biophys. 1988 Apr;262(1):189-98
references.pmid: 3355165
references.citation: Biochemistry. 2004 Mar 9;43(9):2596-604
references.pmid: 14992597