title: Crystal structures of calpain-E64 and -leupeptin inhibitor complexes reveal mobile loops gating the active site.
abstract: The endogenous calpain inhibitor, calpastatin, modulates some patho-physiological aspects of calpain signaling. Excess calpain can escape this inhibition and as well, many calpain isoforms and autolytically generated protease core fragments are not inhibited by calpastatin. There is a need, therefore, to develop specific, cell-permeable calpain inhibitors to block uncontrolled proteolysis and prevent tissue damage during brain and heart ischemia, spinal-cord injury and Alzheimer's diseases. Here, we report the first high-resolution crystal structures of rat mu-calpain protease core complexed with two traditional, low molecular mass inhibitors, leupeptin and E64. These structures show that access to a slightly deeper, but otherwise papain-like active site is gated by two flexible loops. These loops are divergent among the calpain isoforms giving a potential structural basis for substrate/inhibitor selectivity over other papain-like cysteine proteases and between members of the calpain family.
authors.affiliation: Department of Biochemistry, Queen's University, Kingston, Ont. K7L 3N6, Canada.
authors.forename: T
authors.initials: T
authors.lastname: Moldoveanu
authors.forename: R L
authors.initials: RL
authors.lastname: Campbell
authors.forename: D
authors.initials: D
authors.lastname: Cuerrier
authors.forename: P L
authors.initials: PL
authors.lastname: Davies
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors / Q000737:chemistry*; D020134:Catalytic Domain; D056657:Cathepsin K; D002403:Cathepsins / Q000737:chemistry / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D007930:Leucine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D007976:Leupeptins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010206:Papain / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: D015853:Cysteine Proteinase Inhibitors; D006023:Glycoproteins; D007976:Leupeptins; C039304:calpain inhibitors; C032425:N-(N-(3-carboxyoxirane-2-carbonyl)leucyl)isoamylamine; D002403:Cathepsins; D002154:Calpain; D010206:Papain; D056657:Cathepsin K; C534133:Ctsk protein, rat; D007930:Leucine; C032854:leupeptin