Crystal Structures of Calpain–E64 and –Leupeptin Inhibitor Complexes Reveal Mobile Loops Gating the Active Site

• Published in: Journal of molecular biology 2004-11, Vol.343 (5), p.1313-1326
• Main Authors: Moldoveanu, T., Campbell, R.L., Cuerrier, D., Davies, P.L.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; calcium; calpain; Calpain - antagonists & inhibitors; Calpain - chemistry; Catalytic Domain; Cathepsin K; Cathepsins - chemistry; Cathepsins - metabolism; Crystallization; Crystallography, X-Ray; cysteine protease inhibitors; Cysteine Proteinase Inhibitors - chemistry; Cysteine Proteinase Inhibitors - metabolism; Glycoproteins - chemistry; Glycoproteins - metabolism; Leucine - analogs & derivatives; Leucine - chemistry; Leucine - metabolism; Leupeptins - chemistry; Leupeptins - metabolism; Molecular Sequence Data; Papain - chemistry; Papain - metabolism; Protein Structure, Tertiary; Rats; X-ray crystallography
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2004.09.016

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 μ-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.