PDBsum entry 3bow

Hydrolase/hydrolase inhibitor

PDB id: 3bow

Contents

Protein chains

680 a.a.

174 a.a.

65 a.a.

Metals

_CA ×10

Waters ×176

References listed in PDB file

Key reference

Title

Calcium-Bound structure of calpain and its mechanism of inhibition by calpastatin.

Authors

R.A.Hanna, R.L.Campbell, P.L.Davies.

Ref.

Nature, 2008, 456, 409-412. [DOI no: 10.1038/nature07451]

PubMed id

19020623

Abstract

Calpains are non-lysosomal calcium-dependent cysteine proteinases that selectively cleave proteins in response to calcium signals and thereby control cellular functions such as cytoskeletal remodelling, cell cycle progression, gene expression and apoptotic cell death. In mammals, the two best-characterized members of the calpain family, calpain 1 and calpain 2 (micro-calpain and m-calpain, respectively), are ubiquitously expressed. The activity of calpains is tightly controlled by the endogenous inhibitor calpastatin, which is an intrinsically unstructured protein capable of reversibly binding and inhibiting four molecules of calpain, but only in the presence of calcium. To date, the mechanism of inhibition by calpastatin and the basis for its absolute specificity have remained speculative. It was not clear how this unstructured protein inhibits calpains without being cleaved itself, nor was it known how calcium induced changes that facilitated the binding of calpastatin to calpain. Here we report the 2.4-A-resolution crystal structure of the calcium-bound calpain 2 heterodimer bound by one of the four inhibitory domains of calpastatin. Calpastatin is seen to inhibit calpain by occupying both sides of the active site cleft. Although the inhibitor passes through the active site cleft it escapes cleavage in a novel manner by looping out and around the active site cysteine. The inhibitory domain of calpastatin recognizes multiple lower affinity sites present only in the calcium-bound form of the enzyme, resulting in an interaction that is tight, specific and calcium dependent. This crystal structure, and that of a related complex, also reveal the conformational changes that calpain undergoes on binding calcium, which include opening of the active site cleft and movement of the domains relative to each other to produce a more compact enzyme.

Figure 1.
Figure 1: Overview of calpastatin domain 4 (CAST4) bound to calpain 2. The overall structure of CAST4 (purple) bound to the inactive C105S mutant of calpain 2. CAST4, which is unstructured in the absence of calpain, forms three -helices when in complex with the enzyme. Helices in subdomains A and C, which are in contact with DIV (yellow) and DVI (orange), and the helix in subdomain B, which is in contact with the protease core DI and DII (blue and light blue, respectively) are shown in ribbon representation. DIII is coloured green. Gaps in the electron density of CAST4 are indicated by missing residues between D589 and K594, and between N629 and P652.

Figure 2.
Figure 2: Specific interactions of calpastatin with calpain entering and leaving the active-site cleft. a–d, The 27-residue B-peptide^7 is coloured as follows: the residues that make the loop out of the active site are coloured yellow, the residues N-terminal to the loop are purple, and the residues C-terminal to the loop are green. Other calpastatin residues are coloured dark grey. Hydrogen-bond interactions of calpastatin with calpain (coloured as in Fig. 1) are shown by black dashed lines. O and N atoms are coloured red and blue, respectively. a, Overview of calpain binding at the active site of calpain. b, Close-up view of the calpastatin at the unprimed side of the active site. c, Close-up view of calpastatin looping away from the catalytic residue. d, Close-up view of calpastatin at the primed side of the active site.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2008, 456, 409-412) copyright 2008.