Calcium-bound structure of calpain and its mechanism of inhibition by calpastatin

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Abstract

Calpains are non-lysosomal calcium-dependent cysteine proteinases that selectively cleave proteins in response to calcium signals1 and thereby control cellular functions such as cytoskeletal remodelling, cell cycle progression, gene expression and apoptotic cell death2,3,4. In mammals, the two best-characterized members of the calpain family, calpain 1 and calpain 2 (µ-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 calcium5,6. To date, the mechanism of inhibition by calpastatin and the basis for its absolute specificity have remained speculative7,8,9. 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-Å-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 complex10, 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.

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Figure 1: Overview of calpastatin domain 4 (CAST4) bound to calpain 2.
Figure 2: Specific interactions of calpastatin with calpain entering and leaving the active-site cleft.
Figure 3: Close-up views of calpastatin at the active site.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Structure factors and coordinates have been deposited in the RCSB Protein Data Bank with the accession number 3BOW.

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Acknowledgements

This work was funded by the Canadian Institutes for Health Research. R.A.H. is the recipient of a Bauman Fellowship and an R.J. Wilson Fellowship. P.L.D. holds a Canada Research Chair in Protein Engineering. We are grateful to S. Gauthier for technical assistance, to M. Kuiper for the simulation of the conformational change in calpain on binding Ca2+, to the Queen’s University Protein Function Discovery (PFD) Facility and D. McLeod for mass spectrometry, Z. Jia for access to data collection on a home source and to M. Allaire at Brookhaven National Laboratory for assistance with data collection on beam line X6A.

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Correspondence to Peter L. Davies.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-9 with Legends and Supplementary Tables 1-5 (PDF 2862 kb)

Supplementary Movie 1

Supplementary Movie 1 shows a simulation of the change from the calcium-free structure to the calcium-bound structure of calpain. Ten calcium ions are shown docking to the enzyme, the anchor helix is released and the domains shift relative to each other to produce the more compact, active enzyme. (MOV 10216 kb)

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Hanna, R., Campbell, R. & Davies, P. Calcium-bound structure of calpain and its mechanism of inhibition by calpastatin. Nature 456, 409–412 (2008) doi:10.1038/nature07451

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