Concerted multi-pronged attack by calpastatin to occlude the catalytic cleft of heterodimeric calpains

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Abstract

The Ca2+-dependent cysteine proteases, calpains, regulate cell migration1, cell death2, insulin secretion3, synaptic function4 and muscle homeostasis5. Their endogenous inhibitor, calpastatin, consists of four inhibitory repeats, each of which neutralizes an activated calpain with exquisite specificity and potency6. Despite the physiological importance of this interaction, the structural basis of calpain inhibition by calpastatin is unknown7. Here we report the 3.0 Å structure of Ca2+-bound m-calpain in complex with the first calpastatin repeat, both from rat, revealing the mechanism of exclusive specificity. The structure highlights the complexity of calpain activation by Ca2+, illustrating key residues in a peripheral domain that serve to stabilize the protease core on Ca2+ binding. Fully activated calpain binds ten Ca2+ atoms, resulting in several conformational changes allowing recognition by calpastatin. Calpain inhibition is mediated by the intimate contact with three critical regions of calpastatin. Two regions target the penta-EF-hand domains of calpain and the third occupies the substrate-binding cleft, projecting a loop around the active site thiol to evade proteolysis.

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Figure 1: Complex between Ca 2+ -bound m-calpain and calpastatin.
Figure 2: Binding and inhibition of calpain by region B of calpastatin.
Figure 3: Stabilization of active Ca 2+ -bound calpain by DIII.
Figure 4: Calpain–calpastatin proteolytic system.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The sequences and three-dimensional coordinates for the calcium-dependent complex between m-calpain and calpastatin have been deposited in the PDB database under accession number 3DF0.

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Acknowledgements

We thank P. L. Davies for facilitating the initial stages of this work and for sharing the coordinates for the calpastatin repeat 4–calpain complex, M. Osborne for help with the NMR analysis, B. Schulman for collecting the ALS synchrotron data, the staff at Brookhaven National Light Source beam X29 and APS SERCAT for help with data collection, S. White and D. Miller for advice and sharing of synchrotron time, and C. Hosfield, A. Tocilj and R. Kriwacki for reviewing our manuscript. Funding was provided by the US National Institute of Health and National Cancer Institute, the Canadian Institute of Health Research and the American Lebanese Syrian Associated Charities.

Author Contributions T.M. performed the experimental work and data analysis. T.M., K.G. and D.R.G. were involved with the project planning and wrote the paper.

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Correspondence to Douglas R. Green.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-13 with Legends, Supplementary Tables 1-3 and Supplementary References (PDF 3856 kb)

Supplementary Movie

This Supplementary Movie file shows Calpain activation by Ca2+. The free and Ca2+- bound calpains were overlapped based on DIII and are colored as in Fig. 3. Morphing coordinates were generated using LSQMAN16 to produce the individual frames of the movie. The catalytic triad in DI-II is highlighted in orange. (AVI 6301 kb)

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Moldoveanu, T., Gehring, K. & Green, D. Concerted multi-pronged attack by calpastatin to occlude the catalytic cleft of heterodimeric calpains. Nature 456, 404–408 (2008) doi:10.1038/nature07353

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