Binding-induced folding transitions in calpastatin subdomains A and C.
Calpastatin, the endogenous inhibitor of calpain, is an intrinsically unstructured protein proposed to undergo folding transitions upon binding to the enzyme. As this feature has never been experimentally tested, we have set out to characterize the conformation of two peptides corresponding to its conserved subdomains, A and C, known to interact with calpain in a Ca(2+)-dependent manner. The peptides are disordered in water but show a high propensity for alpha-helical conformation in the presence of trifluoroethanol. The conformational transition is sensitive to Ca(2+), and is clearly seen upon binding of the peptides to the enzyme. Secondary-structure prediction of all calpastatin sequences shows that the helix-forming potential within these regions is a conserved feature of the inhibitor. Furthermore, quantitative data on the binding strength of calpastatin fragments reveal that binding of the inhibitor is accompanied by a large decrease in its configurational entropy. Taken together, these observations point to significant binding-induced local folding transitions in calpastatin, in a way that ensures highly specific, yet reversible, action of the inhibitor.
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002154:Calpain / Q000737:chemistry; D002417:Cattle; D002942:Circular Dichroism; D019295:Computational Biology; D030562:Databases, Protein; D019277:Entropy; D006801:Humans; D051379:Mice; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D014270:Trifluoroethanol / Q000737:chemistry; D014867:Water / Q000737:chemistry
D002135:Calcium-Binding Proteins; D010455:Peptides; D011994:Recombinant Proteins; D014867:Water; D014270:Trifluoroethanol; C033668:calpastatin; D002154:Calpain; D002118:Calcium