posted on 2012-10-24, 00:00authored byHyunil Jo, Nataline Meinhardt, Yibing Wu, Swapnil Kulkarni, Xiaozhen Hu, Kristin E. Low, Peter
L. Davies, William F. DeGrado, Doron C. Greenbaum
We have designed a highly specific inhibitor of calpain
by mimicking
a natural protein–protein interaction between calpain and its
endogenous inhibitor calpastatin. To enable this goal we established
a new method of stabilizing an α-helix in a small peptide by
screening 24 commercially available cross-linkers for successful cysteine
alkylation in a model peptide sequence. The effects of cross-linking
on the α-helicity of selected peptides were examined by CD and
NMR spectroscopy, and revealed structurally rigid cross-linkers to
be the best at stabilizing α-helices. We applied this strategy
to the design of inhibitors of calpain that are based on calpastatin,
an intrinsically unstable polypeptide that becomes structured upon
binding to the enzyme. A two-turn α-helix that binds proximal
to the active site cleft was stabilized, resulting in a potent and
selective inhibitor for calpain. We further expanded the utility of
this inhibitor by developing irreversible calpain family activity-based
probes (ABPs), which retained the specificity of the stabilized helical
inhibitor. We believe the inhibitor and ABPs will be useful for future
investigation of calpains, while the cross-linking technique will
enable exploration of other protein–protein interactions.