Binding Epitopes and Interaction Structure of the Neuroprotective Protease Inhibitor Cystatin C with β-Amyloid Revealed by Proteolytic Excision Mass Spectrometry and Molecular Docking Simulation

Human cystatin C (HCC) is a protease inhibitor with a propensity to form beta-amyloid (A beta)-like fibrils and to coassociate with amyloidogenic proteins. Recently, a specific interaction between HCC and A beta has been found. Here, we report the identification of the A beta and HCC binding epitopes in the A beta-HCC complex, using a combination of selective proteolytic excision and high resolution mass spectrometry. Proteolytic excision of A beta(1-40) on sepharose-immobilized HCC and MALDI-MS identified the epitope A beta(17-28). On immobilized A beta(1-40), affinity MS of HCC fragments identified a specific C-terminal epitope, HCC(101-117). Binding specificities of both epitopes were ascertained by ELISA and surface plasmon resonance and by direct electrospray MS of the HCC-A beta epitope peptide complexes. A structure model of the HCC-A beta complex by molecular docking simulation showed full agreement with the identified A and HCC epitopes. Inhibition studies in vitro revealed A beta-fibril inhibiting activity of the HCC(101-117)-epitope. The A beta-HCC interacting epitopes provide lead structures of neuroprotective inhibitors for AD and HCC amyloidosis therapy.