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

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