Interaction structure of the complex between neuroprotective factor humanin and Alzheimer's β-amyloid peptide...

Humanin (HN) is a linear 24-aa peptide recently detected in human Alzheimer's disease (AD) brain. HN specifically inhibits neuronal cell death in vitro induced by ß-amyloid (Aß) peptides and by amyloid precursor protein and its gene mutations in familial AD, thereby representing a potential therapeutic lead structure for AD; however, its molecular mechanism of action is not well understood. We report here the identification of the binding epitopes between HN and Aß(1-40) and characterization of the interaction structure through a molecular modeling study. Wild-type HN and HN-sequence mutations were synthesized by SPPS and the HPLC-purified peptides characterized by MALDI-MS. The interaction epitopes between HN and Aß(1-40) were identified by affinity-MS using proteolytic epitope excision and extraction, followed by elution and mass spectrometric characterization of the affinity-bound peptides. The affinity-MS analyses revealed HN(5-15) as the epitope sequence of HN, whereas Aß(17-28) was identified as the Aß interaction epitope. The epitopes and binding sites were ascertained by ELISA of the complex of HN peptides with immobilized Aß(1-40) and by ELISA with Aß(1-40) and Aß-partial sequences as ligands to immobilized HN. The specificity and affinity of the HN-Aß interaction were characterized by direct ESI-MS of the HN-Aß(1-40) complex and by bioaffinity analysis using a surface acoustic wave biosensor, providing a K(D) of the complex of 610 nm. A molecular dynamics simulation of the HN-Aß(1-40) complex was consistent with the binding specificity and shielding effects of the HN and Aß interaction epitopes. These results indicate a specific strong association of HN and Aß(1-40) polypeptide and provide a molecular basis for understanding the neuroprotective function of HN.