Interaction structure of the complex between neuroprotective factor humanin and Alzheimer's β-amyloid peptide revealed by affinity mass spectrometry and molecular modeling

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 KD of the complex of 610 n m. 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. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd. The interaction epitopes between the neuroprotective peptide humanin and ß‐amyloid(1–40) were identified to reside in the humanin(5–15) and Aß(17–28) sequences by affinity mass spectrometry, using proteolytic excision and extraction. The interaction structure was ascertained and characterized by mass spectrometry, molecular dynamics simulation and affinity quantification. The interaction epitopes and specific association of humanin with ß‐amyloid provide a molecular basis for understanding the neuroprotective function of humanin and for the design of new therapeutic peptides.