title: Identification of an inhibitor-binding site to HIV-1 integrase with affinity acetylation and mass spectrometry.
abstract: We report a methodology that combines affinity acetylation with MS analysis for accurate mapping of an inhibitor-binding site to a target protein. For this purpose, we used a known HIV-1 integrase inhibitor containing aryl di-O-acetyl groups (Acetylated-Inhibitor). In addition, we designed a control compound (Acetylated-Control) that also contained an aryl di-O-acetyl group but did not inhibit HIV-1 integrase. Examination of the reactivity of these compounds with a model peptide library, which collectively contained all 20 natural amino acids, revealed that aryl di-O-acetyl compounds effectively acetylate Cys, Lys, and Tyr residues. Acetylated-Inhibitor and Acetylated-Control exhibited comparable chemical reactivity with respect to these small peptides. However, these two compounds differed markedly in their interactions with HIV-1 integrase. In particular, Acetylated-Inhibitor specifically acetylated K173 at its inhibitory concentration (3 microM) whereas this site remained unrecognized by Acetylated-Control. Our data enabled creation of a detailed model for the integrase:Acetylated-Inhibitor complex, which indicated that the inhibitor selectively binds at an architecturally critical region of the protein. The methodology reported herein has a generic application for systems involving a variety of ligand-protein interactions.
authors.affiliation: Ohio State University Health Sciences Center, College of Pharmacy, Center for Retrovirus Research and Comprehensive Cancer Center, Columbus, OH 43210, USA.
authors.forename: Nick
authors.initials: N
authors.lastname: Shkriabai
authors.forename: Sachindra S
authors.initials: SS
authors.lastname: Patil
authors.forename: Sonja
authors.initials: S
authors.lastname: Hess
authors.forename: Scott R
authors.initials: SR
authors.lastname: Budihas
authors.forename: Robert
authors.initials: R
authors.lastname: Craigie
authors.forename: Terrence R
authors.initials: TR
authors.lastname: Burke
authors.forename: Stuart F J
authors.initials: SF
authors.lastname: Le Grice
authors.forename: Mamuka
authors.initials: M
authors.lastname: Kvaratskhelia
mesh_terms: D000107:Acetylation; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000378:metabolism*; D019427:HIV Integrase / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry / Q000201:enzymology*; D010455:Peptides / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D004791:Enzyme Inhibitors; D010455:Peptides; D019427:HIV Integrase