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 M) 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.H IV-1 encodes three enzymes that are essential for virus replication: reverse transcriptase, protease, and integrase (IN). To date, only the first two proteins have been exploited as therapeutic targets. The continuing emergence of new HIV-1 variants resistant to current treatments, together with cytotoxicity problems, makes the search for new anti-HIV-1 drugs imperative. HIV-1 IN is an attractive antiviral drug target that has no known human counterparts. Furthermore, the use of a common active site for 3Ј end processing and DNA strand transfer constrain the range of mutations that can contribute to drug resistance without compromising catalytic activity.HIV-1 IN catalyzes integration of the viral DNA, made by reverse transcription, into the host chromosome in a two-step reaction (reviewed in ref. 1). In the first step, called 3Ј processing, two nucleotides are removed at each 3Ј end of the viral DNA. In the next step, called DNA strand transfer, concerted transesterification reactions integrate the viral DNA ends into the host genome. In vivo, the enzyme acts in the context of a large nucleoprotein complex termed the ''preintegration complex'' (PIC). A number of viral proteins and host factors assemble within the PIC to orchestrate the integration process (2-10).HIV-1 IN is composed of three distinct structural and functional domains: the N-terminal domain (residues 1-50), which contains the HHCC zinc-binding motif; the core domain (residues 51-212), which contains the catalytic site; and the Cterminal domain (residues 213-270), which is thought to provide a platform for DNA binding. Crystallographic or NMR structural data are available for each of the individual domains (11-15). In addition,...