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.

Fig. 1.

Schematic representation of methodology. An aryl di-O-acetyl-containing inhibitor is incubated with target protein at ≈IC₅₀ concentration. Formation of a specific complex results in covalent modification of the protein at the inhibitor-binding site. Subsequent SDS/PAGE purification ensures unfolding of the protein, which is essential for effective proteolysis. Finally, specifically acetylated peptide fragments and individual residues are identified from MS and MS/MS analysis, respectively.

Fig. 2.

Structures of Acetylated-Inhibitor and Acetylated-Control. Acetylated-Inhibitor impairs both HIV-1 integrase activities (3′ end processing and strand transfer) with very similar IC₅₀ values of ≈3 μM. In contrast, no detectable inactivation of HIV-1 integrase activity was observed with 300 μM Acetylated-Control.

Fig. 3.

MALDI-TOF data on acetylation of peptide 1 (P1). (A) MS data of the original peptide before (Upper) and after (Lower) treatment with Acetylated-Inhibitor. Incubation of P1 with Acetylated-Inhibitor resulted in the formation of new peptide peaks: P1 plus 42, P1 plus 84, and P1 plus 126, corresponding to the addition of one, two, and three acetyl groups, respectively. The original peptide preparation contained small amounts of sodium adduct (P1 plus Na) and an oxidized species (P1 plus 2O), which were also susceptible to acetylation (see Lower). (B) PSD data of the unmodified peptide: P1 (Upper) and the acetylated peak P1 plus 126 (Lower). The “b” and “y” ions peaks are derived from internal fragmentation of peptide bonds and provide amino acid sequence information read from the peptide N terminus to C terminus and from the C terminus to N terminus, respectively. The PSD data confirm the known amino acid sequence of this commercially available peptide. After acetylation (Lower) b1, b2, and b3 ions were shifted by 42, 84, and 126 Da, respectively. These data indicate acetylation of the N terminus, Cys and Lys. The 126-Da gap persisted for b4, b5, and the parent ion, indicating that there are only three acetylation sites in the peptide. The fact that the y2 ion remained unchanged confirms that two C-terminal Trp residues were not acetylated.

Fig. 4.

MALDI-TOF data demonstrating similar chemical reactivities of Acetylated-Inhibitor and Acetylated-Control with respect to model peptide 4 (P4). (A) P4 plus 500 μM Acetylated-Inhibitor. (B) P4 plus 3 μm Acetylated-Inhibitor. (C) P4 plus 500 μM Acetylated-Control. (D) P4 plus 3 μm Acetylated-Control. PSD analysis revealed acetylation of Lys in the P4 plus Ac peak (data not shown). P4 plus Na and P4 plus Na plus Ac are sodium adducts of the original and acetylated peptides, respectively.

Fig. 5.

MS data showing specific acetylation of HIV-1 IN with Acetylated-Inhibitor. (A) IN plus 3 μm Acetylated-Inhibitor. (B) IN plus 3 μm Acetylated-Control. (C) Free IN. MS spectra illustrate that treatment of IN with 3 μM Acetylated-Inhibitor resulted in a single new peak: 167–185 plus Acetyl (A). The molecular mass of this triply charged ion peak [741.03³⁺ = (741.03 - 1.008) × 3 = 2,220.13 Da] corresponds to that of the 167–185 peptide (molecular mass = 2,178.12 Da) plus one acetyl group (molecular mass = 42.03 Da). Importantly, this acetylated peptide peak was not formed in the presence of Acetylated-Control (B). C1, C2, and C3 represent unmodified tryptic peptides of HIV-1 IN.

Fig. 6.

MS/MS data on the acetylated IN peptide (167–185 plus Acetyl). Internal fragmentation of the peptide provided its amino acid sequence information. Sequential fragmentation of peptide bonds from the peptide N and C terminus resulted in “b” and “y” ions, respectively (Upper). ^*, These peaks were generated due to the loss of the NH₃ group from fragments. ++, Doubly charged ions (all other fragments possessed a single charge). The mass increment between “b6” and “b7” ions corresponds to an acetylated lysine (K173 plus Acetyl). Moreover, the masses of ions preceding and following “b” or “y” ions assign perfectly to the IN sequence only when K173 is considered to be acetylated.

Fig. 7.

A model of IN:Acetylated-Inhibitor complex. The spacefilling model shows that the inhibitor (colored red) binds at the protein dimer interface contacting residues K173, T174, and M178 (all colored in blue). Separate protein monomers are colored in yellow and white.