title: Mass spectrometric analysis of the HIV-1 integrase-pyridoxal 5'-phosphate complex reveals a new binding site for a nucleotide inhibitor.
abstract: HIV-1 integrase (IN) is an important target for designing new antiviral therapies. Screening of potential inhibitors using recombinant IN-based assays has revealed a number of promising leads including nucleotide analogs such as pyridoxal 5'-phosphate (PLP). Certain PLP derivatives were shown to also exhibit antiviral activities in cell-based assays. To identify an inhibitory binding site of PLP to IN, we used the intrinsic chemical property of this compound to form a Schiff base with a primary amine in the protein at the nucleotide binding site. The amino acid affected was then revealed by mass spectrometric analysis of the proteolytic peptide fragments of IN. We found that an IC(50) concentration (15 mum) of PLP modified a single IN residue, Lys(244), located in the C-terminal domain. In fact, we observed a correlation between interaction of PLP with Lys(244) and the compound's ability to impair formation of the IN.DNA complex. Site-directed mutagenesis studies confirmed an essential role of Lys(244) for catalytic activities of recombinant IN and viral replication. Molecular modeling revealed that Lys(244) together with several other DNA binding residues provides a plausible pocket for a nucleotide inhibitor-binding site. To our knowledge, this is the first report indicating that a small molecule inhibitor can impair IN activity through its binding to the protein C terminus. At the same time, our findings highlight the importance of structural analysis of the full-length protein.
authors.affiliation: Ohio State University Health Sciences Center, College of Pharmacy, Center for Retrovirus Research and Comprehensive Cancer Center, Columbus, Ohio 43210, USA.
authors.forename: Kerry L
authors.initials: KL
authors.lastname: Williams
authors.forename: Yijun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Nick
authors.initials: N
authors.lastname: Shkriabai
authors.forename: Rajeshri G
authors.initials: RG
authors.lastname: Karki
authors.forename: Marc C
authors.initials: MC
authors.lastname: Nicklaus
authors.forename: Nana
authors.initials: N
authors.lastname: Kotrikadze
authors.forename: Sonja
authors.initials: S
authors.lastname: Hess
authors.forename: Stuart F J
authors.initials: SF
authors.lastname: Le Grice
authors.forename: Robert
authors.initials: R
authors.lastname: Craigie
authors.forename: Vinay K
authors.initials: VK
authors.lastname: Pathak
authors.forename: Mamuka
authors.initials: M
authors.lastname: Kvaratskhelia
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D019427:HIV Integrase / Q000737:chemistry*; D006801:Humans; D020128:Inhibitory Concentration 50; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011732:Pyridoxal Phosphate / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D003432:Cross-Linking Reagents; D004279:DNA, Viral; D010455:Peptides; D011994:Recombinant Proteins; D011732:Pyridoxal Phosphate; D004247:DNA; D019427:HIV Integrase; D008239:Lysine