ABSTRACT
The structure of the C-terminal DNA-binding domain of human immunovirus-1 integrase has been refined using nuclear magnetic resonance spectroscopy. The protein is a dimer in solution and shows a well-defined dimer interface. The folding topology of the monomer consists of a five-stranded beta-barrel that resembles that of Src homology 3 domains. Compared with our previously reported structure, the structure is now defined far better. The final 42 structures display a back-bone root mean square deviation versus the average of 0.46 A. Correlation of the structure with recent mutagenesis studies suggests two possible models for DNA binding. Proteins 1999;36:556-564.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , HIV Integrase/chemistry , HIV-1/enzymology , Nuclear Magnetic Resonance, Biomolecular , Binding Sites , DNA-Binding Proteins/genetics , Dimerization , HIV Integrase/genetics , HIV Integrase/metabolism , Models, Molecular , Mutation , Protein Structure, Secondary , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sensitivity and Specificity , Solutions , src Homology DomainsABSTRACT
Integration of human immunodeficiency virus (HIV) DNA into the human genome requires the virus-encoded integrase (IN) protein, and therefore the IN protein is a suitable target for antiviral strategies. To find a potent HIV IN inhibitor, we screened a "synthetic peptide combinatorial library." We identified a hexapeptide with the sequence HCKFWW that inhibits IN-mediated 3'-processing and integration with an IC50 of 2 microM. The peptide is active on IN proteins from other retroviruses such as HIV-2, feline immunodeficiency virus, and Moloney murine leukemia virus, supporting the notion that a conserved region of IN is targeted. The hexapeptide was also tested in the disintegration reaction. This phosphoryl-transfer reaction can be carried out by the catalytic core of IN alone, and the peptide HCKFWW was found to inhibit this reaction, suggesting that the hexapeptide acts at or near the catalytic site of IN. Identification of an IN hexapeptide inhibitor provides proof of concept for the approach, and, moreover, this peptide may be useful for structure-function analysis of IN.
Subject(s)
DNA Nucleotidyltransferases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , HIV-1/enzymology , Oligopeptides/pharmacology , Virus Integration/drug effects , Amino Acid Sequence , Drug Evaluation, Preclinical , HIV-1/genetics , HIV-2/drug effects , HIV-2/enzymology , HIV-2/genetics , Immunodeficiency Virus, Feline/drug effects , Immunodeficiency Virus, Feline/genetics , Integrases , Molecular Sequence Data , Moloney murine leukemia virus/drug effects , Moloney murine leukemia virus/enzymology , Moloney murine leukemia virus/genetics , Oligopeptides/chemical synthesis , Retroviridae/drug effects , Retroviridae/enzymology , Retroviridae/genetics , Structure-Activity RelationshipABSTRACT
On the basis of molecular dynamics and free-energy perturbation approaches, the Glu46Gln (E46Q) mutation in the guanine-specific ribonuclease T1 (RNase T1) was predicted to render the enzyme specific for adenine. The E46Q mutant was genetically engineered and characterized biochemically and crystallographically by investigating the structures of its two complexes with 2'AMP and 2'GMP. The ribonuclease E46Q mutant is nearly inactive towards dinucleoside phosphate substrates but shows 17% residual activity towards RNA. It binds 2'AMP and 2'GMP equally well with dissociation constants of 49 microM and 37 microM, in contrast to the wild-type enzyme, which strongly discriminates between these two nucleotides, yielding dissociation constants of 36 microM and 0.6 microM. These data suggest that the E46Q mutant binds the nucleotides not to the specific recognition site but to the subsite at His92. This was confirmed by the crystal structures, which also showed that the Gln46 amide is hydrogen bonded to the Phe100 N and O atoms, and tightly anchored in this position. This interaction may either have locked the guanine recognition site so that 2'AMP and 2'GMP are unable to insert, or the contribution to guanine recognition of Glu46 is so important that the E46Q mutant is unable to function in recognition of either guanine and adenine.
