title: Structural implications of spectroscopic characterization of a putative zinc finger peptide from HIV-1 integrase.
abstract: The N-terminal domain of human immunodeficiency virus (HIV-1) integrase (IN) contains the sequence motif His-Xaa3-His-Xaa23-Cys-Xaa2-Cys, which is strongly conserved in all retroviral and retrotransposon IN proteins. This structural motif constitutes a putative zinc finger in which a metal ion may be coordinately bound by the His and Cys residues. A recombinant peptide, IN(1-55), composed of the N-terminal 55 amino acids of HIV-1 IN was expressed in Escherichia coli and purified. Utilizing a combination of techniques including UV-visible absorption, circular dichroism, Fourier transform infrared, and fluorescence spectroscopies, we have demonstrated that metal ions (Zn2+, Co2+, and Cd2+) are bound with equimolar stoichiometry by IN(1-55). The liganded peptide assumes a highly ordered structure with increased alpha-helical content and exhibits remarkable thermal stability. UV-visible difference spectra of the peptide-Co2+ complexes directly implicate thiols in metal coordination, and Co2+ d-d transitions in the visible range indicate that Co2+ is tetrahedrally coordinated. Mutant peptides containing conservative substitutions of one of the conserved His or either of the Cys residues displayed no significant Zn(2+)-induced conformational changes as monitored by CD and fluorescence spectra. We conclude that the N terminus of HIV-1 IN contains a metal-binding domain whose structure is stabilized by tetrahedral coordination of metal by histidines 12 and 16 and cysteines 40 and 43. A preliminary structural model for this zinc finger is presented.
authors.affiliation: Department of Pharmaceutical Research, Merck Research Laboratories, West Point, Pennsylvania 19486.
authors.forename: C J
authors.initials: CJ
authors.lastname: Burke
authors.forename: G
authors.initials: G
authors.lastname: Sanyal
authors.forename: M W
authors.initials: MW
authors.lastname: Bruner
authors.forename: J A
authors.initials: JA
authors.lastname: Ryan
authors.forename: R L
authors.initials: RL
authors.lastname: LaFemina
authors.forename: H L
authors.initials: HL
authors.lastname: Robbins
authors.forename: A S
authors.initials: AS
authors.lastname: Zeft
authors.forename: C R
authors.initials: CR
authors.lastname: Middaugh
authors.forename: M G
authors.initials: MG
authors.lastname: Cordingley
mesh_terms: D000595:Amino Acid Sequence; D002712:Chlorides / Q000494:pharmacology; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003035:Cobalt / Q000494:pharmacology; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D019426:Integrases; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013816:Thermodynamics; D015032:Zinc / Q000494:pharmacology; D017967:Zinc Compounds; D016335:Zinc Fingers
chemical_list: D002712:Chlorides; D011994:Recombinant Proteins; D017967:Zinc Compounds; D003035:Cobalt; C016837:zinc chloride; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C018021:cobaltous chloride; D015032:Zinc