Structural implications of spectroscopic characterization of a putative zinc finger peptide from HIV-1 integrase

J Biol Chem. 1992 May 15;267(14):9639-44.

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.

MeSH terms

  • Amino Acid Sequence
  • Chlorides / pharmacology
  • Chromatography, Gel
  • Cloning, Molecular
  • Cobalt / pharmacology
  • DNA Nucleotidyltransferases / chemistry*
  • DNA Nucleotidyltransferases / genetics
  • DNA Nucleotidyltransferases / metabolism
  • HIV-1 / enzymology*
  • HIV-1 / genetics
  • Hydrogen-Ion Concentration
  • Integrases
  • Kinetics
  • Models, Structural
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Spectrometry, Fluorescence
  • Spectrophotometry
  • Thermodynamics
  • Zinc / pharmacology
  • Zinc Compounds*
  • Zinc Fingers*

Substances

  • Chlorides
  • Recombinant Proteins
  • Zinc Compounds
  • Cobalt
  • zinc chloride
  • DNA Nucleotidyltransferases
  • Integrases
  • cobaltous chloride
  • Zinc