Structural implications of spectroscopic characterization of a putative zinc finger peptide from HIV-1 integrase.
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.
Burke
C J
CJ
Department of Pharmaceutical Research, Merck Research Laboratories, West Point, Pennsylvania 19486.
Sanyal
G
G
Bruner
M W
MW
Ryan
J A
JA
LaFemina
R L
RL
Robbins
H L
HL
Zeft
A S
AS
Middaugh
C R
CR
Cordingley
M G
MG
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
D002712:Chlorides; D011994:Recombinant Proteins; D017967:Zinc Compounds; D003035:Cobalt; C016837:zinc chloride; D004254:DNA Nucleotidyltransferases; D019426:Integrases; C018021:cobaltous chloride; D015032:Zinc