Zn2+ promotes the self-association of human immunodeficiency virus type-1 integrase in vitro.
It has been recently demonstrated that the Mg(2+)-dependent 3'-processing activity of purified human immunodeficiency virus type-1 (HIV-1) integrase is stimulated by the addition of exogenous Zn2+ [Lee, S. P., & Han, M. K. (1996) Biochemistry 35, 3837-3844]. This activation was hypothesized to result from integrase self-association. In this report, we examine the Zn2+ content of purified HIV-1 integrase by atomic absorption spectroscopy and by application of a thiol modification reagent, p-(hydroxymercuri)benzenesulfonate, with a metallochromic indicator, 4-(2-pyridylazo)resorcinol. We find that the Zn2+ content of HIV-1 integrase varies from 0.1 to 0.92 equiv of Zn2+ per monomer depending on the conditions of protein purification. In vitro activity assays, time-resolved fluorescence emission anisotropy, and gel filtration chromatographic analyses all indicate that EDTA yields an apoprotein which is predominantly monomeric and less active with Mg2+. Further, sedimentation equilibrium studies reveal that reconstitution of the apoprotein with Zn2+ results in a monomer-tetramer-octamer transition. These results suggest that Zn2+ promotes a conformation with enhanced oligomerization and thereby stimulates Mg(2+)-dependent 3'-processing. This may also imply that multimers larger than dimers (tetramers and possibly octamers) are required for in vitro activity of integrase in the presence of Zn2+ and Mg2+. It should be noted, however, that the content of Zn2+ did not significantly affect the 3'-processing and strand transfer reactions with Mn2+ in vitro.
Lee
S P
SP
Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, DC 20007, USA.
Xiao
J
J
Knutson
J R
JR
Lewis
M S
MS
Han
M K
MK
D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D019426:Integrases / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D010663:Phenylmercury Compounds / Q000378:metabolism; D012118:Resorcinols / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics; D013054:Spectrophotometry, Atomic; D013439:Sulfhydryl Reagents / Q000378:metabolism; D014461:Ultracentrifugation; D015032:Zinc / Q000032:analysis / Q000494:pharmacology*
D005456:Fluorescent Dyes; D010663:Phenylmercury Compounds; D012118:Resorcinols; D013439:Sulfhydryl Reagents; C002910:4-hydroxymercuribenzenesulfonate; D019426:Integrases; D008274:Magnesium; D015032:Zinc; C028398:4-(2-pyridylazo)resorcinol