A cooperative and specific DNA-binding mode of HIV-1 integrase depends on the nature of the metallic cofactor and involves the zinc-containing N-terminal domain.
HIV-1 integrase catalyzes the insertion of the viral genome into chromosomal DNA. We characterized the structural determinants of the 3'-processing reaction specificity--the first reaction of the integration process--at the DNA-binding level. We found that the integrase N-terminal domain, containing a pseudo zinc-finger motif, plays a key role, at least indirectly, in the formation of specific integrase-DNA contacts. This motif mediates a cooperative DNA binding of integrase that occurs only with the cognate/viral DNA sequence and the physiologically relevant Mg(2+) cofactor. The DNA-binding was essentially non-cooperative with Mn(2+) or using non-specific/random sequences, regardless of the metallic cofactor. 2,2'-Dithiobisbenzamide-1 induced zinc ejection from integrase by covalently targeting the zinc-finger motif, and significantly decreased the Hill coefficient of the Mg(2+)-mediated integrase-DNA interaction, without affecting the overall affinity. Concomitantly, 2,2'-dithiobisbenzamide-1 severely impaired 3'-processing (IC(50) = 11-15 nM), suggesting that zinc ejection primarily perturbs the nature of the active integrase oligomer. A less specific and weaker catalytic effect of 2,2'-dithiobisbenzamide-1 is mediated by Cys 56 in the catalytic core and, notably, accounts for the weaker inhibition of the non-cooperative Mn(2+)-dependent 3'-processing. Our data show that the cooperative DNA-binding mode is strongly related to the sequence-specific DNA-binding, and depends on the simultaneous presence of the Mg(2+) cofactor and the zinc effector.
Carayon
Kevin
K
LBPA, CNRS, Ecole Normale Supérieure de Cachan, 61 av. du Président Wilson, 94235 Cachan, France.
Leh
Hervé
H
Henry
Etienne
E
Simon
Françoise
F
Mouscadet
Jean-François
JF
Deprez
Eric
E
D020816:Amino Acid Motifs; D001483:Base Sequence; D001549:Benzamides / Q000494:pharmacology; D020134:Catalytic Domain; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D019427:HIV Integrase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D049691:History, Medieval; D008274:Magnesium / Q000737:chemistry; D013058:Mass Spectrometry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015032:Zinc / Q000737:chemistry*; D016335:Zinc Fingers
D001549:Benzamides; C100805:DIBA-1; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D019427:HIV Integrase; D008274:Magnesium; D015032:Zinc; C514801:p31 integrase protein, Human immunodeficiency virus 1