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

Abstract

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 Mg2+ cofactor. The DNA-binding was essentially non-cooperative with Mn2+ 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 Mg2+-mediated integrase-DNA interaction, without affecting the overall affinity. Concomitantly, 2, 2′-dithiobisbenzamide-1 severely impaired 3′-processing (IC50 = 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 Mn2+-dependent 3′-processing. Our data show that the cooperative DNA-binding mode is strongly related to the sequencespecific DNA-binding, and depends on the simultaneous presence of the Mg2+ cofactor and the zinc effector. © The Author(s) 2010. Published by Oxford University Press.