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

Nucleic Acids Res. 2010 Jun;38(11):3692-708. doi: 10.1093/nar/gkq087. Epub 2010 Feb 17.

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 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.

Publication types

  • Historical Article
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Base Sequence
  • Benzamides / pharmacology
  • Catalytic Domain
  • DNA, Viral / chemistry*
  • DNA, Viral / metabolism
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / drug effects
  • DNA-Binding Proteins / metabolism
  • HIV Integrase / chemistry*
  • HIV Integrase / drug effects
  • HIV Integrase / metabolism
  • History, Medieval
  • Magnesium / chemistry
  • Mass Spectrometry
  • Protein Binding
  • Protein Structure, Tertiary
  • Zinc / chemistry*
  • Zinc Fingers

Substances

  • Benzamides
  • DIBA-1
  • DNA, Viral
  • DNA-Binding Proteins
  • HIV Integrase
  • Magnesium
  • Zinc
  • p31 integrase protein, Human immunodeficiency virus 1