THE CATALYTIC DOMAIN OF AVIAN-SARCOMA VIRUS INTEGRASE - CONFORMATION OF THE ACTIVE-SITE RESIDUES IN THE PRESENCE OF DIVALENT-CATIONS

Background: Members of the structurally-related superfamily of enzymes that includes RNase H, RuvC resolvase, MuA transposase, and retrovira l integrase require divalent cations for enzymatic activity. So far, c ation positions are reported in the X-ray crystal structures of only t wo of these proteins, E. coli and human immunodeficiency virus 1 (HIV- 1) RNase H. Details of the placement of metal ions in the active site of retroviral integrases are necessary for the understanding of the ca talytic mechanism of these enzymes. Results: The structure of the enzy matically active catalytic domain (residues 52-207) of avian sarcoma v irus integrase (ASV IN) has been solved in the presence of divalent ca tions (Mn2+ or Mg2+) at 1.7-2.2 Angstrom resolution. A single ion of e ither type interacts with the carboxylate groups of the active site as partates and uses four water molecules to complete its octahedral coor dination. The placement of the aspartate side chains and metal ions is very similar to that observed in the RNase H members of this superfam ily; however, the conformation of the catalytic aspartates in the acti ve site of ASV IN differs significantly from that reported for the ana logous residues in HIV-1 IN. Conclusions: Binding of the required meta l ions does not lead to significant structural modifications in the ac tive site of the catalytic domain of ASV IN. This indicates that at le ast one metal-binding site is preformed in the structure, and suggests that the observed constellation of the acidic residues represents a c atalytically competent active site, Only a single divalent cation was observed even at extremely high concentrations of the metals. We concl ude that either only one metal ion is needed for catalysis, or that a second metal-binding site can only exist in the presence of substrate and/or other domains of the protein. The unexpected differences betwee n the active sites of ASV IN and HIV-1 IN remain unexplained; they may reflect the effects of crystal contacts on the active site of HIV-1 I N, or a tendency for structural polymorphism.