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Volume 272, Number 29, Issue of July 18, 1997 pp. 18161-18168
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Binding of Different Divalent Cations to the Active Site of Avian Sarcoma Virus Integrase and Their Effects on Enzymatic Activity

(Received for publication, March 4, 1997, and in revised form, April 22, 1997)

Grzegorz Bujacz ^§ , Jerry Alexandratos and Alexander Wlodawer

From the  Macromolecular Structure Laboratory, NCI-Frederick Cancer Research and Development Center, ABL-Basic Research Program, Frederick, Maryland 21702, and the ^§ Faculty of Food Chemistry and Biotechnology, Technical University of ód, ód, Poland

George Merkel , Mark Andrake , Richard A. Katz and Anna Marie Skalka

From the Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

Retroviral integrases (INs) contain two known metal binding domains. The N-terminal domain includes a zinc finger motif and has been shown to bind Zn²⁺, whereas the central catalytic core domain includes a triad of acidic amino acids that bind Mn²⁺ or Mg²⁺, the metal cofactors required for enzymatic activity. The integration reaction occurs in two distinct steps; the first is a specific endonucleolytic cleavage step called "processing," and the second is a polynucleotide transfer or "joining" step. Our previous results showed that the metal preference for in vitro activity of avian sarcoma virus IN is Mn²⁺ > Mg²⁺ and that a single cation of either metal is coordinated by two of the three critical active site residues (Asp-64 and Asp-121) in crystals of the isolated catalytic domain. Here, we report that Ca²⁺, Zn²⁺, and Cd²⁺ can also bind in the active site of the catalytic domain. Furthermore, two zinc and cadmium cations are bound at the active site, with all three residues of the active site triad (Asp-64, Asp-121, and Glu-157) contributing to their coordination. These results are consistent with a two-metal mechanism for catalysis by retroviral integrases. We also show that Zn²⁺ can serve as a cofactor for the endonucleolytic reactions catalyzed by either the full-length protein, a derivative lacking the N-terminal domain, or the isolated catalytic domain of avian sarcoma virus IN. However, polynucleotidyl transferase activities are severely impaired or undetectable in the presence of Zn²⁺. Thus, although the processing and joining steps of integrase employ a similar mechanism and the same active site triad, they can be clearly distinguished by their metal preferences.

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