CRYSTALLOGRAPHIC STUDIES OF ACTIVE AND INACTIVE RETROVIRAL INTEGRASE

Alexander Wlodawer¹, Jacek Lubkowski¹, Fan Yang¹, Jerry Alexandratos¹, George Merkel², and Anna Marie Skalka²

¹Macromolecular Structure Laboratory, NCI-Frederick Cancer Research and Development Center, ABL-Basic Research Program, Frederick, MD 21702
²Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111

Keywords: Integrase, active site, inactivating mutation, AIDS

Crystallographic studies of the catalytic core domain of avian sarcoma virus integrase (ASV IN) have provided the most detailed picture so far of the active site of this enzyme, which belongs to an important class of targets for designing drugs agains AIDS. Recently, crystals of an inactive D64N mutant, ASVIN64, have been obtained under conditions identical to those used for the native enzyme. Data were collected on the crystals of ASVIN64 at pH 5.0 in citrate buffer, at pH 6.0 in acetate buffer,and under the latter conditions in the presence of Mn²⁺, Zn²⁺, or Mg²⁺ cations. Data were also collected at low pH for the crystals of native ASV IN core domain, and a very high resolution data set (1.42 A) was measured at pH 6.0 using a synchrotron source.

In the structures of native ASV IN at pH 6.0 and below, as well as in all structures of the D64N mutants, the side chain of the active site residue Asx 64 is rotated by ~150° around the CA-CB bond, compared to the structures at higher pH. In the new structures, the residue makes hydrogen bonds with the amide group of Asn 160 and thus the usual metal-binding site consisting of Asp 64 - Asp 121 - Glu 157 is disrupted. Surprisingly, though, a single Zn²⁺ cation can still bind to Asp 121 in the mutant, without restoration of the activity of the enzyme. These structures have elucidated an unexpected mechanism of inactivation of the enzyme by lowering pH or by mutation, in which a protonated side chain of Asx 64 changes its orientation and interaction partner. We also noticed that the loop which covers the active site in the high resolution structure has adopted a new conformation, much better ordered than in any previously determined structures. The new conformation of that loop suggests a mechanism of its mobility, which may be required to maintain the activity of the enzyme.