Substitute for another bond. Docking simulations of two potent inhibitors that bear the 1,2,3-triazole moiety produced two conformations of approximately equal energy. Further analysis of the protease by X-ray crystallography solved the ambiguity of the binding mode and revealed that the triazole ring is an effective amide surrogate and retains all the hydrogen bonds in the active site (see figure).

The HIV-1 matrix protein p17, excised proteolytically from the N terminus of the Gag polyprotein, forms a protective shell attached to the inner surface of the plasma membrane of the virus. During the late stages of the HIV-1 replication cycle, the N-terminally myristoylated p17 domain targets the Gag polyprotein to the host-cell membrane for particle assembly. In the early stages of HIV-1 replication, however, some p17 molecules dissociate from the viral membrane to direct the preintegration complex to the host-cell nucleus. These two opposing targeting functions of p17 require that the protein be capable of reversible membrane interaction. It is postulated that a significant structural change in p17 triggered by proteolytic cleavage of the Gag polyprotein sequesters the N-terminal myristoyl group, resulting in a weaker membrane binding by the matrix protein than the Gag precursor. To test this “myristoyl switch” hypothesis, we obtained highly purified synthetic HIV-1 p17 of 131 amino acid residues and its N-myristoylated form in large quantity. Both forms of p17 were characterized by circular dichroism spectroscopy, protein chemical denaturation, and analytical centrifugal sedimentation. Our results indicate that although N-myristoylation causes no spectroscopically discernible conformational change in p17, it stabilizes the protein by 1 kcal/mol and promotes protein trimerization in solution. These findings support the premise that the myristoyl switch in p17 is triggered not by a structural change associated with proteolysis, but rather by the destabilization of oligomeric structures of membrane-bound p17 in the absence of downstream Gag subdomains.

Six crystal structures of the core domain of integrase (IN) from avian sarcoma virus (ASV) and its active-site derivative containing an Asp64 --> Asn substitution have been solved at atomic resolution ranging 1.02-1.42 A. The high-quality data provide new structural information about the active site of the enzyme and clarify previous inconsistencies in the description of this fragment. The very high resolution of the data and excellent quality of the refined models explain the dynamic properties of IN and the multiple conformations of its disordered residues. They also allow an accurate description of the solvent structure and help to locate other molecules bound to the enzyme. A detailed analysis of the flexible active-site region, in particular the loop formed by residues 144-154, suggests conformational changes which may be associated with substrate binding and enzymatic activity. The pH-dependent conformational changes of the active-site loop correlates with the pH vs activity profile observed for ASV IN.

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 against AIDS. Recently, cryst als of an inactive D64N mutant were obtained under conditions identical to those used for the native enzyme. Data were collected at different pH values and in the presence of divalent cations, Data were also collected at low pH for the crystals of the native ASV IN core domain. 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 (Asx denotes Asn or Asp) is rotated by similar to 150 degrees around the C alpha-C beta bond, compared with the structures at higher pH. In the new structures, this residue makes hydrogen bands with the amide group of Asn-160, and thus, the usual metal-binding site, consisting of Asp-64, Asp-121, and Glu-157, is disrupted. Surprisingly, however, a single Zn2+ 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 the pH or by mutation, in which a protonated side chain of Asx-64 changes its orientation and interaction partner.

The x-ray structures of an inhibitor complex of the catalytic core domain of avian sarcoma virus integrase (ASV IN) were solved at 1.9- to 2.0-A resolution at two pH values, with and without Mn2+ cations. This inhibitor (Y-3), originally identified in a screen for inhibitors of the catalytic activity of HIV type 1 integrase (HIV-1 IN), was found in the present study to be active against ASV IN as well as HIV-1 IN. The Y-3 molecule is located in close proximity to the enzyme active site, interacts with the flexible loop, alters loop conformation, and affects the conformations of active site residues. As crystallized, a Y-3 molecule stacks against its symmetry-related mate. Preincubation of IN with metal cations does not prevent inhibition, and Y-3 binding does not prevent binding of divalent cations to IN. Three compounds chemically related to Y-3 also were investigated, but no binding was observed in the crystals. Our results identify the structural elements of the inhibitor that likely determine its binding properties.

Over the time, study has resulted in a general understanding of the enzymatic mechanism of retroviral integrase (IN), but analyses that are more detailed have been hampered by the lack of precise structural information. It is a virus-encoded enzyme that catalyzes nonspecific insertion of viral DNA into multiple sites on host DNA. As DNA integration is an essential step in the retroviral replication cycle, this enzyme is an attractive target for inhibition of human immunodeficiency virus (HIV). Research in this field took a subsequent leap when the crystal structures of the catalytic domains of both HIV-1 IN and avian sarcoma virus (ASV) IN became available. Precise data on the interaction of these enzymes and the essential ligands are necessary for understanding the structural basis of the reaction mechanism and for guiding rational drug design. Details of the location of metal ions, required for their enzymatic activity, in the active site of retroviral integrases can enhance the understanding of the catalytic mechanism of these enzymes and their relationship to that of other members of the superfamily. This chapter presents the structure of ASV IN catalytic domain with the essential cations Mg2+ or Mn2+ bound in the active site. In addition, the chapter presents the structure of an inactive complex of the catalytic domain of ASV IN with Zn2+. The structure of the catalytic domain of ASV IN complexed with three different divalent cations is presented. These results clearly show that the active site of this enzyme is preformed, in that only relatively small movements of side chains and no shifts of the main chain are needed to provide an environment suitable for cation binding. This is in contrast with the related core HIV-1 IN. However, antibody-binding experiments have shown that HIV-1 IN undergoes a conformational change when incubated with divalent cation cofactors.