Structural Basis for Inactivating Mutations and pH-dependent Activity of Avian Sarcoma Virus Integrase

Abstract

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, crystals 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 ϳ150°around the C␣OC␤ bond, compared with the structures at higher pH. In the new structures, this residue makes hydrogen bonds 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 Zn 2؉ 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. Integrase (IN)1 (1) is one of only four enzymes encoded by retroviruses, such as human immunodeficiency virus type 1 and avian sarcoma virus (ASV), and it is absolutely essential for the support of the viral life cycle. For these reasons, IN is currently a target for the design of antiretroviral drugs. Retroviral INs contain approximately 300 amino acids, organized into three domains: zinc-binding N terminus, catalytic core domain, and DNA-binding C terminus. IN catalyzes the incorporation of the reverse-transcribed viral DNA into the host genome in two steps, processing and joining (1-3), both of which involve a nucleophilic attack by a hydroxyl group on a DNA phosphate. In the processing step, a water molecule attacks near the end of the viral DNA, two nucleotides from the 3Ј-ends of both viral DNA strands. In the joining step, the exposed viral DNA deoxyribose 3Ј-OH is activated to attack the host DNA at a relatively nonspecific location, thereby inserting viral DNA into the host genome. In vitro, these reactions require only virus-like DNA, IN, and metal cations.Although the isolated ASV IN catalytic core domain is defective for the processing and joining activities, it retains two activities: disintegration, the reverse of the joining step that uses a preformed DNA substrate, and an endonuclease activity that cleaves between the highly conserved C and A (Ϫ3 activity) at the termini of the viral DNA (CATT-3Ј) (4). The endonuclease activity is distinct from the normal processing activity of IN, which cleaves between the A and T (Ϫ2 activity). Both the endonuclease and disintegration activities of the catalytic domain are dependent on the D, D(35)E catalytic triad, corresponding to Asp-...

Atomic Resolution Structures of the Core Domain of Avian Sarcoma Virus Integrase and Its D64N Mutant,

“…Because this pH range coincides with the known pH vs activity assay profile for IN (22), there might be a direct relationship between the pH-dependent changes in the conformation of the active-site loop and the enzymatic activity of IN. As previously described (22), a change of protonation followed by a conformational change is observed in the core domain of native ASV IN for the Asp64 side chain around pH 6.2. Thus, we can see that pH-triggered structural changes of this protein are quite extensive, especially around its active site (Figure 3).…”

“…Thus, we can see that pH-triggered structural changes of this protein are quite extensive, especially around its active site (Figure 3). Studies of enzymatic activity as a function of pH (22) indicated that the catalytic activity of ASV IN decreases dramatically at pH below 6.0. Therefore, it is very likely that some or all of the structural changes observed in the region of the active site are responsible for the changes in the enzymatic activity.…”

“…In our previous reports (21,22), we noted a new conformation of the active-site loop, as well as vastly different conformations of the side chain of Asp64; therefore, in the present study, we sought to minimize model bias. The refinement was cross-validated by the R free index (24), which was calculated using 10% of all reflections.…”

Characterization of the Self Association of Avian Sarcoma Virus Integrase by Analytical Ultracentrifugation

“…However, partial proteolysis experiments, deletion mutation results, and sequence similarity with other retroviral integrases, retrotransposons, and bacterial insertion elements suggest that there are three distinct domains in full-length ASV IN: the N-terminal, central core, and C-terminal domains (3). Structural analysis reveals that both the central core and C-terminal domains form dimers (9,10). All three domains have been implicated in the self-association of the full-length proteins.…”

An integrase of endogenous retrovirus is involved in maternal mitochondrial DNA inheritance of the mouse

“…Though integrase has exo-and endonuclease activity [9][10][11][12][13][14][15][16][17][18], in spite of the recombinant full-length Eri15 protein that developed in the E. coli lacking part of the integrase core domain and DNA binding domain, endonuclease activity was discovered which could digest mtDNA (Fig. 3A).…”