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 approximately 150 degrees around the Calpha---Cbeta 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 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.
Highlights
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
We report the structural basis of the inactivation of the ASV IN catalytic core domain resulting from a single conservative active site mutation (D64N), as well as from the decrease of pH, and we correlate the inactivation with the ability of the enzyme to bind divalent cations
In the previously reported structures of ASV IN [6, 7, 9], with the sole exception of the low pH complexes with an inhibitor [8], the side chain of Asp-64 points toward the side chain of Asp-121, forming hydrogen bonds mediated by a water molecule
Summary
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. 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. 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]. In previous studies of the D, D(35)E motif, it was shown that conservative active site substitutions, such as Asp 3 Glu or Glu 3 Asp, reduced activity by 10-fold or more, and other substitutions, such as Asp 3 Ala, abolished it completely [5] These residues were proposed to directly bind the required metal cofactors. It is clear from the nearly perfect coordination distances of metal cations (2.0 –2.2 Å for O . . . Mn2ϩ) that any substitution of the active site residues would disrupt metal binding by displacing the acidic oxygens
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