Three new structures of the core domain of HIV-1 integrase: an active site that binds magnesium

Abstract

HIV-1 integrase is an essential enzyme in the life cycle of the virus, responsible for catalyzing the insertion of the viral genome into the host cell chromosome; it provides an attractive target for antiviral drug design. The previously reported crystal structure of the HIV-1 integrase core domain revealed that this domain belongs to the superfamily of polynucleotidyltransferases. However, the position of the conserved catalytic carboxylic acids differed from those observed in other enzymes of the class, and attempts to crystallize in the presence of the cofactor, Mg2+, were unsuccessful. We report here three additional crystal structures of the core domain of HIV-1 integrase mutants, crystallized in the presence and absence of cacodylate, as well as complexed with Mg2+. These three crystal forms, containing between them seven independent core domain structures, demonstrate the unambiguous extension of the previously disordered helix alpha4 toward the amino terminus from residue M154 and show that the catalytic E152 points in the general direction of the two catalytic aspartates, D64 and D116. In the vicinity of the active site, the structure of the protein in the absence of cacodylate exhibits significant deviations from the previously reported structures. These differences can be attributed to the modification of C65 and C130 by cacodylate, which was an essential component of the original crystallization mixture. We also demonstrate that in the absence of cacodylate this protein will bind to Mg2+, and could provide a satisfactory platform for binding of inhibitors.

Figure 1

Ribbon diagram of HIV-1 integrase core domain from crystal form II. Two short β-strands not present in previous structures are designated β6 and β7. Essential catalytic residues are shown as ball-and-stick models.

Figure 2

(A) Difference omit map in the region of helix α4 and the preceding loop, contoured at 1.2 σ level (crystal form II). (B) Superposition of the active site loops of HIV-1 integrase crystal form II (blue), F185H mutant of HIV-1 integrase (19) (red), and ASV integrase (18) (green). Numbering in parentheses corresponds to ASV integrase. The positions of E152 are consistent in all the new structures of HIV-1 integrase and are close to that of the corresponding E157 of ASV integrase.

Figure 3

Effect of covalent modification of cysteines by cacodylate on the conformation of adjacent protein regions. Ribbon diagram in green depicts crystal form I, the one in red, crystal form II. Positions of modified cysteines and two of the catalytic residues, D64 and D116, are shown.

Figure 4

Binding of magnesium ion by the core domain of HIV-1 integrase. (A) Difference omit map, contoured at 3.0 σ level. (B) Coordination of Mg²⁺ ion by D64 and D116.