Structure of a two-domain fragment of HIV-1 integrase: implications for domain organization in the intact protein

Abstract

Retroviral integrase, an essential enzyme for replication of human immunodeficiency virus type-1 (HIV-1) and other retroviruses, contains three structurally distinct domains, an N-terminal domain, the catalytic core and a C-terminal domain. To elucidate their spatial arrangement, we have solved the structure of a fragment of HIV-1 integrase comprising the N-terminal and catalytic core domains. This structure reveals a dimer interface between the N-terminal domains different from that observed for the isolated domain. It also complements the previously determined structure of the C-terminal two domains of HIV-1 integrase; superposition of the conserved catalytic core of the two structures results in a plausible full-length integrase dimer. Furthermore, an integrase tetramer formed by crystal lattice contacts bears structural resemblance to a related bacterial transposase, Tn5, and exhibits positively charged channels suitable for DNA binding.

Fig. 1. Ribbon diagram of HIV-1 IN^1–212. Two orthogonal views of the IN^1–212 dimer. The A subunit is colored green, and the B subunit yellow. Disordered loops are indicated by the dotted lines.

Fig. 2. Conservation of the catalytic core domain structure. (A) Superposition of the C_α traces of the AB dimer of IN^1–212 with the dimer of IN^50–212 (PDB code 1BIS) (B) Superposition of AB and CD dimers of IN^1–212 in one asymmetric unit. (C) The interface between the N-terminal and catalytic core domain. Residues 18–38 and 184–209 are shown in ribbon diagrams and the residues involved specifically in domain interactions are shown in ball-and-stick.

Fig. 3. Analysis of domain interactions. (A) Ribbons diagram of the N-terminal domain structure as an isolated entity determined by NMR (PDB code 1WJA) and (B) in the context of IN^1–212 by X-ray crystallography after superimposing the yellow subunits of the two structures. Residue numbers at the N- and C-termini are labeled. (C) A near orthogonal view from that of (B) looking down the 2-fold axis. Residues involved in the dimerization, which are rather hydrophobic, are shown in ball-and-stick. Zn²⁺ ions are shown as red spheres. (D) The N-terminal domain is oriented differently relative to the core domain in the A and B subunit of IN^1–212 as revealed by superposition of the C_α traces of the core domains.

Fig. 4. The active site. (A) Stereo view of the phosphate ion bound near the active site composed of three conserved carboxylates, D64, D116 and E152. The D subunit, which is chosen for the active site representation, is completely exposed to solvent and is not affected by lattice contacts even though E152 of this subunit is more mobile compared with E152 of the A, B and C subunits. A 2F_o – F_c electron density map contoured at 1.0σ is superimposed on the final refined structure represented by the stick-and-ball model. Two water molecules are also shown; one is associated with the phosphate and the other stabilizes the main chain configuration at the active site. (B) Comparison of the loop containing D116 in the IN^1–212 D subunit, shown in gold, and the IN^52–210 structures (PDB code 1EXQ), shown in purple. The phosphate ion is ∼7 Å away from the mid-point between D64 and D116.

Fig. 5. Location of the C-terminal domain. Ribbons diagram of SIV IN^50–293 (PDB code 1C6V), HIV-1 IN^52–288 (PDB code 1EX4) and RSV IN^49–286 (PDB code 1C0M) after superposition of the catalytic core of each structure. Each dimer is colored green and yellow. The C-terminal domains of the green subunit of SIV, RSV and HIV-1 integrase are located similarly relative to the catalytic core dimer. Superposition of the catalytic cores of HIV-1 IN^52–288 and IN^1–212 structures positions the N-terminal domain dimer between the C-terminal domains without any steric clash. The modeled N-terminal domain dimer is colored red and blue for the yellow and green subunits, respectively.

Fig. 6. The ABCD tetramer of IN^1–212. (A) Ribbons diagram of the AB and CD dimers from one asymmetric unit. A, B, C and D subunits are colored green, yellow, blue and red, respectively. Purple and green spheres represent zinc and potassium ions bound to the protein. Balls and sticks represent the ordered phosphates located near the active sites. (B) Details of the upper half of the dimer-of-dimer interface. The N-terminal domain, residues 186–195 of the D subunit and residues 149–197 of the B subunit are shown as ribbon diagrams. Side chains contributing to the interface are shown in ball-and-stick models. The lower half of the interface is essentially a repeat of the upper one. (C) Molecular surface of the ABCD tetramer viewed with a slight rotation from the view shown in (A). Positive and negative electrostatic potentials are shown in blue and red (saturation at ∼15 kT/e). The image was created with GRASP (Nicholls et al., 1991). Viral DNA ends are superimposed on the ABCD tetramer guided by the phosphate, active site and Q148. The black arrow points to the ordered inorganic phosphate in our structure, and the yellow arrow points to the scissile phosphate. The most positively charged region is the central hinge between the AB and CD dimers, which would accommodate target DNA in this model. The negatively charged patch centered around the active site would normally be neutralized by divalent metal ions. Binding of viral DNA would be stabilized by a C-terminal domain serving as a ‘clamp’ (not shown), and possibly by a positively charged patch on the side of the core domain.

Fig. 7. Comparison between the composite integrase tetramer and Tn5 transposase in complex with DNA substrate. (A) Orthogonal views of a Ribbons diagram of the integrase tetramer with the C-terminal domain modeled in. The integrase full-length tetramer is composed by superposition of the catalytic core domain of the HIV-1 IN^52–288 structure (PDB code 1EX4) onto the catalytic domains of the AB and CD dimers of IN^1–212. The four subunits are colored the same as in Figure 6A. (B) Orthogonal views of the Tn5 transposase–DNA complex (PDB code 1F3I). The protein dimer is shown in yellow and red, and the two DNA duplexes associated with Tn5 transposase are shown in green and blue ribbons. All figures except those displaying molecular surface were created with Ribbons (Carson, 1987).