Metal binding by the D,DX35E motif of human immunodeficiency virus type 1 integrase: selective rescue of Cys substitutions by Mn2+ in vitro

Abstract

The D,DX(35)E motif characteristic of retroviral integrase enzymes (INs) is expected to bind the required metal cofactors (Mg(2+) or Mn(2+)), but direct evidence for a catalytic role has been lacking. Here we used a metal rescue strategy to investigate metal binding. We established conditions for analysis of an activity of IN, disintegration, in both Mg(2+) and Mn(2+), and tested IN mutants with cysteine substitutions in each acidic residue of the D,DX(35)E motif. Mn(2+) but not Mg(2+) can bind tightly to Cys, so if metal binding at the acidic residues is mechanistically important, it is expected that the Cys-substituted enzymes would be active in the presence of Mn(2+) only. Of the three acidic residues, a strong metal rescue effect was obtained for D116C, a weaker rescue was seen for D64C, and no rescue was seen with E152C. Modest rescue could also be detected for D116C in normal integration in vitro. Comparison to Ser and Ala substitutions at D116 established that the rescue was selective for Cys. Further studies of the response to pH suggest that the metal cofactor may stabilize the deprotonated nucleophile active in catalysis, and studies of the response to NaCl titrations disclose an additional role for the metal cofactor in stabilizing the IN-DNA complex.

FIG. 1.

(A) DNA breaking and joining reactions involved in integration. Two nucleotides are removed from the viral cDNA end in the terminal cleavage step (left), then the recessed 3′ hydroxyl generated by cleavage is used to attack a phosphodiester in the target DNA, joining the viral DNA end and cleaving the target (right). Disintegration is the reversal of the strand transfer step. (B) Diagram of the microtiter plate assay for disintegration. The disintegration reaction covalently links a biotin-modified DNA strand to digoxigenin-modified strand. The resulting strand can be captured on a streptavidin-coated microtiter plate and quantified with an antidigoxigenin ELISA. HRP, horseradish peroxidase.

FIG. 2.

Activities of IN mutants containing substitutions of the D,DX₃₅E motif acidic residues with Cys. In each panel, the metal used in the test reactions is indicated below the graph. (A) Activities of wild-type IN and mutants in the strand transfer assay quantified by the microtiter ELISA assay. The wild-type activity is set at 1; the mutants studied are indicated below the graph. (B) Disintegration activities of mutant and wild-type IN. Markings are as described for panel A. The values (arbitrary PhosphorImager units) were as follows: wild-type, 1874; D64C, 159; D116C, 928; E152C, 11. (C) Strand transfer activity of wild-type and Cys substitution IN mutants measured with end-labeled substrates. Left, activities with a blunt-ended LTR substrate. Right, activities with the postcleavage LTR substrate. (D) Disintegration activities measured with end-labeled substrates. (E) Terminal cleavage activities measured with end-labeled substrates.

FIG. 3.

Response of the disintegration reaction to different pHs in the presence of Mn²⁺ or Mg²⁺. The pH values are indicated along the bottom of the graph; the peak activity of wild-type IN in Mn²⁺ is set at 1.

FIG. 4.

Terminal cleavage by wild-type (WT) IN monitored with end-labeled substrates in the presence of different concentrations of NaCl and Mg²⁺ or Mn²⁺. The concentrations of NaCl in each reaction mixture are shown at the top; the metal cofactor present is shown at the bottom.

FIG. 5.

Mapping functional interactions on the IN catalytic domain. (A) Space-filling model of the IN catalytic domain (amino acid residues 50 to 212). The acidic residues of the D,DX₃₅E motif are shown in red. Other colored residues are as follows: 119 (target DNA binding), green; 159 (LTR DNA binding), blue; 148, 143, and 62 (LTR overhang binding), yellow. (B) The IN catalytic domain with basic residues shown blue and acidic residues shown in red. (C) Candidate model of the IN catalytic domain with viral DNA and target DNA docked in locations suggested by mutagenesis and biochemical studies. The viral DNA is shown in cyan, and the target DNA is shown in magenta.