Zinc folds the N-terminal domain of HIV-1 integrase, promotes multimerization, and enhances catalytic activity

Abstract

The N-terminal domain of HIV-1 integrase contains a pair of His and Cys residues (the HHCC motif) that are conserved among retroviral integrases. Although His and Cys residues are often involved in binding zinc, the HHCC motif does not correspond to any recognized class of zinc binding domain. We have investigated the binding of zinc to HIV-1 integrase protein and find that it binds zinc with a stoichiometry of one zinc per integrase monomer. Analysis of zinc binding to deletion derivatives of integrase locates the binding site to the N-terminal domain. Integrase with a mutation in the HHCC motif does not bind zinc, consistent with coordination of zinc by these residues. The isolated N-terminal domain is disordered in the absence of zinc but, in the presence of zinc, it adopts a secondary structure with a high alpha helical content. Integrase bound by zinc tetramerizes more readily than the apoenzyme and is also more active than the apoenzyme in in vitro integration assays. We conclude that binding of zinc to the HHCC motif stabilizes the folded state of the N-terminal domain of integrase and bound zinc is required for optimal enzymatic activity.

Figure 1

Coelution of zinc with HIV-1 integrase in gel filtration. Integrase was dialyzed against buffer A containing zinc chloride as described and applied to a Superdex 75 column equilibrated with buffer A not containing zinc. The protein concentration in each of the collected fractions was determined by measuring the optical density at 280 nm, and the zinc concentration in the same fractions was determined by atomic absorption spectroscopy. (A) Full-length integrase (IN^1–288/F185K/C280S). (B) Integrase with a C-terminal deletion (IN^1–212/F185K). (C) Integrase with an N-terminal deletion (IN^50–288/F185K/C280S). (D) The central domain of integrase (IN^50–212/F185K). (E) Integrase with the His residues of the HHCC motif changed to Asn (IN^1–288/H12N/H16N/F185K/C280S).

Figure 2

Folding of the N-terminal domain of HIV-1 integrase in the presence of zinc. Far UV CD spectra of the N-terminal domain of integrase (IN^1–55) in the absence (solid line) and presence (dotted line) of bound zinc.

Figure 3

Zinc-binding alters the CD spectrum of HIV-1 integrase in the near UV region. (A) Near UV CD spectra of integrase (IN^1–288/F185K/C280S) in the absence (solid line) and presence (dotted line) of bound zinc. (B) Near UV CD spectra of IN^1–288/H12N/H16N/F185K/C280S) in the absence (solid line) and presence (dotted line) of bound zinc. The substitution of Asn for His in the HHCC motif abolishes the effect of zinc on the near UV CD spectrum.

Figure 4

Zinc binding promotes tetramerization of HIV-1 integrase. (A) Gel filtration of integrase (IN^1–288/F185K/C280S) on Superdex 200 after dialysis against buffer A containing EDTA (solid line) or zinc (dotted line). The zinc-dialyzed protein eluted at the expected position for tetramers (relative to globular protein standards). The EDTA-dialyzed protein eluted at the expected position for dimers. The peak elution times of chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), bovine serum albumin (67 kDa), and aldolase (158 kDa) standards are indicated. (B) Integrase lacking the C-terminal domain (IN^1–212/F185K) eluted exclusively as dimers after dialysis against EDTA or zinc. (C) Integrase lacking the N-terminal domain (IN^50–288/F185K/C280S) eluted predominantly as dimers, with a trailing edge toward the tetramer position, after dialysis against EDTA or zinc.

Figure 5

Zinc binding stimulates both the 3′-processing and DNA strand transfer activities of HIV integrase. (A) Autoradiogram of 3′-processing products of reactions catalyzed by integrase dialyzed against buffer A containing EDTA or zinc. Reaction mixtures containing 20 nM DNA substrate and 0.94 μM integrase were incubated at 37°C for 1 hr. The migration positions of the unreacted DNA substrate (S) and 3′-processing products (P) are indicated. (B) Quantitation of the 3′-processing time course shown in A. ○, Integrase dialyzed against zinc; •, integrase dialyzed against EDTA. (C) Autoradiogram of DNA strand transfer products of reactions catalyzed by integrase dialyzed against buffer A containing EDTA or zinc. Reaction conditions and labeling are the same as indicated for A, except that the concentration of DNA substrate was 150 μM. (D) Quantitation of the DNA strand transfer time course shown in C. ○, Integrase dialyzed against zinc; •, integrase dialyzed against EDTA.