Crystal engineering of HIV-1 reverse transcriptase for structure-based drug design

Abstract

HIV-1 reverse transcriptase (RT) is a primary target for anti-AIDS drugs. Structures of HIV-1 RT, usually determined at approximately 2.5-3.0 A resolution, are important for understanding enzyme function and mechanisms of drug resistance in addition to being helpful in the design of RT inhibitors. Despite hundreds of attempts, it was not possible to obtain the structure of a complex of HIV-1 RT with TMC278, a nonnucleoside RT inhibitor (NNRTI) in advanced clinical trials. A systematic and iterative protein crystal engineering approach was developed to optimize RT for obtaining crystals in complexes with TMC278 and other NNRTIs that diffract X-rays to 1.8 A resolution. Another form of engineered RT was optimized to produce a high-resolution apo-RT crystal form, reported here at 1.85 A resolution, with a distinct RT conformation. Engineered RTs were mutagenized using a new, flexible and cost effective method called methylated overlap-extension ligation independent cloning. Our analysis suggests that reducing the solvent content, increasing lattice contacts, and stabilizing the internal low-energy conformations of RT are critical for the growth of crystals that diffract to high resolution. The new RTs enable rapid crystallization and yield high-resolution structures that are useful in designing/developing new anti-AIDS drugs.

Figure 1.

Iterative approach to crystal engineering.

Figure 2.

Mutagenesis of RT. (A) Schematic showing the three binding sites (arrows) of the 2′-O-methylated primers used in MOE-LIC. Locations of specific restriction enzyme cut sites are also indicated. (B) Annealed duplex of the primer terminated insert and vector with 2′-O-methyl nucleotides indicated by Me. (C) Cartoon of RT color-coded by the p66 subdomains. All mutations made in this study are indicated as spheres. The beneficial mutations are colored yellow and labeled. (D) Flowchart showing the generation of the mutants, which are color-coded to show X-ray diffraction resolution of the crystals. Stars denote those mutants for which the unliganded crystals show improved resolution. (E) Diagram of RT1A, RT52A and RT69A.

Figure 3.

Crystal Structure of RT52A with TMC278 at 1.8 Å resolution. (A) Simulated annealed Fo–Fc omit map (3σ contours) for TMC278. (B) Typical 1B1-RT/NNRTI residues involved in crystal packing (pdb code: 1S9E). Residues involved in crystal contacts of HIV-1 RT are shown as space filled (residues within 4.5 Å of the asymmetric unit). (C) RT52A/TMC278 complex residues involved in crystal contacts. (D) Unliganded RT69A residues involved in crystal contacts.

Figure 4.

Stereo view of electron density in the RNase H domain of apo-RT69A. Stereo view of the 3Fo–2Fc map (calculated at 1.85 Å resolution and contoured at 2.5σ) surrounding Tyr532 in the RNase H domain of RT69A.

Figure 5.

Comparison of unit cell and X-ray diffraction resolution of mutants. Plot of unit cell (Matthews coefficient) and X-ray diffraction resolution (Å) of the mutants that produced crystals that diffracted X-rays to better than 4 Å resolution. The legend of the table indicates the mutations and the parental template for each of the mutants. RT69A and RT97A are plotted based on crystals with RNHIs bound; all others were complexed with NNRTIs. RT35A is highlighted in bold and RT52A and RT69A are boxed for emphasis.