C. Ross

High resolution structures of HIV-1 RT from four RT-inhibitor complexes.

We have determined the structures of four complexes of HIV-1 reverse transcriptase with non-nucleoside inhibitors, three fully refined at high resolution. The highest resolution structure is of the RT-nevirapine complex which has an R-factor of 0.186 and a root-mean-square bond length deviation of 0.015 Å for all data to 2.2 Å. The structures reveal a common mode of binding for these chemically diverse compounds. The common features of binding are largely hydrophobic interactions and arise from induced shape complementarity achieved by conformational rearrangement of the enzyme and conformational/ conf igurational rearrangement of the compounds.

Mechanism of inhibition of HIV-1 reverse transcriptase by non-nucleoside inhibitors

The structure of unliganded HIV-1 reverse transcriptase has been determined at 2.35 Å resolution and refined to an R-factor of 0.219 (for all data) with good stereochemistry. The unliganded structure was produced by soaking out a weak binding non-nucleoside inhibitor, HEPT, from pregrown crystals. Comparison with the structures of four different RT and non-nucleoside inhibitor complexes reveals that only minor domain rearrangements occur, but there is a significant repositioning of a three-stranded β-sheet in the p66 subunit (containing the catalytic aspartic acid residues 110, 185 and 186) with respect to the rest of the polymerase site. This suggests that NNIs inhibit RT by locking the polymerase active site in an inactive conformation, reminiscent of the conformation observed in the inactive p51 subunit.

The structure of HIV-1 reverse transcriptase complexed with 9-chloro-TIBO: lessons for inhibitor design.

BACKGROUND HIV reverse transcriptase (RT) is a key target of anti-AIDS therapies. Structural studies of HIV-1 RT, unliganded and complexed with different non-nucleoside inhibitors (NNIs), have pointed to a common mode of binding and inactivation through distortion of the polymerase catalytic site by NNIs containing two hinged rings. The mode of binding of the TIBO family of inhibitors is of interest because these compounds do not fit the two-hinged-ring model. RESULTS The structure of HIV-1 RT complexed with 9-chloro-TIBO (R82913) has been determined at 2.6 A resolution. As reported for the lower resolution analysis of another TIBO compound, this inhibitor binds at the same site as other NNIs, but our higher resolution study reveals the Cl-TIBO is distorted from the conformation seen in crystals of the inhibitor alone. This allows Cl-TIBO to mimic the binding of NNIs containing two hinged rings. Inhibitor-protein interactions are again predominantly hydrophobic and the protein conformation corresponds to that seen in complexes with other tight-binding NNIs. CONCLUSIONS Although Cl-TIBO is chemically very different from other NNIs, it achieves remarkable spatial equivalence and shape complementarity with other NNIs on binding to RT. Comparison of the different RT-NNI complexes suggests modifications to the TIBO group of inhibitors which might enhance their binding and hence, potentially, their therapeutic efficacy.

Continuous and discontinuous changes in the unit cell of HIV-1 reverse transcriptase crystals on dehydration.

A crystal form of HIV-1 reverse transcriptase (RT) complexed with inhibitors showed diffraction to a high-resolution limit of 3.7 A. Instability in the unit-cell dimensions of these crystals was observed during soaking experiments, but the range of this variability and consequent change in lattice order was revealed by a chance observation of dehydration. Deliberately induced dehydration results in crystals having a variety of unit cells, the best-ordered of which show diffraction to a minimum Bragg spacing of 2.2 A. In order to understand the molecular basis for this phenomenon, the initial observation of dehydration, the data sets from dehydrated crystals, the crystal packing and the domain conformation of RT are analysed in detail here. This analysis reveals that the crystals undergo remarkable changes following a variety of possible dehydration pathways: some changes occur gradually whilst others are abrupt and require significant domain rearrangements. Comparison of domain arrangements in different crystal forms gives insight into the flexibility of RT which, in turn, may reflect the internal motions allowing this therapeutically important enzyme to fulfill its biological function.

The growth and characterization of crystals of human immunodeficiency virus (HIV) reverse transcriptase

Abstract Extensive studies on the crystallization of HIV-1 reverse transcriptase (RT) have yielded several crystal forms, two of which show diffraction to minimum Bragg spacings of 6 A or better. Type 1 crystals belong to the space group P2 1 2 1 2 1 with unit cell dimensions a = 147 A , b = 190 A and c = 182 A . Crystal density measurement indicate a very high crystal solvent content of 77% consistent with the presence of two RT heterodimers (66k/51k) per crystallographic asymmetric unit. These crystals are suitable for a low resolution determination of the apoenzyme structure. The second well ordered crystal form (space group P4 2 22 with unit cell dimensions a = b = 120 A , c = 320 A ) results from the co-crystallization of RT heterodimer and a double-stranded DNA oligonucleotide. Crystal density measurements again yield a relatively high value for the solvent content (7%; one RT heterodimer per crystallographic asymmetric unit) and elemental analysis indicates that one DNA oligonucleotide is associated with each RT heterodimer. This is consistent with each heterodimer possessing a single, competent, active site.