Crystal structures of 8-Cl and 9-Cl TIBO complexed with wild-type HIV-1 RT and 8-Cl TIBO complexed with the Tyr181Cys HIV-1 RT drug-resistant mutant

Das Kalyan, Jianping Ding, Yu Hsiou, Arthur D. Clark, Henri Moereels, Luc Koymans, Koen Andries, Rudi Pauwels, Paul A.J. Janssen, Paul L. Boyer, Patrick Clark, Richard H. Smith, Marilyn B. Kroeger Smith, Christopher J. Michejda, Stephen H. Hughes, Edward Arnold

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Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is an important target for chemotherapeutic agents used in the treatment of AIDS; the TIBO compounds are potent non-nucleoside inhibitors of HIV-1 RT (NNRTIs). Crystal structures of HTV-1 RT complexed with 8-Cl TIBO (R86183, IC50 = 4.6 nM) and 9-Cl TIBO (R82913, IC50 = 33 nM) have been determined at 3.0 Å resolution. Mutant HIV-1 RT, containing Cys in place of Tyr at position 181 (Tyr181Cys), is highly resistant to many NNRTIs and HIV-1 variants containing this mutation have been selected in both cell culture and clinical trials. We also report the crystal structure of Tyr181Cys HIV-1 RT in complex with 8-Cl TIBO (IC50 = 130 nM) determined at 3.2 Å resolution. Averaging of the electron density maps computed for different HIV-1 RT/NNRTI complexes and from diffraction datasets obtained using a synchrotron source from frozen (-165 °C) and cooled (-10 °C) crystals of the same complex was employed to improve the quality of electrondensity maps and to reduce model bias. The overall locations and conformations of the bound inhibitors in the complexes containing wild-type HIV-1 RT and the two TIBO inhibitors are very similar, as are the overall shapes and volumes of the non-nucleoside inhibitor-binding pocket (NNIBP). The major differences between the two wild-type HIV-1 RT/TIBO complexes occur in the vicinity of the TIBO chlorine substituents and involve the polypeptide segments around the β5-β6 connecting loop (residues 95 to 105) and the β13-β14 hairpin (residues 235 and 236). In all known structures of HIV-1 RT/NNRTI complexes, including these two, the position of the β12-β13 hairpin or the 'primer grip' is significantly displaced relative to the position in the structure of HIV-1 RT complexed with a double-stranded DNA and in unliganded HIV-1 RT structures. Since the primer grip helps to position the template-primer, this displacement suggests that binding of NNRTIs would affect the relative positions of the primer terminus and the polymerase active site. This could explain biochemical data showing that NNRTI binding to HIV-1 RT reduces efficiency of the chemical step of DNA polymerization, but does not prevent binding of either dNTPs or DNA. When the structure of the Tyr181Cys mutant HIV-1 RT in complex with 8-Cl TIBO is compared with the corresponding structure containing wild-type HIV-1 RT, the overall conformations of Tyr181Cys and wild-type HIV-1 RT and of the 8-Cl TIBO inhibitors are very similar. Some positional changes in the polypeptide backbone of the β6-β10-β9 sheet containing residue 181 are observed when the Tyr181Cys and wild-type complexes are compared, particularly near residue Val179 of β9. In the p51 subunit, the Cys181 side-chain is oriented in a similar direction to the Tyr181 side-chain in the wild-type complex. However, the electron density corresponding to the sulfur of the Cys181 side-chain in the p66 subunit is very weak, indicating that the thiol group is disordered, presumably because there is no significant interaction with either 8-Cl TIBO or nearby amino acid residues. In the mutant complex, there are slight rearrangements of the side-chains of other amino acid residues in the NNIBP and of the flexible dimethylallyl group of 8-Cl TIBO; these conformational changes could potentially compensate for the interactions that were lost when the relatively large tyrosine at position 181 was replaced by a less bulky cysteine residue. In the corresponding wild-type complex, Tyr181 in the p66 subunit has significant interactions with the bound. inhibitor and the position of the Tyr181 side-chain is well defined in both subunits. Apparently the Tyr181→Cys mutation eliminates favorable contacts of the aromatic ring of the tyrosine and the bound inhibitor, reducing the stability of NNRTI binding. This is consistent with the observation that the Tyr181Cys mutant HIV-1 RT is more resistant to NNRTIs that have extensive interactions with the Tyr181 side-chain This interpretation is supported by a recent biochemical study which found that an NNRTI dissociates from its complex with Tyr181Cys HIV-1 RT faster than it does from a complex with wild-type HIV-1 RT.

Original languageEnglish (US)
Pages (from-to)1085-1100
Number of pages16
JournalJournal of molecular biology
Issue number5
StatePublished - Dec 20 1996

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology


  • Antiviral inhibitors
  • Drug design
  • Drug resistance
  • Protein structure
  • Reverse transcriptase

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    Kalyan, D., Ding, J., Hsiou, Y., Clark, A. D., Moereels, H., Koymans, L., Andries, K., Pauwels, R., Janssen, P. A. J., Boyer, P. L., Clark, P., Smith, R. H., Kroeger Smith, M. B., Michejda, C. J., Hughes, S. H., & Arnold, E. (1996). Crystal structures of 8-Cl and 9-Cl TIBO complexed with wild-type HIV-1 RT and 8-Cl TIBO complexed with the Tyr181Cys HIV-1 RT drug-resistant mutant. Journal of molecular biology, 264(5), 1085-1100.