title: Efavirenz binding to HIV-1 reverse transcriptase monomers and dimers.
abstract: Efavirenz (EFV) is a nonnucleoside reverse transcriptase inhibitor (NNRTI) of HIV-1 reverse transcriptase (RT) used for the treatment of AIDS. RT is a heterodimer composed of p66 and p51 subunits; p51 is produced from p66 by C-terminal truncation by HIV protease. The monomers can form p66/p66 and p51/p51 homodimers as well as the p66/p51 heterodimer. Dimerization and efavirenz binding are coupled processes. In the crystal structure of the p66/p51-EFV complex, the drug is bound to the p66 subunit. The binding of efavirenz to wild-type and dimerization-defective RT proteins was studied by equilibrium dialysis, tryptophan fluorescence, and native gel electrophoresis. A 1:1 binding stoichiometry was determined for both monomers and homodimers. Equilibrium dissociation constants are approximately 2.5 microM for both p66- and p51-EFV complexes, 250 nM for the p66/p66-EFV complex, and 7 nM for the p51/p51-EFV complex. An equilibrium dissociation constant of 92 nM for the p66/p51-EFV complex was calculated from the thermodynamic linkage between dimerization and inhibitor binding. Binding and unbinding kinetics monitored by fluorescence were slow. Progress curve analyses revealed a one-step, direct binding mechanism with association rate constants k(1) of approximately 13.5 M(-1) s(-1) for monomers and heterodimer and dissociation rate constants k(-1) of approximately 9 x 10(-5) s(-1) for monomers. A conformational selection mechanism is proposed to account for the slow association rate. These results show that efavirenz is a slow, tight-binding inhibitor capable of binding all forms of RT and suggest that the NNRTI binding site in monomers and dimers is similar.
authors.affiliation: Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA.
authors.forename: Valerie A
authors.initials: VA
authors.lastname: Braz
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Holladay
authors.forename: Mary D
authors.initials: MD
authors.lastname: Barkley
mesh_terms: D000480:Alkynes; D019380:Anti-HIV Agents / Q000737:chemistry* / Q000494:pharmacology*; D048588:Benzoxazines / Q000737:chemistry* / Q000494:pharmacology*; D001665:Binding Sites; D003521:Cyclopropanes; D019281:Dimerization; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D011487:Protein Conformation; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D000480:Alkynes; D019380:Anti-HIV Agents; D048588:Benzoxazines; D003521:Cyclopropanes; D018894:Reverse Transcriptase Inhibitors; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; C098320:efavirenz