Efavirenz Binding to HIV-1 Reverse Transcriptase Monomers and Dimers

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 similar to 2.5 mu M 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 4 of similar to 13.5 m(-1) s(-1) for monomers and heterodimer and dissociation rate constants k(-1) of similar to 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.