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.

Scheme 1

Thermodynamic Linkage of NNRTI Binding and Subunit Dimerization

Scheme 2

Mechanisms of Slow Binding Inhibition

Figure 1

Equilibrium dialysis data for p51. (A) wild-type p51 equilibrated with efavirenz for 5 days: (○) experimental values [Ibound]_exp, and (•) calculated values [Ibound]_calc from Dialfit using eqs A1a–Alc. (B) p51^W401A equilibrated with efavirenz for 30 h: (○) [Ibound]_exp, and (•) [Ibound]_calc from Dialfit using eq A1a. Insets show residuals ([Ibound]_exp – [Ibound]_calc) versus total protein concentration [Protein]_Tot in each measurement.

Figure 2

Structure of HIV-1 RT complexed with efavirenz (1FK9): p66 (purple), p51 (green), efavirenz (cyan), and tryptophans (red).

Figure 3

Association of efavirenz to (—) p66/p51 and () p51 monitored by tryptophan fluorescence at 5 °C. λ_ex = 295 nm, λ_em = 340 nm. 20 μM p66/p51 (83% heterodimer) diluted to 2 μM prior to adding efavirenz; 4.5 μM p51 (97% monomer). [EFV]:[protein] 2:1.

FIGURE 4

Progress curves for p51^W401A binding to efavirenz monitored by tryptophan fluorescence at 5 °C. λ_ex = 295 nm, λ_em = 340 nm. 2.5 μM p51^W401A; () 5 μM, () 8 μM, () 11 μM, and (—) 14 μM efavirenz. Data acquired at 30 s intervals for first 4–5 h, then at 5 min intervals. Data were fit to eq 2a to obtain k_obs.

FIGURE 5

Progress curve analysis of efavirenz binding to (▲) p66/p51, () p51^W401A, () p66^W401A. Lines are fits to eq 3. Error bars give the range of average values from 2 experiments.

FIGURE 6

Dissociation of () p51^W401A—EFV and () p66^W401A—EFV complexes monitored by tryptophan fluorescence at 5 °C. λ_ex = 295 nm, λ_em = 340 nm. Black curves are fits to eq 2b. Data acquired at 30 s intervals for first 4–5 h, then at 5 min intervals.

FIGURE 7

Blue Native polyacrylamide gel electrophoresis of p66 in the absence and presence of NNRTIs. (A) Monomer and homodimer binding to [¹⁴C] EFV: (lane 1) 2 μM p66^W401A incubated 2 h with [¹⁴C] EFV, (lane 2) 5 μM p66 incubated 2 h with [¹⁴C] EFV, and (lane 3) 5 μM p66 incubated 1 wk with [¹⁴C] EFV. (B) wild-type p66 incubated 1 wk in the absence and presence of NNRTI 1 wk: (lane 1) Native Markers; (lane 2) 0.8 μM p66; (lane 3) 3 μM p66, 25 μM EFV; and (lane 4) 3 μM p66, 25 μM NVP.

FIGURE 8

Structures of HIV-1 (upper) RT (1DLO) and (lower) RT—EFV complex (1FK9). Polymerase domains: fingers (blue), palm (red), thumb (green), and connection (orange) subdomains; RNase H domain (magenta). Efavirenz (yellow) and contact residues (grey) are shown using the van der Waals radii.