HIV-1 Reverse Transcriptase Monomers Adopt Multiple Conformations in Solution

Abstract

HIV-1 reverse transcriptase (RT) is a key enzyme in HIV infection and an important therapeutic target. The enzyme is an asymmetric heterodimer of p66 and p51 subunits. Although each subunit has an N-terminal polymerase domain with identical amino acid sequence (440 residues), in the heterodimer the polymerase domain of p66 adopts an open conformation while that of p51 is in a closed conformation. The p66 and p51 monomers are folded proteins of unknown structure that also form asymmetric homodimers, indicating that both subunits can assume open and closed conformations. The linkage between conformational and binding equilibria is a key factor in the assembly of complex biomolecular systems such as RT.Comparison of hydrogen/deuterium exchange patterns indicates that the secondary structure of the polymerase domains of p66 and p51 monomers is almost identical, but that both p66 and p51 are less structured as free monomers than they are in the heterodimer. Small angle X-ray scattering (SAXS) was employed to study the conformations of the monomers in solution. Scattering curves were not well fit by monomeric structures derived from the crystal structure of the heterodimer. Better fits were obtained using rigid body modeling and allowing the subdomains to rotate with respect to each other to optimize fits to the scattering data. Multiple optimizations yielded ensembles of 500 structures per monomer. The p51 clusters could be grouped in 2 clusters of closely related conformations; the p66 structures showed greater variability with 20 conformational clusters. The 2 clusters for p51 resemble the closed and open conformations seen in the crystal structure. The 20 clusters of p66 comprise a variety of open and closed conformations. This conformational heterogeneity is consistent with the conformational selection mechanism proposed to explain the slow subunit binding and inhibitor binding kinetics.