Conformational Dynamics of the HIV-1 Trans-Activation Response Element RNA Hairpin Bound to a Lab-Evolved Peptide

Abstract

We report microsecond-length classical molecular dynamics simulations of a lab-evolved peptide bound to the HIV-1 trans-activation response element (TAR) RNA hairpin. An essential 5′-structure in all HIV-1 mRNAs, TAR interacts with HIV-1 Tat to promote HIV-1 genome transcription, and is considered a promising therapeutic target. Using yeast display, a U1A-derived TAR binding protein (TBP) was selected that binds TAR with subnanomolar affinity and extraordinarily high specificity over the native U1A U1hpII target, thereby preventing Tat-dependent transcription in vitro. A co-crystal structure of TBP in complex with TAR reveals the basis for its unprecedented affinity and specificity. The complex exhibits a Watson-Crick C30-G34 base pair in the six-nucleotide apical loop. From chemical modification experiments and sequence conservation, this base pair has been posited as a prerequisite for Tat binding. We investigated the role of a short TBP peptide - responsible for the preponderance of TAR readout - in stabilizing the observed TAR structure using molecular dynamics simulations of TAR in the apo state and in complex with the full TBP protein and the short TBP peptide. To define discrete conformational states, we performed principal component analysis and clustered trajectories based on the resulting principal components. We observed that the apical loop C30-G34 base pair and a major groove U23-A27-U38 base triple are stable features of peptide-bound TAR on the microsecond timescale. In agreement with NMR studies of free TAR, dissolution of the major groove base triple occurs in trajectories of the apo state. The occupancy of contacts between the protein and the TAR major groove is greater for the arginine residues whose mutation to alanine has a greater impact on the binding affinity, as measured by isothermal calorimetry.