Structural Characterization of a Peptide Derived from a Lab-Evolved Protein that Targets HIV-1 TAR RNA

Abstract

Innovative approaches are needed to create new therapeutics that target HIV, especially those that act on novel facets of the viral life cycle. The HIV-1 TAR (trans-activation response) element RNA is a validated drug target that resists mutations to maintain interactions with the viral protein Tat and the host pTEFb complex, giving rise to a protein-RNA complex that is essential for efficient proviral transcription. So far, TAR has evaded discovery of compounds with sufficient affinity and selectivity to warrant pharmaceutical development. To meet this challenge, we pursued a ‘semi-design and protein evolution’ approach that yielded many high-affinity TAR Binding Proteins (TBPs) derived from RRM1 of the U1A spliceosomal protein. Here we describe the lab-evolution results and present the structural analysis of TBP6.7 in complex with TAR at 1.8Å. TBP6.7 recognizes conserved guanines in the TAR major groove using a constellation of arginines within the beta2-beta3 loop. We demonstrate the feasibility of reducing TBPs to short a cyclic peptide (peptide 1) that retains TAR binding and inhibit Tat-mediated transcription in HeLa cell nuclear lysate. In addition, TBP6.7(Q48T) shows that a mutation in the beta2-beta3 loop yields additional interactions between K20, K22 and N15 of the β1-α1 loop and the TAR bulge. Concurrently, we also present a crystallization module validated by isothermal titration calorimetry (ITC), which will be used for co-crystallizing TAR RNA with peptides and small molecules. Future efforts will involve optimization of peptide 1, biophysical characterization of optimized peptides and co-crystallization with TAR RNA.