RNA-protein interfaces control key replication events during the HIV-1 lifecycle. The viral trans-activator of transcription (Tat) protein uses an archetypal ARM (arginine-rich motif) to recruit the host positive transcription elongation factor b (pTEFb) complex onto the viral trans-activation response (TAR) RNA, leading to activation of HIV transcription. Efforts to block this interaction have stimulated production of biologics designed to disrupt this essential RNA-protein interface. Here, we present four co-crystal structures of lab-evolved TAR-binding proteins (TBPs) in complex with HIV-1 TAR. Our results reveal that high-affinity binding requires a distinct sequence and spacing of arginines within a specific β2-β3 hairpin loop that arose during selection. Although loops with as many as five arginines were analyzed, only three arginines could bind simultaneously with major-groove guanines. Amino acids that promote backbone interactions within the β2-β3 loop were also observed to be important for high-affinity interactions. Based on structural and affinity analyses, we designed two cyclic peptide mimics of the TAR-binding β2-β3 loop sequences present in two high-affinity TBPs (KD values of 4.2 ± 0.3 nM and 3.0 ± 0.3 nM). Our efforts yielded low molecular weight compounds that bind TAR with low micromolar affinity (KD values ranging from 3.6-22 μM). Significantly, one cyclic compound within this series blocked binding of the Tat-ARM peptide to TAR in solution assays, whereas its linear counterpart did not. Overall, this work provides insight into protein-mediated TAR recognition and lays the ground for the development of cyclic peptide inhibitors of a vital HIV-1 RNA-protein interaction.

Highlights d b-NADH binding to hVDAC-1 does not change conformation of N-terminal helices d b-NADH binding leads to low VDAC conductance by sterically blocking anion flux d Elevated dynamics of the N-terminal helices are present in the ground state d Movements of helix a2 are important for VDAC gating

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RNA recognition by proteins is central to biology. Here we demonstrate the existence of a recurrent structural motif, the "arginine fork", that codifies arginine readout of cognate backbone and guanine nucleobase interactions in a variety of protein− RNA complexes derived from viruses, metabolic enzymes, and ribosomes. Nearly 30 years ago, a theoretical arginine fork model was posited to account for the specificity between the HIV-1 Tat protein and TAR RNA. This model predicted that a single arginine should form four complementary contacts with nearby phosphates, yielding a two-pronged backbone readout. Recent highresolution structures of TAR−protein complexes have unveiled new details, including (i) arginine interactions with the phosphate backbone and the major-groove edge of guanine and (ii) simultaneous cation−π contacts between the guanidinium group and flanking nucleobases. These findings prompted us to search for arginine forks within experimental protein−RNA structures retrieved from the Protein Data Bank. The results revealed four distinct classes of arginine forks that we have defined using a rigorous but flexible nomenclature. Examples are presented in the context of ribosomal and nonribosomal interfaces with analysis of arginine dihedral angles and structural (suite) classification of RNA targets. When arginine fork chemical recognition principles were applied to existing structures with unusual arginine−guanine recognition, we found that the arginine fork geometry was more consistent with the experimental data, suggesting the utility of fork classifications to improve structural models. Software to analyze arginine−RNA interactions has been made available to the community.

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