Co-crystal structures of HIV TAR RNA bound to lab-evolved proteins show key roles for arginine relevant to the design of cyclic peptide TAR inhibitors

Abstract

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.

Macrocyclization strategies for cyclic peptides and peptidomimetics

“…of NEt 3 in H 2 O/THF at room temperature over several hours. Encouraging results were obtained when this methodology was applied to insulin, 85 the 58-residue protein bovine pancreatic trypsin inhibitor, 86 adrenomedullin analogues, 87 peptide mimetics binding to the HIV trans -activation response RNA, 88 and in the chemical synthesis of the protein interleukin-2. 89 …”

Affinity and Structural Analysis of the U1A RNA Recognition Motif with Engineered Methionines to Improve Experimental Phasing

“…The main strategy has been documented. TAR RNA in complex with TAR binding protein 6.9 6xh0 MR g n/a [52] a Single-wavelength anomalous diffraction. b Multiwavelength anomalous diffraction.…”

“…(Right) Cartoon drawing of the double-mutant protein (dm)U1A-hpII 21-mer co-crystal structure [56], emphasizing protein recognition of the hpII loop (PDB entry 1urn); the duplex region (white) is not involved in hpII binding and can be replaced by a target sequence to promote RNA crystallization. (b) (Left) Lab-evolved TBP6.9 recognizes TAR RNA at the internal bulged loop, which includes a U23 A27-U40 base triple and bulged A35 within helical stem S1b [51,52]. An arginine fork is a key determinant of recognition at the G26 major-groove edge and the U23 backbone [59].…”

Statistical and thermodynamic analysis of the binding of trans-activation response–binding proteins to HIV-1 TAR RNA

“…In a recent article ( 1 ), the authors examine the binding of lab-evolved trans-activation response (TAR)–binding proteins (TBPs) to HIV-1 TAR RNA. Here, we show our analysis of the thermodynamic data of the binding that identifies three quantitative features of the binding, which may provide further insight into the interactions.…”

“…This suggests that the rest structure of TBPs other than the TAR-binding β2-β3 loop, which is absent in the two outliers, contributes to the binding. The Δ G ° values of the six TBPs at 310.15 K calculated using Equation 1 are normally distributed, according to the Shapiro–Wilk normality test (W = 0.933, p = 0.607): where Δ H ° and Δ S ° are experimentally measured values reported in the original paper ( 1 ). Based on the statistical parameters, probability density of the Δ G ° can be generated using Equation 2 : where σ = 1.9407 kJ/mol and Δ G ° mean = –41.3 kJ/mol.…”