The crystal structure of the Rev binding element of HIV-1 reveals novel base pairing and conformational variability

Abstract

The crystal and molecular structure of an RNA duplex corresponding to the high affinity Rev protein binding element (RBE) has been determined at 2.1-Å resolution. Four unique duplexes are present in the crystal, comprising two structural variants. In each duplex, the RNA double helix consists of an annealed 12-mer and 14-mer that form an asymmetric internal loop consisting of G-G and G-A noncanonical base pairs and a flipped-out uridine. The 12-mer strand has an A-form conformation, whereas the 14-mer strand is distorted to accommodate the bulges and noncanonical base pairing. In contrast to the NMR model of the unbound RBE, an asymmetric G-G pair with N2-N7 and N1-O6 hydrogen bonding, is formed in each helix. The G-A base pairing agrees with the NMR structure in one structural variant, but forms a novel watermediated pair in the other. A backbone flip and reorientation of the G-G base pair is required to assume the RBE conformation present in the NMR model of the complex between the RBE and the Rev peptide.T he Rev response element (RRE) is a 244-nt region in the env gene of HIV-1 that mediates transport of viral mRNA from the nucleus to the cytoplasm. Initially, the Rev protein binds with high affinity and specificity to a highly structured 30-residue region of the stem-loop IIB domain often termed the Rev binding element (RBE). The RBE-Rev affinity has been determined to be approximately 10 Ϫ11 M. Subsequently, the fulllength RRE appears to bind four Rev monomers (1) through protein-protein and protein-RNA interactions. Expression of the late viral mRNAs and their protein products depends on Rev-RRE mediated transport; therefore this interaction is critical to the life cycle of HIV-1. Several ligands have been identified (2, 3) that bind tightly to the RBE and compete with the Rev protein. It is expected that interference in the Rev-RBE interaction may disrupt HIV-1 replication. Thus, the RBE is an attractive target for structure-based drug design.Several NMR studies have examined the solution structure of the RBE, both in the ''free'' form (4, 5) and in complex with the Rev peptide (5-7). These structural studies have been supplemented by a molecular dynamics simulation of the RBE (8) and combined NMR-molecular dynamics studies of a complex of the Rev peptide with an RNA aptamer selected for high-affinity binding (9). The studies agree that the RBE internal loop forms G-A and G-G noncanonical base pairs separated by a uridine that loops out from the continuous helix into solution. These noncanonical pairs open up the RNA major groove to present a unique binding site for the Rev peptide or protein, which in turn induces a conformational change in the RBE backbone on binding. Materials and MethodsDesign, Synthesis, Purification, and Crystallization. Although the high-affinity RBE originally was identified as a stem-loop structure, the binding later was observed to arise from interaction of the Rev protein with the asymmetric internal loop region of the stem. Because earlier crystallographic studies had indicat...

Noncanonical G(syn)–G(anti) base pairs stabilized by sulphate anions in two X-ray structures of the (GUGGUCUGAUGAGGCC) RNA duplex

“…The G(syn)-G(anti) base pair has been observed in RNA in only a few cases: in the 16S rRNA of the Escherichia coli ribosome (Schuwirth et al 2005), in the HIV Rev response element hairpin (Ippolito and Steitz 2000) and its duplex model (Hung et al 2000), and in synthetic RNA duplexes studied by X-ray (Timsit and Bombard 2007) and by NMR methods (Burkard and Turner 2000). Despite being embedded in different sequence and structural contexts, the above structures share similar G-G base-pair geometry, which includes G(carbonyl)-G(N1) and G(N7)-G(exoamino) hydrogen bonds.…”

Fluorescence‐based methods for evaluating the RNA affinity and specificity of HIV‐1 Rev–RRE inhibitors

“…Indeed, the RRE is a highly dynamic molecule that can adopt alternate conformations upon ligand binding. 51 The abil-…”

RNA structural motifs: building blocks of a modular biomolecule

“…Thus, these sequences may be broadly distributed phylogenetically and may even have several variants within a genome, but they do not serve general architectural roles in RNA folding and tertiary structure stabilization and do not interact with many different types of ligands. Examples of specific recognition motifs are TAR (Richter et al 2002) and RBE (RRE) (Hung et al 2000;Lesnik et al 2002) motifs of HIV-1 and the IRE motifs found in mRNAs related to iron metabolism (Theil, 2000 ;Pantopoulos, 2004). Many riboregulators, riboswitches, and aptamers form potentially highly structured regions for protein or small molecule interaction, and mutations in these structures may result in a loss of regulation and a corresponding disease state (Sobczak & Krzyzosiak, 2002 ;Wong et al 2005).…”