Journal of Molecular Biology
Water, Shape Recognition, Salt Bridges, and Cation–Pi Interactions Differentiate Peptide Recognition of the HIV Rev-Responsive Element
Introduction
Protein–RNA recognition is essential to many fundamental biological processes and complex because RNA molecules can adopt a wide variety of secondary and tertiary structures. Among the many classes of RNA-binding motifs, arginine-rich motifs (ARMs)1 are particularly interesting as they are relatively short in length, have little sequence similarity aside from containing many arginine residues, and exhibit diverse structures.2, 3, 4, 5 For example, in bovine immunodeficiency virus Tat, human immunodeficiency virus (HIV) Rev, bacteriophage λ N, HIV Tat, and human T cell leukemia virus type 1 Rex peptides, the RNA-binding peptides adopt a variety of different structures upon association with the RNA target,6, 7 including a β-hairpin,8 an α-helix,9, 10 a distorted α-helix,11 an extended conformation,12 and an S-shaped conformation,13 respectively.
In HIV, the Rev protein recognizes a sequence of RNA known as the Rev-responsive element (RRE).14, 15, 16 Binding of Rev to RRE is an essential step in the HIV life cycle and regulates nuclear export of unspliced and partially spliced RNAs17, 18 via the CRM1 export pathway.19, 20 The RRE is a 351-nucleotide sequence found in the env gene21 of the HIV genome and is found in all incompletely spliced HIV mRNA transcripts.22, 23 The full-length Rev protein is composed of 116 amino acids24 and binds a high-affinity binding site as a monomer followed by cooperative, sequential binding of Rev monomers to lower-affinity sites on RRE stem I.14, 16, 23, 25, 26, 27, 28 A minimal Rev–RRE complex has been localized to a small arginine-rich peptide15, 25, 29 and stem IIB of RRE.14, 16, 30 Critical residues have been determined (Fig. 1) by a combination of in vitro selection,31 chemical modification,32, 34 and mutagenesis studies.10, 29, 33 Biochemical assays indicate that Rev residues Thr34, Asn40 and four arginine residues (Arg35, Arg38, Arg39, and Arg44) are required for specific Rev–RRE binding.10 In the RNA, two essential major groove purine–purine base pairs, G47-A73 and G48-G71,26, 31, 40, 35 are separated by the conformationally flexible U72.
A number of structural studies have characterized the minimal Rev–RRE complex. CD spectroscopy10, 33 and an NMR study of the Rev peptide employing selective 15N-labeling41 indicate that the Rev peptide forms an α-helix upon complexation with the RRE. The complete solution structure of the Rev–RRE complex9 reveals that the Rev peptide binds as an α-helix to a widened RRE major groove formed by the G48-G71 and G47-A73 purine–purine base pairs. Due to the dynamic nature of the arginine side chains and limitations of refinement techniques, some of the peptide–RNA interactions were not resolved. However, the NMR structure9 does indicate that Rev binds to RRE, employing a combination of purine–purine base pair shape recognition,31 base-specific hydrogen bonding, and some van der Waals interactions. Structural studies have revealed that the RNA also adapts upon binding the Rev peptide. NMR studies of RRE unbound42 indicate that the RNA undergoes a conformational change, in which G71 rotates from a syn to an anti conformation. Comparison of the x-ray crystal structure of the RRE alone43, 44 to the Rev–RRE complex shows that the RNA the major groove widens by more than 5 Å, transitioning from a slightly more narrow than A-form RNA major groove width to a much wider conformation upon complexation.
A synthetic arginine-rich peptide developed from in vitro selection studies, known as RSG 1.2, binds to RRE IIB RNA with 7-fold higher affinity and 15-fold greater specificity than the Rev peptide.36, 37 The synthetic peptide RSG 1.2 completely displaces the native Rev peptide from RRE in competition assays and forms a partial α-helix in complex with RRE.37 A high-resolution NMR structure of RSG 1.2 bound to an RRE construct of stem IIB (RRE IIB) has been solved by NMR.38 The RRE IIB construct, previously employed in Rev–RRE NMR studies,42 only differs from RRE by a change in the stabilizing tetraloop sequence, from GCAA to UUCG, and the deletion of the G53-C65 base pair, which is not required for peptide recognition.40 In the RSG 1.2–RRE IIB NMR structure, RSG 1.2 binds to a widened major groove of RRE similar to the Rev peptide, including binding to the noncanonical G47-A73 and G48-G71 base pairs. The U72 base is turned into the major groove and appears to interact with Arg15, whereas in the Rev–RRE structure, it is rotated out into solution. While this difference leads to little change in the RRE backbone relative to the Rev–RRE complex, in the RSG 1.2 complex with RRE IIB, a deep major groove pocket is established in the RRE, allowing for intimate contact between RSG 1.2 amino acid side chains and the RNA.
Molecular dynamics (MD) simulations can provide atomic-level detail of biomolecules, specifically providing insight into intermolecular interactions and structure.45, 46, 47, 48, 49 Time-dependent structural attributes and dynamic processes such as water and ion binding, hydrogen bonding, and intermolecular interactions can all be examined through MD simulations.50, 51, 52, 53, 54, 55 In other systems, computational studies have also led to a better understanding of the protein–RNA interface47, 56 and contributions to binding free energies.57, 58 When considered in concert with experimental results, computations can be invaluable in both explaining and predicting biological phenomena.
Here, MD studies of the two peptide–RNA complexes are presented, providing a more detailed explanation for the enhanced binding affinity of RSG 1.2 for RRE over the Rev peptide. The role of water, electrostatic stabilization, and shape recognition explains how two very similar peptides recognize the same sequence of RNA in dramatically different manners.
Section snippets
Simulation stability
MD simulations of the Rev and RSG 1.2 peptides bound to the RRE and RRE IIB sequences, respectively, were simulated in explicit solvent according to procedures outlined in Methodology. In order to assess the stability and convergence of the simulation, we monitored the root-mean-square (RMS) deviation from average and starting structures (Supplementary Material). As well, helical parameters59, 60 of the RNA stem regions were also monitored to confirm values fluctuated around constant values.
Discussion
Analysis of hydrogen-bonding patterns in the Rev–RRE complex indicates that while Rev does interact with the RRE through some base-specific contacts, the majority of the interactions occur through amino acid side-chain contacts to the sugar-phosphate backbone. A similar but not identical distribution of hydrogen bonds is also observed in Battiste et al., with 11 of the phosphates interacting with Rev side chains and only 6 interactions occurring directly between the peptide and the bases. Taken
Construction of systems
To better understand peptide–RNA recognition, we performed simulations of Rev–RRE and RSG 1.2–RRE IIB complexes (Table 1). The 15N and 13C NMR studies of Rev–RRE indicated appreciable side-chain dynamics.9 For this reason, three lowest-energy structures, as calculated with a generalized Born model 88, 89, 90 in the presence of a dielectric continuum with an ɛ of 78.3, from Battiste et al. [Protein Data Bank (PDB) ID: 1ETG]9 were initially examined (Supplementary Material), from which model 8
Acknowledgements
The authors would like to thank C. Simmerling for critical reading of the manuscript. This material is based upon work supported by the National Science Foundation under Grant Nos. CHE-0521063, CHE-0821581, DUE-0431664, and TG-CHE070021N. Additional support was provided by Research Corporation (CC6553).
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