Binding of a cyclic BIV beta-Tat peptide with its TAR RNA construct

The ability of RNA structures to adopt diverse yet complex tertiary structures has resulted in numerous fascinating RNA-protein recognition events. It was recently reported that a close relative of the HIV Rev peptide, namely a 17 residue Tat peptide from bovine immuno-deficiency virus (BIV), is able to bind to the 28 nucleotide BIV TAR RNA construct. Here we report that by simply converting the 17 residue beta-ribbon peptide structure to a 19 residue cyclopeptide, the binding affinity (Kd) of the resulting cyclopeptide to the TAR RNA target, observed by fluorescence binding study, was enhanced approximately 5-fold.     

Structural model of the HIV-1 Tat(46-58)-TAR complex

The trans-activator protein (Tat) of human immunodeficiency virus type 1 (HIV-1) binds to an uridine-rich bulge of an RNA target (TAR; trans-activation responsive element) predominantly via its basic sequence domain. The structure of the Tat(46-58)-TAR complex has been determined by a novel modeling approach relying on structural information about one crucial arginine residue and crosslink data. The strategy described here solely uses this experimental data without additional "modeling" assumptions about the structure of the complex in order to avoid human bias. Model building was performed in a fashion similar to structure calculations from nuclear magnetic resonance (NMR)-spectroscopic data using restrained molecular dynamics. The resulting set of structures of Tat(46-58) in its complex with TAR reveals that all models have converged to a common fold, showing a backbone root mean square deviation (RMSD) of 1.36A. Analysis of the calculated structures suggests that HIV-I Tat forms a hairpin loop in its complex with TAR that shares striking similarity to the hairpin formed by the structure of the bovine immunodeficiency virus Tat protein after TAR binding as determined by NMR studies. The outlined approach is not limited to the Tat-TAR complex modeling, but is also applicable to all molecular complexes with sufficient biochemical and biophysical data available.     

Conformational dynamics of RNA-peptide binding: a molecular dynamics simulation study

Molecular dynamics simulations of the binding of the heterochiral tripeptide KkN to the transactivation responsive (TAR) RNA of HIV-1 is presented, using an all-atom force field with explicit water. To obtain starting structures for the TAR-KkN complex, semirigid docking calculations were performed that employ an NMR structure of free TAR RNA. The molecular dynamics simulations show that the starting structures in which KkN binds to the major groove of TAR (as it is the case for the Tat-TAR complex of HIV-1) are unstable. On the other hand, the minor-groove starting structures are found to lead to several binding modes, which are stabilized by a complex interplay of stacking, hydrogen bonding, and electrostatic interactions. Although the ligand does not occupy the binding position of Tat protein, it is shown to hinder the interhelical motion of free TAR RNA. The latter is presumably necessary to achieve the conformational change of TAR RNA to bind Tat protein. Considering the time evolution of the trajectories, the binding process is found to be ligand-induced and cooperative. That is, the conformational rearrangement only occurs in the presence of the ligand and the concerted motion of the ligand and a large part of the RNA binding site is necessary to achieve the final low-energy binding state.     

Observation of an A-DNA to B-DNA transition in a nonhelical nucleic acid hairpin molecule using molecular dynamics.

One of the truly challenging problems for molecular dynamics (MD) simulations is demonstrating that the trajectories can sample not only in the vicinity of an experimentally determined structure, but also that the trajectories can find the correct experimental structure starting from some other structure. Frequently these transitions to the correct structure require that the simulations overcome energetic barriers to conformational change. Here we present unrestrained molecular dynamics simulations of the DNA analogs of the RNA 5'-GGACUUCGGUCC-3' hairpin tetraloop. In one simulation we have used deoxyuracil residues, and in the other we have used the native DNA deoxythymine residues. We demonstrate that, on a nanosecond time scale, MD is able to simulate the transitions of both of the A-DNA stems to B-DNA stems within the constraints imposed by the four-base loop that caps the helix. These results suggest that we are now in a position to use MD to address the nature of sequence-dependent structural effects in nonduplex DNA structures.     

Structural Model of the HIV-1 Tat(46–58)-TAR Complex

Abstract

The trans-activator protein (Tat) of human immunodeficiency virus type 1 (HIV-1>) binds to an uridine-rich bulge of an RNA target (TAR; trans-activation responsive element) predominantly via its basic sequence domain. The structure of the Tat(46–58)-TAR complex has been determined by a novel modeling approach relying on structural information about one crucial arginine residue and crosslink data. The strategy described here solely uses this experimental data without additional “modeling” assumptions about the structure of the complex in order to avoid human bias. Model building was performed in a fashion similar to structure calculations from nuclear magnetic resonance (NMR)-spectroscopic data using restrained molecular dynamics.

The resulting set of structures of Tat(46–58) in its complex with TAR reveals that all models have converged to a common fold, showing a backbone root mean square deviation (RMSD) of 1.36Å. Analysis of the calculated structures suggests that HIV-1 Tat forms a hairpin loop in its complex with TAR that shares striking similarity to the hairpin formed by the structure of the bovine immunodeficiency virus Tat protein after TAR binding as determined by NMR studies. The outlined approach is not limited to the Tat-TAR complex modeling, but is also applicable to all molecular complexes with sufficient biochemical and biophysical data available.     

Molecular dynamics simulations and MM–PBSA calculations of the lectin from snowdrop (Galanthus nivalis)

Galanthus nivalis agglutinin (GNA), a mannose-specific lectin from snowdrop bulbs, is a member of the monocot mannose-specific lectin family and exhibits antiviral activity toward HIV. In the present study, molecular dynamics (MD) simulations were performed to study the interaction between GNA and its carbohydrate ligand over a specific time span. By analysis of the secondary structures, it was observed that the GNA conformation maintains rather stable along the trajectories and the high fluctuations were only centered on the carbohydrate recognition domains. Our MD simulations also reproduced most of the hydrogen bonds observed in the x-ray crystal structure. Furthermore, the obtained MD trajectories were used to estimate the binding free energy of the complex using the molecular mechanics/Poisson Boltzmann surface area (MM-PBSA) method. It was revealed by the inspection of the binding free energy components that the major contributions to the complex stability arose from electrostatic interactions.     

Multivalent binding oligomers inhibit HIV Tat–TAR interaction critical for viral replication

We describe the development of a new type of scaffold to target RNA structures. Multivalent binding oligomers (MBOs) are molecules in which multiple sidechains extend from a polyamine backbone such that favorable RNA binding occurs. We have used this strategy to develop MBO-based inhibitors to prevent the association of a protein–RNA complex, Tat–TAR, that is essential for HIV replication. In vitro binding assays combined with model cell-based assays demonstrate that the optimal MBOs inhibit Tat–TAR binding at low micromolar concentrations. Antiviral studies are also consistent with the in vitro and cell-based assays. MBOs provide a framework for the development of future RNA-targeting molecules.     

Structural basis of the highly efficient trapping of the HIV Tat protein by an RNA aptamer

An RNA aptamer containing two binding sites exhibits extremely high affinity to the HIV Tat protein. We have determined the structure of the aptamer complexed with two argininamide molecules. Two adjacent U:A:U base triples were formed, which widens the major groove to make space for the two argininamide molecules. The argininamide molecules bind to the G bases through hydrogen bonds. The binding is stabilized through stacking interactions. The structure of the aptamer complexed with a Tat-derived arginine-rich peptide was also characterized. It was suggested that the aptamer structure is similar for both complexes and that the aptamer interacts with two different arginine residues of the peptide simultaneously at the two binding sites, which could explain the high affinity to Tat.     

Development of a functional backbone cyclic mimetic of the HIV-1 Tat arginine-rich motif

We have used the backbone cyclic proteinomimetics approach to develop peptides that functionally mimic the arginine-rich motif (ARM) of the HIV-1 Tat protein. This consensus sequence serves both as a nuclear localization signal (NLS) and as an RNA binding domain. Based on the NMR structure of Tat, we have designed and synthesized a backbone cyclic ARM mimetic peptide library. The peptides were screened for their ability to mediate nuclear import of the corresponding BSA conjugates in permeabilized cells. One peptide, designated "Tat11," displayed active NLS properties. Nuclear import of Tat11-BSA was found to proceed by the same distinct pathway used by the Tat-NLS and not by the common importin alpha pathway, which is used by the SV40-NLS. Most of the Tat-derived backbone cyclic peptides display selective inhibitory activity as demonstrated by the inhibition of the nuclear import mediated by the Tat-NLS and not by the SV40-NLS. The Tat-ARM-derived peptides, including Tat-11, also inhibited binding of the HIV-1 Rev-ARM to its corresponding RNA element (Rev response element) with inhibition constants of 5 nm. Here we have shown for the first time (a) a functional mimetic of a protein sequence, which activates a nuclear import receptor and (b) a mimetic of a protein sequence with a dual functionality. Tat11 is a lead compound which can potentially inhibit the HIV-1 life cycle by a dual mechanism: inhibition of nuclear import and of RNA binding.     

Correlation between biological activity and binding energy in systems of integrin with cyclic RGD-containing binders: a QM/MM molecular dynamics study

We here report a combined quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) study on the binding interactions between the α(V)β(3) integrin and eight cyclic arginine-glycine-aspartate (RGD) containing peptides. The initial conformation of each peptide within the binding site of the integrin was determined by docking the ligand to the reactive site of the integrin crystal structure with the aid of docking software FRED. The subsequent QM/MM MD simulations of the complex structures show that these eight cyclic RGD-peptides have a generally similar interaction mode with the binding site of the integrin to the cyclo(RGDf-N[M]V) analog found in the crystal structure. Still, there are subtle differences in the interactions of peptide ligands with the integrin, which contribute to the different inhibition activities. The averaged QM/MM protein-ligand interaction energy (IE) is remarkably correlated to the biological activity of the ligand. The IE, as well as a three-variable model which is somewhat interpretable, thus can be used to predict the bioactivity of a new ligand quantitatively, at least within a family of analogs. The present study establishes a helpful protocol for advancing lead compounds to potent inhibitors.     

RNA binding assays for Tat-derived peptides: implications for specificity.

RNA recognition by the HIV Tat protein is mediated in part by an arginine- and lysine-rich basic subdomain implicated as a signature element in proteins that bind RNA. Relative RNA binding affinities for a 14-residue peptide derived from Tat that spans the basic region are determined using a competition protocol. Binding specificity is compared with complexation by a 38-residue model for the RNA binding domain of Tat using the same approach. Binding strength for the minimal (14 residue) peptide is correlated with that for the longer peptide: both peptides recognize a short, bulged duplex. However, the shorter peptide dissociates more rapidly from the wild-type site and discriminates less well between nonspecific (double-stranded RNA) and specific sites. Relative dissociation constants for 38-residue peptide determined from direct partition and competition assays differ; the former assay consistently predicts stronger discrimination against RNAs with mutations in the stems flanking the bulge. Differences between the two assays are reconciled in terms of contributions from labile binding which is unstable to native gel electrophoresis. Kinetic stability may constitute a major specificity determinant for basic subdomain-mediated recognition of RNA.