Molecular modeling of the HIV-1 tat protein - TAR RNA complex and the design of an inhibitor of the Tat/TAR RNA interaction

HIV-1 Tat protein (trans-acting transcriptional activator) regulates transc ription in the virus upon binding to the transactivating responsive (TAR) R NA sequence, which is located in the HIV-1 long terminal repeat. Tat is a s mall protein of 86 residues. Mutation data, binding studies and the use of chemical probes have shown that specific binding occurs between the region centered on a U-rich bulge of TAR RNA and the arginine rich basic region ( residues 49-57) of Tat. The fact that Tat protein plays a critical role in HIV-1 replication suggests that small compounds that disrupt the Tat - TAR interaction will inhibit viral replication. In order to design such a compo und, the first step is to model the Tat- TAR complex. The NMR structure of a 29-mer of TAR RNA that contains the minimal RNA regi on recognised by the Tar protein is known. However, the structure of the ba sic region of Tat is not well established. We have, therefore carried out a conformational analysis of the 9-residue long basic region that has the se quence RKKRRQRRR. The results indicate that this region has an extended str ucture. This result is consistent with CD spectroscopic data. We have also carried out a conformational analysis of six synthetic 9-residue peptides w hose transactivation abilities have been measured. A superposition of the e nergy minimized structures of the synthetic peptides with the 9 - residue T ar peptide shows that the r.m.s. deviations of the residues located in the positions 5-8 of the highly active peptides from the corresponding region i n Tar are about 0.8 Angstrom, whereas the r.m.s. deviations are higher (abo ut 1.5 Angstrom) in the case of the peptides with low activities. Thus the conformation of the tetrapeptides is found to correlate well with activity data. This conclusion is supported by molecular modelling of the complex of the 9-residue Tat with the averaged NMR structure of the 29-mer TAR RNA. I t is observed that the maximum number of interactions of the Tat peptide wi th the recognition site on TAR are with residues 5-8 (RQRR) of the Tat pept ide. Based on these observations and using the folding rules for dehydro-alanine (Delta -Ala) derived from our earlier studies, we have designed a tetrapep tide with a sequence Arg- Delta Ala - Delta Ala Arg. Energy minimization an d molecular modelling of the interaction of the designed tetrapeptide with the TAR recogniton site indicate that the tetrapeptide not only has interac tions with the recognition site on TAR but also has interactions with addit ional bases of TAR in the upper stem region as compared to those with Tat. We therefore expect the designed peptide to be an effective inhibitor of th e Tat/TAR RNA interaction.