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How binding of small molecule and peptide ligands to HIV-1 TAR alters the RNA motional landscape.

ABSTRACT: The HIV-1 TAR RNA represents a well-known paradigm to study the role of dynamics and conformational change in RNA function. This regulatory RNA changes conformation in response to binding of Tat protein and of a variety of peptidic and small molecule ligands, indicating that its conformational flexibility and intrinsic dynamics play important roles in molecular recognition. We have used (13)C NMR relaxation experiments to examine changes in the motional landscape of HIV-1 TAR in the presence of three ligands of different affinity and specificity. The ligands are argininamide, a linear peptide mimic of the Tat basic domain and a cyclic peptide that potently inhibits Tat-dependent activation of transcription. All three molecules induce the same motional characteristics within the three nucleotides bulge that represents the Tat-binding site. However, the cyclic peptide has a unique motional signature in the apical loop, which represents a binding site for the essential host co-factor cyclin T1. These results suggest that all peptidic mimics of Tat induce the same dynamics in TAR within this protein binding site. However, the new cyclic peptide mimic of Tat represents a new class of ligands with a unique effect on the dynamics and the structure of the apical loop.

SUBMITTER: Bardaro MF

PROVIDER: S-EPMC2655691 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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How binding of small molecule and peptide ligands to HIV-1 TAR alters the RNA motional landscape.

Bardaro Michael F MF Shajani Zahra Z Patora-Komisarska Krystyna K Robinson John A JA Varani Gabriele G

Nucleic acids research 20090112 5

The HIV-1 TAR RNA represents a well-known paradigm to study the role of dynamics and conformational change in RNA function. This regulatory RNA changes conformation in response to binding of Tat protein and of a variety of peptidic and small molecule ligands, indicating that its conformational flexibility and intrinsic dynamics play important roles in molecular recognition. We have used (13)C NMR relaxation experiments to examine changes in the motional landscape of HIV-1 TAR in the presence of ...[more]

PMID: 19139066

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Project description:The regulator of expression of virion (Rev) protein binds specifically to the Rev-responsive element (RRE) RNA in order to regulate the expression of the human immunodeficiency virus (HIV)-1 genes. Fluorescence indicator displacement assays have been used to identify ligands that can inhibit the Rev–RRE interaction; however, the small fluorescence indicators cannot fully replace the Rev peptide or protein. As a result, a single rhodamine B labeled Rev (RB-Rev) model peptide was utilized in this study to develop a direct and efficient Rev–RRE inhibitor screening model. Due to photon-induced electron transfer quenching of the tryptophan residue on the RB fluorophore, the fluorescence of RB in Rev was weakened and could be dramatically reactivated by interaction with RRE RNA in ammonium acetate buffer (approximately six times). The interaction could reduce the electron transfer between tryptophan and RB, and RRE could also increase RB fluorescence. The inhibitor screening model was evaluated using three known positive Rev–RRE inhibitors, namely, proflavin, 6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine (ICR 191), and neomycin, as well as a negative drug, arginine. With the addition of the positive drugs, the fluorescence of the Rev–RRE decreased, indicating the displacement of RB-Rev. This was confirmed using atomic force microscopy (AFM) and the fluorescence was essentially unaffected by the addition of arginine. The results demonstrated that RB-Rev can be used as a fluorescent probe for recognizing small ligands that target RRE RNA. The Rev–RRE inhibitor screening model offers a novel approach to evaluating and identifying long-acting Rev inhibitors. Graphical abstract Image 1 Highlights • Fluorescence enhancement of RB-Rev after interaction with RRE was discovered.• RB-Rev was used as a fluorescent probe for recognizing small ligands that target RRE RNA.• A direct fluorescence “turn on” Rev–RRE inhibitor screening model was developed.

Dataset Information

How binding of small molecule and peptide ligands to HIV-1 TAR alters the RNA motional landscape.

Publications

How binding of small molecule and peptide ligands to HIV-1 TAR alters the RNA motional landscape.

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