5 research outputs found
Active Site Similarity between PrgI and Bcl-xL.
<p>(A) CPASS alignment of the <i>S. typhimurium</i> PrgI active-site complexed to DDAB with the active-site of human Bcl-2 protein (Bcl-xL) complexed with acyl-sulfonamide-based inhibitor. The residues aligned by CPASS are labeled and colored blue in the structures. The active site sequence alignment is also shown below the structures. The ligands are colored yellow. (B) Overlay of the human Bcl-2 protein (red) with <i>S. typhimurium</i> PrgI (turquoise) based on a DaliLite <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007442#pone.0007442-Holm1" target="_blank">[29]</a> alignment. (C) Multiple-sequence alignment of the three known T3SS structures of <i>S. typhimurium</i> PrgI, <i>B. pseudomallei</i> BsaL, and <i>S. flexneri</i> MxiH with the human Bcl-2 protein (Bcl-xL). The reliability of the each amino acid alignment is color-coded from blue (poor) to red (good) using the CORE index <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007442#pone.0007442-Notredame1" target="_blank">[35]</a>. The consensus alignment received a score of 69, where a perfect alignment receives a score of 100.</p
Verification that the Bcl-xL inhibitor chelerythrine also binds PrgI.
<p>(A). Expanded overlay of the 2D <sup>15</sup>N-<sup>1</sup>H HSQC spectra for free PrgI (black) and PrgI bound to chelerythrine (blue). CSPs greater than one standard deviation are boxed. (B) An AutoDock/ADF docked structure of PrgI complexed with chelerythrine based on the observed CSPs from (A). (C) The Bcl-xL region shown to bind chelerythrine is highlighted while the remaining protein structure is transparent. Chelerythrine is colored yellow and is drawn with licorice bonds. Side-chains for Y173 and V135 are shown as licorice bonds and colored grey. (D) A ribbon diagram of the AutoDock/ADF docked PrgI-chelerythrine co-structure. The PrgI-chelerythrine binding region that overlaps with Bcl-xL is highlighted. Chelerythrine is colored yellow and is drawn with licorice bonds. Side-chains for Y57 and K15 are shown as licorice bonds and colored grey. (E) An expanded view of the overlay of Bcl-xL (red) with PrgI (blue) illustrating the structural similarity of the chelerythrine binding sites.</p
The two PrgI ligand binding sites identified using FAST-NMR.
<p>The two PrgI ligand binding sites are highlighted on an electrostatic potential surface (blue positive charge, red negative charge) calculated with DelPhi <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007442#pone.0007442-Nicholls1" target="_blank">[79]</a> implemented in Chimera <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007442#pone.0007442-Pettersen1" target="_blank">[80]</a>. The didecyldimethylammonium bromide binding site (A) is found in a region responsible for needle formation while the chelerythrine binding site (B) is found on the opposite face.</p
Identification of PrgI Binding Ligands.
<p>(A) DDAB NMR spectra in the absence (<i>top</i>) and presence (<i>bottom</i>) of PrgI illustrating changes in NMR intensities (boxed) upon binding PrgI. Both free and bound 1D <sup>1</sup>H NMR spectra were normalized to a constant DMSO signal intensity. (B) Expanded view of the superimposed 2D <sup>15</sup>N-<sup>1</sup>H HSQC spectra of the free and DDAB bound PrgI NMR samples. Residues that incur a chemical shift perturbation are boxed. (C) Expanded view of PrgI surface rendered in VMD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007442#pone.0007442-Humphrey1" target="_blank">[78]</a> where residues that incur a chemical shift change are colored blue and DDAB is colored yellow. Co-structure based on NMR determined ligand binding site using AutoDock <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007442#pone.0007442-Morris1" target="_blank">[27]</a> and our AutoDockFilter program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007442#pone.0007442-Stark1" target="_blank">[24]</a>.</p
Development of Small Molecules with a Noncanonical Binding Mode to HIV‑1 Trans Activation Response (TAR) RNA
Small molecules that
bind to RNA potently and specifically are
relatively rare. The study of molecules that bind to the HIV-1 transactivation
response (TAR) hairpin, a cis-acting HIV genomic element, has long
been an important model system for the chemistry of targeting RNA.
Here we report the synthesis, biochemical, and structural evaluation
of a series of molecules that bind to HIV-1 TAR RNA. A promising analogue, <b>15</b>, retained the TAR binding affinity of the initial hit and
displaced a Tat-derived peptide with an IC<sub>50</sub> of 40 μM.
NMR characterization of a soluble analogue, <b>2</b>, revealed
a noncanonical binding mode for this class of compounds. Finally,
evaluation of <b>2</b> and <b>15</b> by selective 2′-hydroxyl
acylation analyzed by primer extension (SHAPE) indicates specificity
in binding to TAR within the context of an in vitro-synthesized 365-nt
HIV-1 5′-untranslated region (UTR). Thus, these compounds exhibit
a novel and specific mode of interaction with TAR, providing important
suggestions for RNA ligand design