Insights into the recruitment of class IIa Histone Deacetylases (HDACs) to the SMRT/NCoR transcriptional repression complex

Class IIa histone deacetylases repress transcription of target genes. However their mechanism of action is poorly understood since they exhibit very low levels of deacetylase activity. The class IIa HDACs are associated with the SMRT / NCoR re pression complexes and this may, at least in part, a ccount for their repressive activity. However, the molecular mechanism of recruitment to co - repressor proteins has yet to be established. Here we show that a repeated peptide motif present in both SMRT and NCoR is sufficient to mediate specific interaction , with micromolar affinity, with all the class IIa HDACs (HDACs 4, 5, 7 & 9). Mutations in the consensus motif abrogate binding. Mutational analysis of HDAC4 suggests that the peptide interacts in the vicinity of the active site of the enzyme and requires the “closed” conformation of the zinc - binding loop on the surface of the enzyme. Together these findings represent the first insights into the molecular mechanism of recruitment of class IIa HDACs to the SMRT / NCoR repression complexes

Urotensin-II Peptidomimetic Incorporating a Non-Reducible 1,5-Triazole Disulfide Bond Reveals a Pseudo-Irreversible Covalent Binding Mechanism to the Urotensin G-Protein Coupled Receptor

The urotensin-II receptor (UTR) is a class A GPCR that predominantly binds to the pleiotropic cyclic peptide urotensin-II (U-II). U-II is constrained by a disulfide bridge that induces a β-turn structure and binds pseudo-irreversibly to UTR and is believed to result in a structural rearrangement of the receptor. However, it is not well understood how U-II binds pseudo-irreversibly and the nature of the reorganization of the receptor that results in G-protein activation. Here we describe a series of U-II peptidomimetics incorporating a non-reducible disulfide bond structural surrogate to investigate the feasibility that native U-II binds to the G protein-coupled receptor through disulfide bond shuffling as a mechanism of covalent interaction. Disubstituted 1,2,3-triazoles were designed with the aid of computational modeling as a non-reducible mimic of the disulfide bridge (Cys5–Cys10) in U-II. Solid phase synthesis using CuAAC or RuAAC as the key macrocyclisation step provided four analogues of U-II(4–11) incorporating either a 1,5-triazole bridge (5, 6) or 1,4-triazole bridge (9, 10). Biological evaluation of compounds 5, 6, 9 and 10 was achieved using in vitro [125I]UII binding and [Ca2+]i assays at recombinant human UTR. Compounds 5 and 6 demonstrated high affinity (KD ∼ 10 nM) for the UTR and were also shown to bind reversibly as predicted and activate the UTR to increase [Ca2+]i. Importantly, our results provide new insight into the mechanism of covalent binding of U-II with the UTR

Binding requirements of the SMRT peptide explored utilising artificial amino acids to replace histidine.

<p>(A) Binding curves obtained by fluorescence polarisation for wild-type peptide and 1-napthyl, 2-napthyl, styryl and homophenylalanine substitutions of histidine1426 of SMRT. The K_d in µM (mean±SEM) is presented to the right of the compound name. (B) Structure of wild-type SMRT peptide bound to the BCL6-POZ domain. Molecular modelling of (C) 1-naphthyl SMRT peptide and (D) 2-naphthyl SMRT peptide.</p

BCL6-POZ domain in complex with rifabutin, 79–6 and the SMRT peptide.

<p>A section of the BCL6-POZ domain structure is shown binding; (A) rifabutin, (B) the SMRT peptide and (C) 79–6.</p

A natural product screen to identify novel inhibitors of BCL6 transcriptional repression.

<p>(A) Schematic of BCL6 showing amino-terminal POZ domain (red), carboxy terminal zinc fingers (yellow) and mid portion containing PEST domains (blue). Different proteins associate with the three portions of BCL6. NCoR, BCoR and SMRT associate with the POZ domain, MTA3 and NuRD with the mid portion and ETO1 with the zinc fingers. (B) Illustration of the screening strategy. BCL6 (green) is shown associating with its binding site cloned upstream of a luciferase reporter gene. Without any compound, or with an inactive compound i.e. one that does not bind BCL6, luciferase output is repressed but in the presence of active compound BCL6 mediated repression is prevented and output of luciferase increases. (C) Screening results for half a plate (40 compounds) from the natural product library. The black bar (furthest left) is the mean negative control i.e. transfected cells without test compound, and the black horizontal line the mean value across the entire screen. The red bar shows rifamycin SV. (D) The effect of rifamycin is due to inhibition of BCL6 transcriptional repression. HEK293T cells were co-transfected with a BCL6 expression construct and a luciferase reporter. Transcriptional repression due to BCL6 was relieved by rifamycin SV (R), but not by an agent that was ineffective in the screen (D).</p

Crystal structure of rifabutin and BCL6-POZ domain.

<p>Electron density corresponding to rifabutin, following refinement, in the context of surrounding electron density demonstrating the proximity of (A) tyrosine58 from one monomer of the POZ dimer and (B) asparagine21 and arginine24 from the other monomer. (C) Surface representation of BCL6-POZ with basic residues (including asparagine21 and arginine24) in blue and acidic residues in red. The napthoquinone rings of rifabutin are in proximity to tyrosine58 whilst the aliphatic bridge is adjacent to the basic surface.</p

Rifamycin SV and its derivative, rifabutin, bind directly to the BCL6-POZ domain.

<p>Schematic diagram to compare the structures of (A) rifamycin SV and (C) rifabutin. These two compounds differ with respect to the side chains on C-3 and C-4. (B) and (D) TROSY ¹H,¹⁵N HSQC spectra of 280 μM BCL6-POZ domain. (B) Chemical shift changes due to rifamycin SV. An overlay of the spectra of BCL6-POZ domain alone (green) and in the presence of a 16∶1 molar ratio of rifamycin (purple). (D) Chemical shift changes due to rifabutin with an overlay of the spectra of BCL6-POZ domain alone (green) and in the presence of 4∶1 (light blue), 8∶1 (red) and 16∶1 (purple) molar ratios of rifabutin.</p

A heme-binding domain controls regulation of ATP-dependent potassium channels

Heme iron has many and varied roles in biology. Most commonly it binds as a prosthetic group to proteins, and it has been widely supposed and amply demonstrated that subtle variations in the protein structure around the heme, including the heme ligands, are used to control the reactivity of the metal ion. However, the role of heme in biology now appears to also include a regulatory responsibility in the cell; this includes regulation of ion channel function. In this work, we show that cardiac KATP channels are regulated by heme. We identify a cytoplasmic heme-binding CXXHX16H motif on the sulphonylurea receptor subunit of the channel, and mutagenesis together with quantitative and spectroscopic analyses of heme-binding and single channel experiments identified Cys628 and His648 as important for heme binding. We discuss the wider implications of these findings and we use the information to present hypotheses for mechanisms of heme-dependent regulation across other ion channels