Eukaryotic transcriptional repressors function by recruiting large co-regulatory complexes that target histone deacetylase enzymes to gene promoters/enhancers. Transcriptional repression complexes, assembled by the co-repressor NCoR, and its homologue SMRT, play critical roles in many processes including development and metabolic physiology. The core repression complex involves the recruitment of three proteins: HDAC3, GPS2 and TBL1 to a highly conserved repression domain within SMRT and NCoR. We have used a variety of structural and functional approaches to gain insight into the assembly, stoichiometry and biological role of this complex. We report the crystal structure of the tetrameric oligomerization domain of TBL1, which interacts with both SMRT and GPS2, and the NMR structure of the interface complex between GPS2 and SMRT. These structures, together with computational docking, mutagenesis and functional assays, reveal the assembly mechanism and stoichiometry of the co-repressor complex.The regulated repression of transcription plays a key role in many biological processes. These include cell fate decisions during development and cellular differentiation, as well as the maintenance of homeostasis. SMRT and NCoR are large homologous co-repressor proteins that were identified through their role in transcriptional repression by many 5 Corresponding author: john.schwabe@le.ac.uk. 4 These authors should be considered co-first authors. Author ContributionsThe contributions of J.O., L.F. & P.W. were critical to the final manuscript and these authors should be considered co-first authors. J.O. (assisted by J.Gooch) performed most of the protein cloning, expression, purification and interaction mapping, although important preliminary experiments were performed by B.K. J.O. prepared the GPS2-SMRT complex for NMR structure determination which was carried out by J-C.Y. and D.N. Crystallizations were performed primarily by J.O., with some later trials by J.Greenwood and L.F. Crystal structure determinations were performed by L.F., J.O. and J.W.R.S. The interaction motifs in GPS2 and SMRT were identified by J.W.R.S. and tested by pull-down experiments by J.O. Fluorescence polarization, co-immunoprecipitation and co-transfection/ purification assays were performed by P.W. The two-hybrid assays, gel filtrations and NMR comparisons of WT and MT TBL1 were performed by P.W., Z.C. and B.G. In silico docking experiments were performed by T.K. The laboratories of L.N. and D.N. provided experimental expertise for transfection and NMR studies respectively. J.W.R.S. planned and supervised the project and prepared the manuscript with assistance from the other authors. Accession Codes1H, 13C and 15N NMR resonance assignments for the SMRT(167-207) -GPS2(53-90) complex have been deposited at the BioMagResBank under accession code 17271, and the coordinates have been deposited under the pdb accession code 215G. The TBL1 X-ray structures have been deposited with the PDB codes (2XTC, 2XTD & 2XTE). When purified from HeLa cell extr...

A central event in the invasion of a host cell by an enveloped virus is the fusion of viral and cell membranes. For many viruses, membrane fusion is driven by specific viral surface proteins that undergo large-scale conformational rearrangements, triggered by exposure to low pH in the endosome upon internalization. Here, we present evidence suggesting that in both class I (helical hairpin proteins) and class II (beta-structure-rich proteins) pH-dependent fusion proteins the protonation of specific histidine residues triggers fusion via an analogous molecular mechanism. These histidines are located in the vicinity of positively charged residues in the prefusion conformation, and they subsequently form salt bridges with negatively charged residues in the postfusion conformation. The molecular surfaces involved in the corresponding structural rearrangements leading to fusion are highly conserved and thus might provide a suitable common target for the design of antivirals, which could be active against a diverse range of pathogenic viruses.

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Dengue and related flaviviruses represent a significant global health threat. The envelope glycoprotein E mediates virus attachment to a host cell and the subsequent fusion of viral and host cell membranes. The fusion process is driven by conformational changes in the E protein and is an essential step in the virus life cycle. In this study, we analyzed the pre-fusion and post-fusion structures of the dengue virus E protein to identify potential novel sites that could bind small molecules, which could interfere with the conformational transitions that mediate the fusion process. We used an in silico virtual screening approach combining three different docking algorithms (DOCK, GOLD and FlexX) to identify compounds that are likely to bind to these sites. Seven structurally diverse molecules were selected to test experimentally for inhibition of dengue virus propagation. The best compound showed an IC(50) in the micromolar range against dengue virus type 2.