Showing papers by "Unit of Virus Host Cell Interactions published in 2017"
TL;DR: Structural basis for blocking PD-1-mediated immune suppression by therapeutic antibody pembrolizumab is found and this work provides a new perspective on the development of antibody-drug conjugate conjugates for immune suppression.
Abstract: Structural basis for blocking PD-1-mediated immune suppression by therapeutic antibody pembrolizumab
97 citations
TL;DR: Fluorescence polarization assays are developed to quantify initiation of cap-primed or unprimed RNA synthesis by recombinant influenza B polymerase bound to the vRNA or cRNA promoter and it is found that initiation at the c RNA promoter is unusually susceptible to inhibition by 2΄F-2΄dNTPs.
Abstract: Influenza polymerase replicates, via a complementary RNA intermediate (cRNA), and transcribes the eight viral RNA (vRNA) genome segments. To initiate RNA synthesis it is bound to the conserved 5΄ and 3΄ extremities of the vRNA or cRNA (the 'promoter'). 5΄-3΄ base-pairing in the distal promoter region is essential to position the template RNA at the polymerase active site, as shown by a new crystal structure with the 3΄ end threading through the template entry tunnel. We develop fluorescence polarization assays to quantify initiation of cap-primed (transcription) or unprimed (replication) RNA synthesis by recombinant influenza B polymerase bound to the vRNA or cRNA promoter. The rate-limiting step is formation of a primed initiation complex with minimally ApG required to stabilize the 3΄ end of the template within the active-site. Polymerase bound to the vRNA promoter initiates RNA synthesis terminally, while the cRNA promoter directs internal initiation at a significantly lower rate. Progression to elongation requires breaking the promoter 5΄-3΄ base-pairing region and favourable compensation by the emerging template-product base-pairs. The RNA synthesis assay is adaptable to high-throughput screening for polymerase inhibitors. In a pilot study, we find that initiation at the cRNA promoter is unusually susceptible to inhibition by 2΄F-2΄dNTPs.
55 citations
TL;DR: It is shown that HDAC7, via its surface zinc ion binding site, binds to a 28 residue stretch in SMRT comprising a GSI motif followed by an alpha helix, which is characterized as intrinsically disordered by NMR spectroscopy.
Abstract: The 2525 amino acid SMRT corepressor is an intrinsically disordered hub protein responsible for binding and coordinating the activities of multiple transcription factors and chromatin modifying enzymes. Here we have studied its interaction with HDAC7, a class IIa deacetylase that interacts with the corepressor complex together with the highly active class I deacetylase HDAC3. The binding site of class IIa deacetylases was previously mapped to an approximate 500 amino acid region of SMRT, with recent implication of short glycine-serine-isoleucine (GSI) containing motifs. In order to characterize the interaction in detail, we applied a random library screening approach within this region and obtained a range of stable, soluble SMRT fragments. In agreement with an absence of predicted structural domains, these were characterized as intrinsically disordered by NMR spectroscopy. We identified one of them, comprising residues 1255-1452, as interacting with HDAC7 with micromolar affinity. The binding site was mapped in detail by NMR and confirmed by truncation and alanine mutagenesis. Complementing this with mutational analysis of HDAC7, we show that HDAC7, via its surface zinc ion binding site, binds to a 28 residue stretch in SMRT comprising a GSI motif followed by an alpha helix.
15 citations
TL;DR: This work identified a soluble expression construct of the IcsA/VirG α-domain using the ESPRIT library screening system and determined its structure to 1.9Å resolution, revealing a new domain, which shares a common fold with the autochaperone domains of various autotransporters.
13 citations