Structure and nuclear import function of the C-terminal domain of influenza virus polymerase PB2 subunit.

European Molecular Biology Laboratory (EMBL) Grenoble Outstation, 6 rue Jules Horowitz, BP181, 38042 Grenoble Cedex 9, France.
Nature Structural & Molecular Biology (Impact Factor: 11.63). 04/2007; 14(3):229-33. DOI: 10.1038/nsmb1212
Source: PubMed

ABSTRACT The trimeric influenza virus polymerase, comprising subunits PA, PB1 and PB2, is responsible for transcription and replication of the segmented viral RNA genome. Using a novel library-based screening technique called expression of soluble proteins by random incremental truncation (ESPRIT), we identified an independently folded C-terminal domain from PB2 and determined its solution structure by NMR. Using green fluorescent protein fusions, we show that both the domain and the full-length PB2 subunit are efficiently imported into the nucleus dependent on a previously overlooked bipartite nuclear localization sequence (NLS). The crystal structure of the domain complexed with human importin alpha5 shows how the last 20 residues unfold to permit binding to the import factor. The domain contains three surface residues implicated in adaptation from avian to mammalian hosts. One of these tethers the NLS-containing peptide to the core of the domain in the unbound state.

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Available from: Julien Boudet, Jul 30, 2015
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    • "In order to find soluble and well-expressing constructs of the MADS domain TF, SEP3, we performed library screening of ;3000 constructs using the ESPRIT random library method, which identifies well-expressing soluble domain constructs in poorly annotated regions (Tarendeau et al., 2007; Yumerefendi et al., 2010). The construct comprising residues 75 to 178 (SEP3 75-178 ) was selected for further studies (Acajjaoui and Zubieta, 2013) (Figure 1B). "
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    • "In the past decade, several importin-a structures in complex with NLS peptides have been solved (Catimel et al., 2001; Chen et al., 2005; Conti and Kuriyan, 2000; Conti et al., 1998; Cutress et al., 2008; Dias et al., 2009; Fontes et al., 2000, 2003a,b; Giesecke and Stewart, 2010; Hirano and Matsuura, 2011; Kobe, 1999; Matsuura and Stewart, 2005; Mynott et al., 2011; Pumroy et al., 2012; Takeda et al., 2011; Tarendeau et al., 2007; Yang et al., 2010). Based on structural homologies of the bound peptide, five peptide classes have been defined, represented by (i) the bipartite peptide nucleoplasmin (Np1), and (ii) the monopartite peptides cmyc (Myc), bound to major and minor site; (iii) pCN, a subgroup of c-myc; (iv) aIBB, bound only to the major site and (v) Nup50, bound only to the minor site (Fig. 2A). "
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    • "Escherichia coli as they were obtained by a high-throughput screening method known as expression of soluble proteins by random incremental truncation (ESPRIT) (Tarendeau et al., 2007; Yumerefendi et al., 2010). HEK293T cells were transfected with these constructs and 24 h later, the proteins were immunoprecipitated with specific anti-eIF4GI or an unrelated antibody and analyzed in Western blots probed with an anti-HA antibody. "
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