Tarendeau, F. et al. Structure and nuclear import function of the C-terminal domain of influenza virus polymerase PB2 subunit. Nat. Struct. Mol. Biol. 14, 229-233

European Molecular Biology Laboratory (EMBL) Grenoble Outstation, 6 rue Jules Horowitz, BP181, 38042 Grenoble Cedex 9, France.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 04/2007; 14(3):229-33. DOI: 10.1038/nsmb1212
Source: PubMed


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.

Download full-text


Available from: Julien Boudet,
  • Source
    • "3.4. CIP2A-ArmRP can form similar interactions as other ArmRP proteins Crystallized ArmRP proteins exhibit a highly conserved binding mode (Reichen et al., 2014), where an extended peptide from the interacting proteins is bound into the central groove of the ArmRP proteins through a combination of electrostatic and backbone interactions (PDB 3QHE, 4OIH, 3L6X/3L6Y, 2JDQ, 1JDH; Graham et al., 2001; Ishiyama et al., 2010; Morishita et al., 2011; Roman et al., 2013; Tarendeau et al., 2007). The bound peptides run antiparallel to the ArmRP motifs and the hydrogen bonds between the conserved Asn residues in the H3 α-helices of ArmRP and the peptide backbone keep the peptide in an extended conformation (Andrade et al., 2001; Conti et al., 1998), while other residues in the binding groove confer specificity by interacting with the side chains of the bound peptide (Reichen et al., 2014). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Cancerous Inhibitor of Protein Phosphatase 2A (CIP2A) is a human oncoprotein, which exerts its cancer-promoting function through interaction with other proteins, for example Protein Phosphatase 2A (PP2A) and MYC. The lack of structural information for CIP2A significantly prevents the design of anti-cancer therapeutics targeting this protein. In an attempt to counteract this fact, we modeled the three-dimensional structure of the N-terminal domain (CIP2A-ArmRP), analyzed key areas and amino acids, and coupled the results to the existing literature. The model reliably shows a stable armadillo repeat fold with a positively charged groove. The fact that this conserved groove highly likely binds peptides is corroborated by the presence of a conserved polar ladder, which is essential for the proper peptide-binding mode of armadillo repeat proteins and, according to our results, several known CIP2A interaction partners appropriately possess an ArmRP-binding consensus motif. Moreover, we show that Arg229Gln, which has been linked to the development of cancer, causes a significant change in charge and surface properties of CIP2A-ArmRP. In conclusion, our results reveal that CIP2A-ArmRP shares the typical fold, protein-protein interaction site and interaction patterns with other natural armadillo proteins and that, presumably, several interaction partners bind into the central groove of the modeled CIP2A-ArmRP. By providing essential structural characteristics of CIP2A, the present study significantly increases our knowledge on how CIP2A interacts with other proteins in cancer progression and how to develop new therapeutics targeting CIP2A.
    Journal of Theoretical Biology 09/2015; 386. DOI:10.1016/j.jtbi.2015.09.010 · 2.12 Impact Factor
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: In plants, MADS domain transcription factors act as central regulators of diverse developmental pathways. In Arabidopsis thaliana, one of the most central members of this family is SEPALLATA3 (SEP3), which is involved in many aspects of plant reproduction, including floral meristem and floral organ development. SEP3 has been shown to form homo and heterooligomeric complexes with other MADS domain transcription factors through its intervening (I) and keratin-like (K) domains. SEP3 function depends on its ability to form specific protein-protein complexes; however, the atomic level determinants of oligomerization are poorly understood. Here, we report the 2.5-Å crystal structure of a small portion of the intervening and the complete keratin-like domain of SEP3. The domains form two amphipathic alpha helices separated by a rigid kink, which prevents intramolecular association and presents separate dimerization and tetramerization interfaces comprising predominantly hydrophobic patches. Mutations to the tetramerization interface demonstrate the importance of highly conserved hydrophobic residues for tetramer stability. Atomic force microscopy was used to show SEP3-DNA interactions and the role of oligomerization in DNA binding and conformation. Based on these data, the oligomerization patterns of the larger family of MADS domain transcription factors can be predicted and manipulated based on the primary sequence. INTRODUCTION
    The Plant Cell 09/2014; 26(9). DOI:10.1105/tpc.114.127910 · 9.34 Impact Factor
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Several binding scaffolds that are not based on immunoglobulins have been designed as alternatives to traditional monoclonal antibodies. Many of them have been developed to bind to folded proteins, yet cellular networks for signaling and protein trafficking often depend on binding to unfolded regions of proteins. This type of binding can thus be well described as a peptide-protein interaction. In this review, we compare different peptide-binding scaffolds, highlighting that armadillo repeat proteins (ArmRP) offer an attractive modular system, as they bind a stretch of extended peptide in a repeat-wise manner. Instead of generating each new binding molecule by an independent selection, preselected repeats - each complementary to a piece of the target peptide - could be designed and assembled on demand into a new protein, which then binds the prescribed complete peptide. Stacked armadillo repeats (ArmR), each typically consisting of 42 amino acids arranged in three α-helices, build an elongated superhelical structure which enables binding of peptides in extended conformation. A consensus-based design approach, complemented with molecular dynamics simulations and rational engineering, resulted in well-expressed monomeric proteins with high stability. Peptide binders were selected and several structures were determined, forming the basis for the future development of modular peptide-binding scaffolds.
    Journal of Structural Biology 08/2013; 185(2). DOI:10.1016/j.jsb.2013.07.012 · 3.23 Impact Factor
Show more