Edited by Charles E. SamuelPhosphoprotein is the main cofactor of the viral RNA polymerase of Mononegavirales. It is involved in multiple interactions that are essential for the polymerase function. Most prominently it positions the polymerase complex onto the nucleocapsid, but also acts as a chaperone for the nucleoprotein. Mononegavirales phosphoproteins lack sequence conservation, but contain all large disordered regions. We show here that Nand C-terminal intrinsically disordered regions account for 80% of the phosphoprotein of the respiratory syncytial virus. But these regions display marked dynamic heterogeneity. Whereas almost stable helices are formed C terminally to the oligomerization domain, extremely transient helices are present in the N-terminal region. They all mediate internal long-range contacts in this non-globular protein. Transient secondary elements together with fully disordered regions also provide protein binding sites recognized by the respiratory syncytial virus nucleoprotein and compatible with weak interactions required for the processivity of the polymerase. Human respiratory syncytial virus (hRSV),3 a member of the family Pneumoviridae (1) and order Mononegavirales (MNV), is the main viral cause of lower respiratory tract illness worldwide, and the main agent responsible for bronchiolitis and pneumonia in infants (2). All children have been infected by the age of two, requiring hospitalization in ϳ5% cases (3). Elderly and immunocompromised adults are also at increased risk. No efficient treatment is presently available for hRSV (4), and vaccination is challenging due to complex immunogenicity (5). The search for hRSV antiviral drugs directed toward specific viral functions is therefore still ongoing (6).The hRSV RNA-dependent RNA complex (RdRp) constitutes a virus-specific target with specific protein-protein interactions that have not all been investigated in detail (7). It uses the nonsegmented single-stranded negative sense RNA genome of hRSV as a template. In infected cells, the viral RdRp is found in specific inclusion bodies (8), which have been shown to be transcription and replication centers for other Mononegavirales, e.g. rabies (9) and vesicular stomatitis viruses (10). The apo RdRp complex is composed a minima of the large catalytic subunit (L) and its essential cofactor, the phosphoprotein (P) (11, 12). The P protein plays a central role in the RdRp by interacting with all main RdRp components. During transcription and replication it tethers the L protein to the nucleocapsid (NC), consisting of the genomic RNA packaged by the nucleoprotein (N), by direct interaction with N (13-16). hRSV P also binds to the transcription antitermination factor M2-1 (17-19). Phosphorylation of P has been proposed to regulate these interactions, although it is not essential for replication (20 -22). P also acts as a chaperone for neo-synthesized N by forming an N 0 ⅐P complex that preserves N in a monomeric and RNA-free state (23). We have shown previously that formation of hRSV NC⅐P and ...

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The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure–function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5′-TGCGT-3′/3′-ACGCA-5′ motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity.

Background: E. coli GlmS is active as a dimer.Results: The dimer is in equilibrium with a hexameric state that is catalytically inactive in vitro. The glucosamine 6-phosphate product completely shifts the equilibrium toward the hexamer. Conclusion: This accounts for a morpheein-type allosteric regulation of E. coli GlmS activity. Significance: The inactive hexamer is a target for developing specific antibacterial agents.

The 19-24 kDa Translationally Controlled Tumor Protein (TCTP) is involved in a wide range of molecular interactions with biological and nonbiological partners of various chemical compositions such as proteins, peptides, nucleic acids, carbohydrates, or small molecules. TCTP is therefore an important and versatile binding platform. Many of these protein-protein interactions have been validated, albeit only few received an in-depth structural characterization. In this chapter, we will focus on the structural analysis of TCTP and we will review the available literature regarding its interaction network from a structural perspective.

The translationally controlled tumor protein (TCTP) is a multifunctional protein that may interact with many other biomolecules, including itself. The experimental determinations of TCTP structure revealed a folded core domain and an intrinsically disordered region, which includes the first highly conserved TCTP signature, but whose role in the protein functions remains to be elucidated. In this work, we combined NMR experiments and MD simulations to characterize the conformational ensemble of the TCTP intrinsically disordered loop, in the presence or not of calcium ions and with or without the phosphorylation of Ser46 and Ser64. Our results show that these changes in the TCTP electrostatic conditions induce significant shifts of its conformational ensemble toward structures more or less extended in which the disordered loop is pulled away or folded against the core domain. Particularly, these conditions impact the transient contacts between the two highly conserved signatures of the protein. Moreover, both experimental and theoretical data show that the interface of the non-covalent TCTP dimerization involves its second signature which suggests that this region might be involved in protein-protein interaction. We also show that calcium hampers the formation of TCTP dimers, likely by favoring the competitive binding of the disordered loop to the dimerization interface. All together, we propose that the TCTP intrinsically disordered region is involved in remodeling the core domain surface to modulate its accessibility to its partners in response to a variety of cellular conditions.

Although RNase Y acts as the key enzyme initiating messenger RNA decay in Bacillus subtilis and likely in many other Gram-positive bacteria, its three-dimensional structure remains unknown. An antibody belonging to the rare immunoglobulin G (IgG) 2b lx isotype was raised against a 12-residue conserved peptide from the N-terminal noncatalytic domain of B. subtilis RNase Y (BsRNaseY) that is predicted to be intrinsically disordered. Here, we show that this domain can be produced as a stand-alone protein called Nter-BsRNaseY that undergoes conformational changes between monomeric and dimeric forms. Circular dichroism and size exclusion chromatography coupled with multiangle light scattering or with small angle x-ray scattering indicate that the Nter-BsRNaseY dimer displays an elongated form and a high content of a-helices, in agreement with the existence of a central coiled-coil structure appended with flexible ends, and that the monomeric state of Nter-BsRNaseY is favored upon binding the fragment antigen binding (Fab) of the antibody. The dissociation constants of the IgG/BsRNaseY, IgG/Nter-BsRNaseY, and IgG/peptide complexes indicate that the affinity of the IgG for Nter-BsRNaseY is in the nM range and suggest that the peptide is less accessible in BsRNaseY than in Nter-BsRNaseY. The crystal structure of the Fab in complex with the peptide antigen shows that the peptide adopts an elongated U-shaped conformation in which the unique hydrophobic residue of the peptide, Leu6, is completely buried. The peptide/Fab complex may mimic the interaction of a microdomain of the N-terminal domain of BsRNaseY with one of its cellular partners within the degradosome complex. Altogether, our results suggest that BsRNaseY may become accessible for protein interaction upon dissociation of its N-terminal domain into the monomeric form. FIGURE 1 Characterization of a monoclonal antibody induced against a 12-residue peptide of Nter-BsRNaseY. (A) Sequence alignment of the N-terminal domains of RNases Y from B. subtilis, Staphylococcus aureus, Streptococcus pyogenes, Listeria monocytogenes, Clostridium perfringens, Lactoccus lactis, Borrelia burgdorferi, and Leadbetterella byssophila was performed with Clustal Omega (67) and rendered with ESPript (68). The predicted N-terminal TM helix (residues 6-24) and the inducing peptide (residues 79-90) are indicated. (B) Recognition of BsRNaseY by IgG RY79-90 as detected by immunoblotting (legend continued on next page) Fab Binding Dissociates RNase Y IDD Biophysical Journal 115, 2102-2113, December 4, 2018 2103Protein stoichiometry analysis of the Nter-BsRNaseY/Fab RY79-90 complex (at a 1:2 ratio) was performed by SEC-MALS using the ''UV extinction from RI peak'' method (28) and the protein conjugate method (29) (see Supporting Materials and Methods). In the first method, the UV 280 extinction coefficients of the peaks are extracted from the SEC-MALS data and compared to the predicted extinction coefficients Fab Binding Dissociates RNase Y IDD (C) Structural models of Nter-BsRNaseY. Top: Two at...

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