Crystal Structure of the Measles Virus Phosphoprotein Domain Responsible for the Induced Folding of the C-terminal Domain of the Nucleoprotein

Johansson, Kenth; Bourhis, Jean-Marie; Campanacci, Valérie; Cambillau, Christian; Canard, Bruno; Longhi, Sonia

doi:10.1074/jbc.m308745200

Cited by 150 publications

(239 citation statements)

References 55 publications

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“…It should be noted that the small negative peak at approximately 224 nm suggests some residual helical content in both proteins. When ERD10 and ERD14 were titrated with trifluoro acetic acid (data not shown), a transition from the disordered state to the helix was observed within the range of 15% to 30% trifluoro acetic acid, which suggests a significant local preference for the helical conformation and a possible induced folding mechanism during interaction with partners (Johansson et al, 2003;Meszaros et al, 2007). In all, these observations unequivocally suggest that ERD10 and ERD14 are highly disordered proteins.…”

Section: Idp Character Of Erd10 and Erd14mentioning

confidence: 49%

Chaperone Activity of ERD10 and ERD14, Two Disordered Stress-Related Plant Proteins

et al. 2008

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ERD10 and ERD14 (for early response to dehydration) proteins are members of the dehydrin family that accumulate in response to abiotic environmental stresses, such as high salinity, drought, and low temperature, in Arabidopsis (Arabidopsis thaliana). Whereas these proteins protect cells against the consequences of dehydration, the exact mode(s) of their action remains poorly understood. Here, detailed evidence is provided that ERD10 and ERD14 belong to the family of intrinsically disordered proteins, and it is shown in various assays that they act as chaperones in vitro. ERD10 and ERD14 are able to prevent the heat-induced aggregation and/or inactivation of various substrates, such as lysozyme, alcohol dehydrogenase, firefly luciferase, and citrate synthase. It is also demonstrated that ERD10 and ERD14 bind to acidic phospholipid vesicles without significantly affecting membrane fluidity. Membrane binding is strongly influenced by ionic strength. Our results show that these intrinsically disordered proteins have chaperone activity of rather wide substrate specificity and that they interact with phospholipid vesicles through electrostatic forces. We suggest that these findings provide the rationale for the mechanism of how these proteins avert the adverse effects of dehydration stresses.

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Section: Idp Character Of Erd10 and Erd14mentioning

confidence: 49%

Chaperone Activity of ERD10 and ERD14, Two Disordered Stress-Related Plant Proteins

et al. 2008

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“…In contrast, residues within helix ␣1 are relatively unperturbed. These observations define the likely location for attachment of the measles N-tail peptide as the surface cleft created by helices ␣2 and ␣3 of P 457-507 , as predicted on the basis of surface hydrophobicity and sequence conser vation among the paramyxoviruses (8).…”

Section: [3]mentioning

confidence: 99%

“…For P 457-507 , we mapped the total chemical shift changes that occur on binding N 477-505 onto the x-ray crystal structure (8). Substantial chemical shift changes occur in the last half of helix ␣2, helix ␣3, and their connecting loop ( Fig.…”

Section: [3]mentioning

confidence: 99%

“…By using isothermal titration calorimetry, it has been shown that measles P 457-507 binds measles N 477-505 with 1:1 stoichiometry and that the binding affinity is weak (K d ϭ 13 M at 20°C and 35 M at 37°C) (9). Data from a series of spectroscopic studies suggest that folding and binding of the tail of N are coupled (8,10,11), and a theoretical model of the interaction has been proposed in which an N-tail peptide binds as a helix into a hydrophobic groove on the surface of P 457-507 (8). A schematic model of polymerase attachment in measles virus is shown in Fig.…”

mentioning

confidence: 99%

“…A coiled-coil domain oligomerizes P (7) while the extreme carboxyl terminus of P is involved in nucleocapsid binding. The structure of this region of measles P (amino acids 459-507) has been recently determined by x-ray crystallography and is a compact bundle of three ␣-helices (8). This region by itself constitutes the nucleocapsid-binding domain of P, being both necessary and sufficient for binding to nucleocapsid-like particles (8,9).…”

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confidence: 99%

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Structural basis for the attachment of a paramyxoviral polymerase to its template

Kingston

Hamel²,

Dahlquist³

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

189

223

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The nucleocapsid of measles virus is the template for viral RNA synthesis and is generated through packaging of the genomic RNA by the nucleocapsid protein (N). The viral polymerase associates with the nucleocapsid through a small, trihelical binding domain at the carboxyl terminus of the phosphoprotein (P). Translocation of the polymerase along the nucleocapsid during RNA synthesis is thought to involve the repeated attachment and release of the binding domain. We have investigated the interaction between the binding domain from measles P (amino acids 457-507) and the sequence it recognizes within measles N (amino acids 477-505). By using both solution NMR spectroscopy and x-ray crystallography, we show that N487-503 binds as a helix to the surface created by the second (␣2) and third (␣3) helices of P457-507, in an orientation parallel to the helix ␣3, creating a four-helix bundle. The binding interface is tightly packed and dominated by hydrophobic amino acids. Binding and folding of N487-503 are coupled. However, when not bound to P, N487-503 does not resemble a statistical random coil but instead exists in a loosely structured state that mimics the bound conformation. We propose that before diffusional encounter, the ensemble of accessible conformations for N487-503 is biased toward structures capable of binding P, facilitating rapid association of the two proteins. This study provides a structural analysis of polymerase-template interactions in a paramyxovirus and presents an example of a protein-protein interaction that must be only transiently maintained as part of its normal function.M easles virus, a member of the paramyxovirus family, causes an acute infectious disease in humans. The virus is enveloped and possesses a negative-sense, single-stranded RNA genome Ϸ15,900 nt in length. Within the virion, the nucleocapsid protein (N) packages the genomic RNA into a helical protein-RNA complex termed the nucleocapsid. In the cytoplasm of an infected cell, the viral RNA polymerase uses the nucleocapsid as a template for both the transcription of messenger RNAs, encoding the individual viral proteins, as well as the replication and encapsidation of full-length copies of the viral genome. Unencapsidated RNA cannot act as a template for the polymerase. The replication of paramyxoviruses has been comprehensively reviewed (1-3).The paramyxoviral polymerase has two components, the large protein and the phosphoprotein (P) (Fig. 1). The catalytic activities of the polymerase reside within the large protein, whereas P is responsible for, among other activities, binding the polymerase to the nucleocapsid (4, 5). P is a modular protein containing a number of functional domains separated by intrinsically disordered sequences (6). A coiled-coil domain oligomerizes P (7) while the extreme carboxyl terminus of P is involved in nucleocapsid binding. The structure of this region of measles P (amino acids 459-507) has been recently determined by x-ray crystallography and is a compact bundle of three ␣-helices (8). This region...

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Site‐Directed Spin Labeling EPR Spectroscopy

Belle

Rouger

Costanzo

et al. 2010

Instrumental Analysis of Intrinsically Disordered Proteins

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6Electron paramagnetic resonance (EPR) spectroscopy is a technique that specifi cally detects unpaired electrons. EPR -sensitive reporter groups (spin labels or spin probes) can be introduced into biological systems via sitedirected spin labeling (SDSL). The basic strategy of SDSL involves the introduction of a paramagnetic group at a selected protein site. This is usually accomplished by cysteine substitution mutagenesis, followed by covalent modifi cation of the unique sulfydryl group with a selective nitroxide reagent, such as the 1 -oxyl -2,2,5,5 -tetramethyl -δ 3 -pyrroline -3 -methyl methane thiosulfonate. SDSL followed by EPR spectroscopy has been extensively used to study conformational changes within structured proteins, as well as folding and unfolding processes of structured proteins in the presence of denaturing agents, such as urea and guanidium chloride. In this chapter, we review the theoretical principles of this approach and illustrate how we successfully applied it to investigate the structural properties and the induced folding of ABSTRACT 132 INSTRUMENTAL ANALYSIS OF INTRINSICALLY DISORDERED PROTEINS the intrinsically disordered C -terminal domain of the measles virus nucleoprotein (N -tail, aa 401 -525) upon binding to the X domain (aa 459 -507) of the viral phosphoprotein (P).

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Crystal Structure of the Measles Virus Phosphoprotein Domain Responsible for the Induced Folding of the C-terminal Domain of the Nucleoprotein

Cited by 150 publications

References 55 publications

Chaperone Activity of ERD10 and ERD14, Two Disordered Stress-Related Plant Proteins

Chaperone Activity of ERD10 and ERD14, Two Disordered Stress-Related Plant Proteins

Structural basis for the attachment of a paramyxoviral polymerase to its template

Site‐Directed Spin Labeling EPR Spectroscopy

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