Intrinsically disordered proteins (IDPs) defy the structure-function paradigm as they fulfill essential biological functions while lacking well-defined secondary and tertiary structures. Conformational and spectroscopic analyses showed that IDPs do not constitute a uniform family, and can be divided into subfamilies as a function of their residual structure content. Residual intramolecular interactions are thought to facilitate binding to a partner and then induced folding. Comprehensive information about experimental approaches to investigate structural disorder and induced folding is still scarce. We herein provide hints to readily recognize features typical of intrinsic disorder and review the principal techniques to assess structural disorder and induced folding. We describe their theoretical principles and discuss their respective advantages and limitations. Finally, we point out the necessity of using different approaches and show how information can be broadened by the use of multiples techniques.

Measles virus is a negative-sense, single-stranded RNA virus within the Mononegavirales order, which includes several human pathogens, including rabies, Ebola, Nipah, and Hendra viruses. The measles virus nucleoprotein consists of a structured N-terminal domain, and of an intrinsically disordered C-terminal domain, N TAIL (aa 401-525), which undergoes induced folding in the presence of the C-terminal domain (XD, aa 459-507) of the viral phosphoprotein. Within N TAIL , an a-helical molecular recognition element (a-MoRE, aa 488-499) involved in binding to P and in induced folding was identified and then observed in the crystal structure of XD. Using small-angle X-ray scattering, we have derived a low-resolution structural model of the complex between XD and N TAIL , which shows that most of N TAIL remains disordered in the complex despite P-induced folding within the a-MoRE. The model consists of an extended shape accommodating the multiple conformations adopted by the disordered N-terminal region of N TAIL , and of a bulky globular region, corresponding to XD and to the C terminus of N TAIL (aa 486-525). Using surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and heteronuclear magnetic resonance, we show that N TAIL has an additional site (aa 517-525) involved in binding to XD but not in the unstructured-to-structured transition. This work provides evidence that intrinsically disordered domains can establish complex interactions with their partners, and can contact them through multiple sites that do not all necessarily gain regular secondary structure.Keywords: measles virus; nucleoprotein; phosphoprotein; intrinsic disorder; induced folding; NMR; CD; SAXS Measles virus (MV) is an enveloped RNA virus within the Morbillivirus genus of the Paramyxoviridae family.Its nonsegmented, negative-sense, single-stranded RNA genome is encapsidated by the viral nucleoprotein (N) within a helical nucleocapsid. This N-RNA complex is used as a template for both transcription and replication. These latter activities are carried out by the viral polymerase complex, which consists of two components, the large protein (L) and the phosphoprotein (P) (for review, see Lamb and Kolakofsky 2001).

Nipah virus (NiV) is a highly pathogenic emergent paramyxovirus causing deadly encephalitis in humans. Its replication requires a constant supply of unassembled nucleoprotein (N(0)) in complex with its viral chaperone, the phosphoprotein (P). To elucidate the chaperone function of P, we reconstituted NiV the N(0)-P core complex and determined its crystal structure. The binding of the N-terminal region of P blocks the polymerization of N by interfering with subdomain exchange between N protomers and keeps N(0) in an open conformation, ready to grasp an RNA molecule. We found that a peptide derived from the N-binding region of P protects cells against viral infection and demonstrated by structure-based mutagenesis that this peptide acts by inhibiting N(0)-P formation. These results provide new insights about the assembly of N along genomic RNA and validate the N(0)-P complex as a target for drug development.

Measles virus (MV) 1 is a member of the Paramyxovirinae sub-family within the Mononegavirales order. Its non-segmented, negative-sense, single-stranded RNA genome is packaged by the viral nucleoprotein (N) within a helical nucleocapsid. Mononegavirales use this N-RNA complex as a template for both transcription and replication. These reactions are carried out by the RNA-dependent RNA polymerase polymerase (L) in conjuction with the phosphoprotein (P) (for a review see Ref.

BackgroundDouble stranded RNA (dsRNA) is widely accepted as an RNA motif recognized as a danger signal by the cellular sentries. However, the biology of non-segmented negative strand RNA viruses, or Mononegavirales, is hardly compatible with the production of such dsRNA.Methodology and Principal FindingsDuring measles virus infection, the IFN-β gene transcription was found to be paralleled by the virus transcription, but not by the virus replication. Since the expression of every individual viral mRNA failed to activate the IFN-β gene, we postulated the involvement of the leader RNA, which is a small not capped and not polyadenylated RNA firstly transcribed by Mononegavirales. The measles virus leader RNA, synthesized both in vitro and in vivo, was efficient in inducing the IFN-β expression, provided that it was delivered into the cytosol as a 5′-trisphosphate ended RNA. The use of a human cell line expressing a debilitated RIG-I molecule, together with overexpression studies of wild type RIG-I, showed that the IFN-β induction by virus infection or by leader RNA required RIG-I to be functional. RIG-I binds to leader RNA independently from being 5-trisphosphate ended; while a point mutant, Q299A, predicted to establish contacts with the RNA, fails to bind to leader RNA. Since the 5′-triphosphate is required for optimal RIG-I activation but not for leader RNA binding, our data support that RIG-I is activated upon recognition of the 5′-triphosphate RNA end.Conclusions/SignificanceRIG-I is proposed to recognize Mononegavirales transcription, which occurs in the cytosol, while scanning cytosolic RNAs, and to trigger an IFN response when encountering a free 5′-triphosphate RNA resulting from a mislocated transcription activity, which is therefore considered as the hallmark of a foreign invader.

In this report, the solution structure of the nucleocapsid-binding domain of the measles virus phosphoprotein (XD, aa 459-507) is described. A dynamic description of the interaction between XD and the disordered C-terminal domain of the nucleocapsid protein, (N(TAIL), aa 401-525), is also presented. XD is an all alpha protein consisting of a three-helix bundle with an up-down-up arrangement of the helices. The solution structure of XD is very similar to the crystal structures of both the free and bound form of XD. One exception is the presence of a highly dynamic loop encompassing XD residues 489-491, which is involved in the embedding of the alpha-helical XD-binding region of N(TAIL). Secondary chemical shift values for full-length N(TAIL) were used to define the precise boundaries of a transient helical segment that coincides with the XD-binding domain, thus shedding light on the pre-recognition state of N(TAIL). Titration experiments with unlabeled XD showed that the transient alpha-helical conformation of N(TAIL) is stabilized upon binding. Lineshape analysis of NMR resonances revealed that residues 483-506 of N(TAIL) are in intermediate exchange with XD, while the 475-482 and 507-525 regions are in fast exchange. The N(TAIL) resonance behavior in the titration experiments is consistent with a complex binding model with more than two states.

The major inducible 70-kDa heat shock protein (hsp72) binds measles virus (MV) nucleocapsids and increases MV gene expression. The cytoplasmic tail of the MV N protein (N(TAIL)) contains three hydrophobic domains (Box-1-3) that are potential targets of hsp72 interaction. Low affinity binding to Box-3 is correlated to hsp72-dependent stimulation of MV minireplicon reporter gene expression whereas interactions between hsp72 and Box-1 and/or -2 have not been documented. The present work showed that virus deficient in Box-3/hsp72 interaction retains the ability to form nucleocapsid/hsp72 complexes, identifying Box-2 but not Box-1 as a mediator of high affinity hsp72 binding. Box-2 is the binding site for the viral P protein X domain (XD), where P tethers the viral polymerase to nucleocapsid in support of transcription and genome replication, and competitive inhibition of XD binding to N(TAIL) by hsp72 was shown. Recognition of a common binding site by P and hsp72 represents a potential mechanism for host cell modulation of viral gene expression.

We used site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy to study the induced folding of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N(TAIL)). Four single-site N(TAIL) mutants (S407C, S488C, L496C, and V517C), located in three conserved regions, were prepared and labeled with a nitroxide paramagnetic probe. We could monitor the gain of rigidity that N(TAIL) undergoes in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol (TFE) or one of its physiological partners, namely, the C-terminal domain (XD) of the viral phosphoprotein. The mobility of the spin label grafted at positions 488, 496, and 517 was significantly reduced upon addition of XD, contrary to that of the spin label bound to position 407, which was unaffected. Furthermore, the EPR spectra of spin-labeled S488C and L496C bound to XD in the presence of 30% sucrose are indicative of the formation of an alpha-helix in the proximity of the spin labels. Such an alpha-helix had been already identified by previous biochemical and structural studies. Using TFE we unveiled a previously undetected structural propensity within the N-terminal region of N(TAIL) and showed that its C-terminal region "resists" gaining structure even at high TFE concentrations. Finally, we for the first time showed the reversibility of the induced folding process that N(TAIL) undergoes in the presence of XD. These results highlight the suitability of site-directed spin-labeling EPR spectroscopy to identify protein regions involved in binding and folding events, while providing insights at the residue level.