The genome of measles virus is encapsidated by multiple copies of the nucleoprotein (N), forming helical nucleocapsids of molecular mass approaching 150 Megadalton. The intrinsically disordered C-terminal domain of N (N TAIL ) is essential for transcription and replication of the virus via interaction with the phosphoprotein P of the viral polymerase complex. The molecular recognition element (MoRE) of N TAIL that binds P is situated 90 amino acids from the folded RNA-binding domain (N CORE ) of N, raising questions about the functional role of this disordered chain. Here we report the first in situ structural characterization of N TAIL in the context of the entire N-RNA capsid. Using nuclear magnetic resonance spectroscopy, small angle scattering, and electron microscopy, we demonstrate that N TAIL is highly flexible in intact nucleocapsids and that the MoRE is in transient interaction with N CORE . We present a model in which the first 50 disordered amino acids of N TAIL are conformationally restricted as the chain escapes to the outside of the nucleocapsid via the interstitial space between successive N CORE helical turns. The model provides a structural framework for understanding the role of N TAIL in the initiation of viral transcription and replication, placing the flexible MoRE close to the viral RNA and, thus, positioning the polymerase complex in its functional environment.is a member of the Paramyxoviridae family of the Mononegavirales order of negative sense, single stranded RNA viruses. The viral genome is encapsidated by multiple copies of the nucleoprotein (N) forming a helical nucleocapsid. Transcription and replication of the viral RNA are initiated by an interaction between N and the polymerase complex, composed of the phosphoprotein (P) and the RNAdependent RNA polymerase (1). N consists of two domains: N CORE (residues 1-400), responsible for the interaction with the viral RNA and for maintaining the nucleocapsid structure, and a long intrinsically disordered domain, N TAIL (residues 401-525) serving as the anchor point for the polymerase complex (2, 3). The molecular recognition element (MoRE) (residues 485-502) of the disordered N TAIL interacts with the C-terminal three-helix bundle domain, XD, of P (residues 459-507) (4) and thereby recruits the polymerase complex onto the nucleocapsid template (5, 6).The realization that intrinsically disordered proteins (IDPs) are functional despite a lack of structure (7-9) has revealed entirely new paradigms that appear to redefine our understanding of the role of conformational flexibility in molecular interactions (10-12). Until now most IDPs have been studied in isolation, or in the presence of a single interaction partner, although it is evident that a real physiological environment could influence the nature and relevance of apparent intrinsic disorder. In this context resolving the question of whether the protein is actually disordered in situ is of paramount importance. In this case the mechanistic role of the extensive disorder present in N TA...

Despite playing important roles throughout biology, molecular recognition mechanisms in intrinsically disordered proteins remain poorly understood. We present a combination of (1)H(N), (13)C', and (15)N relaxation dispersion NMR, measured at multiple titration points, to map the interaction between the disordered domain of Sendai virus nucleoprotein (NT) and the C-terminal domain of the phosphoprotein (PX). Interaction with PX funnels the free-state equilibrium of NT by stabilizing one of the previously identified helical substates present in the prerecognition ensemble in a nonspecific and dynamic encounter complex on the surface of PX. This helix then locates into the binding site at a rate coincident with intrinsic breathing motions of the helical groove on the surface of PX. The binding kinetics of complex formation are thus regulated by the intrinsic free-state conformational dynamics of both proteins. This approach, providing high-resolution structural and kinetic information about a complex folding and binding interaction trajectory, can be applied to a number of experimental systems to provide a general framework for understanding conformational disorder in biomolecular function.

In order to understand the conformational behaviour of Intrinsically Disordered Proteins (IDPs), it is essential to develop a molecular representation of the partially folded state. Due to the very large number of degrees of conformational freedom available to such a disordered system, this problem is highly underdetermined. Characterisation therefore requires extensive experimental data, and novel analytical tools are required to exploit the specific conformational sensitivity of different experimental parameters. In this review we concentrate on the use of nuclear magnetic resonance (NMR) spectroscopy for the study of conformational behaviour of IDPs at atomic resolution. Each experimental NMR parameter is sensitive to different aspects of the structural and dynamic behaviour of the disordered state and requires specific consideration of the relevant averaging properties of the physical interaction. In this review we present recent advances in the description of disordered proteins and the selection of representative ensembles on the basis of experimental data using statistical coil sampling from flexible-meccano and ensemble selection using ASTEROIDS. Using these tools we aim to develop a unified molecular representation of the disordered state, combining complementary data sets to extract a meaningful description of the conformational behaviour of the protein.

Hendra virus (HeV) is a recently emerged severe human pathogen that belongs to the Henipavirus genus within the Paramyxoviridae family. The HeV genome is encapsidated by the nucleoprotein (N) within a helical nucleocapsid. Recruitment of the viral polymerase onto the nucleocapsid template relies on the interaction between the C-terminal domain, NTAIL, of N and the C-terminal X domain, XD, of the polymerase co-factor phosphoprotein (P). Here, we provide an atomic resolution description of the intrinsically disordered NTAIL domain in its isolated state and in intact nucleocapsids using nuclear magnetic resonance (NMR) spectroscopy. Using electron microscopy, we show that HeV nucleocapsids form herringbone-like structures typical of paramyxoviruses. We also report the crystal structure of XD of P that consists of a three-helix bundle. We study the interaction between NTAIL and XD using NMR titration experiments and provide a detailed mapping of the reciprocal binding sites. We show that the interaction is accompanied by α-helical folding of the molecular recognition element of NTAIL upon binding to a hydrophobic patch on the surface of XD. Finally, using solution NMR, we investigate the interaction between intact nucleocapsids and XD. Our results indicate that monomeric XD binds to NTAIL without triggering an additional unwinding of the nucleocapsid template. The present results provide a structural description at the atomic level of the protein-protein interactions required for transcription and replication of HeV, and the first direct observation of the interaction between the X domain of P and intact nucleocapsids in Paramyxoviridae.

The atomic structure of the stable tetramerization domain of the measles virus phosphoprotein shows a tight four-stranded coiled coil. Although at first sight similar to the tetramerization domain of the Sendai virus phosphoprotein, which has a hydrophilic interface, the measles virus domain has kinked helices that have a strongly hydrophobic interface and it lacks the additional N-terminal three helical bundles linking the long helices.

The structural characterization of modular proteins containing long intrinsically disordered regions intercalated with folded domains is complicated by their conformational diversity and flexibility and requires the integration of multiple experimental approaches. Nipah virus (NiV) phosphoprotein, an essential component of the viral RNA transcription/replication machine and a component of the viral arsenal that hijacks cellular components and counteracts host immune responses, is a prototypical model for such modular proteins. Curiously, the phosphoprotein of NiV is significantly longer than the corresponding protein of other paramyxoviruses. Here, we combine multiple biophysical methods, including x-ray crystallography, NMR spectroscopy, and small angle x-ray scattering, to characterize the structure of this protein and provide an atomistic representation of the full-length protein in the form of a conformational ensemble. We show that full-length NiV phosphoprotein is tetrameric, and we solve the crystal structure of its tetramerization domain. Using NMR spectroscopy and small angle x-ray scattering, we show that the long N-terminal intrinsically disordered region and the linker connecting the tetramerization domain to the C-terminal X domain exchange between multiple conformations while containing short regions of residual secondary structure. Some of these transient helices are known to interact with partners, whereas others represent putative binding sites for yet unidentified proteins. Finally, using NMR spectroscopy and isothermal titration calorimetry, we map a region of the phosphoprotein, comprising residues between 110 and 140 and common to the V and W proteins, that binds with weak affinity to STAT1 and confirm the involvement of key amino acids of the viral protein in this interaction. This provides new, to our knowledge, insights into how the phosphoprotein and the nonstructural V and W proteins of NiV perform their multiple functions.

Measles virus RNAg enomes are packaged into helical nucleocapsids (NCs), comprising thousands of nucleoproteins (N) that bind the entire genome.N-RNAprovides the template for replication and transcription by the viral polymerase and is apromising target for viral inhibition. Elucidation of mechanisms regulating this process has been severely hampered by the inability to controllably assemble NCs.Here, we demonstrate self-organization of Ni nto NC-like particles in vitro upon addition of RNA, providing as imple and versatile tool for investigating assembly.R eal-time NMR and fluorescence spectroscopyr eveals biphasic assembly kinetics. Remarkably,a ssembly depends strongly on the RNAsequence,with the genomic 5' end and poly-Adenine sequences assembling efficiently,while sequences such as poly-Uracil are incompetent for NC formation. This observation has important consequences for understanding the assembly process.

NMR shows how an intrinsically disordered protein controls replication of measles virus via a dynamic weakly interacting complex.