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Revealing the role of intrinsically disordered proteins in transcription and replication of measles and sendai paramyxoviruses using nuclear magnetic resonance spectroscopy

Final Report Summary - VIRAL_IDP (Revealing the role of intrinsically disordered proteins in transcription and replication of measles and sendai paramyxoviruses using nuclear magnetic resonance spectroscopy.)

Measles virus (MeV) is a highly contagious human pathogen responsible for approximately 40 million infections each year and 450.000 deaths worldwide. It belongs to the Paramyxoviridae family within the Mononegavirales order, that includes several human pathogens with a strong socio-economical impact, comprising both well characterized and emerging viruses. MeV ranks in the first ten causes of worldwide mortality, representing the main cause of childhood mortality in developing countries. Despite extensive vaccination campaigns, the disease has not yet been eradicated and no effective antiviral treatment exists. The replication complex of MeV is a promising target for antiviral chemotherapy: however, until now this complex and its formation mechanism had not been characterized in detail. The elucidation of the mechanisms of action of the component proteins, as well as their structural characterization (atomic resolution models of the proteins in their free and pre-recognition states and in complex with their partner proteins), is a prerequisite for the identification and the rational design of antiviral agents.
The aim of the VIRAL_IDP project is the characterization at atomic level of of the interactions that govern the mechanism of genome replication in Paramyxoviridiae (Measles, MeV, and Sendai, SeV, viruses) and the formation of the complex between the nucleocapsid-binding X domain (XD) of the phosphoprotein P (a modular polymerase co-factor) and the C-terminal unstructured domain of the nucleoprotein N (NTAIL). In particular, due to the difficult physical-chemical characterization of such interactions and of molecular disorder, the original project aims to develop new experimental and numerical methods for improving the quantitative description of systems that present disorder/high flexibility (intrinsically disordered proteins, IDPs), thus having both a strong biological and methodological relevance.

The quantitative description of transient helical folding and dynamics of NTAIL of MeV/SeV in its free form has been studied by means of spectroscopic methods, completing and extending the work previously performed in the host laboratory that display that such systems exhibit a mixed population of several helical structures. Using NMR spectroscopy it has been possible to measure spin relaxation rates of both the proteins at several fields, thus allowing the characterization of dynamics on the timescale of several nanoseconds and to couple it with longer timescale dynamics characterized by means of residual dipolar couplings (i.e. up to milliseconds).
The spectroscopic studies have been complemented by several atomic level simulation techniques (mainly molecular dynamics and Metadynamics) with the aim of providing information about the kinetics and energetics of interconversion between NTAILs helical populations. This led to the reproduction of experimental data (chemical shifts, spin relaxation, residual dipolar coupling) and to a good description of potential energy surface (PES), as well as to an atomic level description of NTAIL populations in agreement with the ones determined using other methods, thus validating the existing knowledge and moreover providing information about timescales/helical lifetimes of the interconversion processes.
MeV NTAIL spin relaxation and dynamics have been also studied in presence of XD and in the context of the intact MeV nucleocapsid, allowing the finalization of a model that provides a structural framework for understanding the role of NTAIL in the initiation of viral transcription and replication of Paramyxoviridiae. This work has been efficiently coupled to studies of small angle X-rays scattering (SAXS) and electron microscopy in order to provide the widest possible picture of the biologically relevant mechanism of RNA replication.

The aforementioned findings translate into a crucial information both for the explanation of basic biochemical mechanisms that govern Paramyxoviridiae genome transcription machinery and for future drug discovery based projects related to these viruses: the kinetic and energetic characterization of the dynamics of NTAIL is the necessary step before the characterization of the NTAIL-XD complex formation. Hence the present work characterizes the two extremes of the interaction (the free NTAIL, the complex in the contest of the nucleocapsid) that are necessary for the comprehension of the replication mechanism of these viruses. From this work important guidelines emerge for the characterization of the complex formation kinetics and energetics that are already under investigation in the host laboratory.
Moreover, the biophysical characterization methods that have been successfully applied to MeV/SeV NTAILs can be now used for the characterization of other IDPs. Due to the recently discovered relevance of molecular disorders in many high impact diseases (ranging from neurodegenerative disease to cancer) this constitutes a crucial aspect of the project in terms of transfer of knowledge to other projects with respect to the original one, allowing a new set of mixed computational and spectroscopic techniques that allow the atomic level characterization of biomedically relevant interactions involving IDPs. The necessity of testing these methods in a general way allowed their application also to systems different than IDPs (e.g. structured domains with extended flexible regions, proteins in the crystalline form), thus resulting in a high level of dissemination of the work performed in the context of the present project of whom all the Structural Biology community can benefit.