Assessing induced folding within the intrinsically disordered C-terminal domain of the Henipavirus nucleoproteins by site-directed spin labeling EPR spectroscopy

Abstract

This work aims at characterizing structural transitions within the intrinsically disordered C-terminal domain of the nucleoprotein (N_TAIL) from the Nipah and Hendra viruses, two recently emerged pathogens gathered within the Henipavirus genus. To this end, we used site-directed spin labeling combined with electron paramagnetic resonance spectroscopy to investigate the α-helical-induced folding that Henipavirus N_TAIL domains undergo in the presence of the C-terminal X domain of the phosphoprotein (P_XD). For each N_TAIL protein, six positions located within four previously proposed molecular recognition elements (MoREs) were targeted for spin labeling, with three of these positions (475, 481, and 487) falling within the MoRE responsible for binding to P_XD (Box3). A detailed analysis of the impact of the partner protein on the labeled N_TAIL variants revealed a dramatic modification in the environment of the spin labels grafted within Box3, with the observed modifications supporting the formation of an induced α-helix within this region. In the free state, the slightly lower mobility of the spin labels grafted within Box3 as compared to the other positions suggests the existence of a transiently populated α-helix, as already reported for measles virus (MeV) N_TAIL. Comparison with the well-characterized MeV N_TAIL–P_XD system, allowed us to validate the structural models of Henipavirus N_TAIL–P_XD complexes that we previously proposed. In addition, this study highlighted a few notable differences between the Nipah and Hendra viruses. In particular, the observation of composite spectra for the free form of the Nipah virus N_TAIL variants spin labeled in Box3 supports conformational heterogeneity of this partly pre-configured α-helix, with the pre-existence of stable α-helical segments. Altogether these results provide insights into the molecular mechanisms of the Henipavirus N_TAIL–P_XD binding reaction.

Keywords:

• intrinsic disorder
• induced folding
• spin labeling
• EPR spectroscopy
• Nipah
• Hendra
• nucleoprotein
• phosphoprotein

Acknowledgments

We wish to thank Nicolas Armstrong and the mass spectrometry platform of the IFR48 of Marseille for mass spectrometry analyses, and David Blocquel (AFMB) for help with the SEC-MALLS analyses of the synthetic NiV P_XD sample. We also thank Jean-Pierre Andrieu (Laboratoire d’Enzymologie Moléculaire, Institut de Biologie Structurale, Grenoble, France) and the proteomic platform of the IBS for the amino acid compositions analyses. The authors are also grateful to the EPR facilities of the national TGE RPE and Aix-Marseille University EPR platform, and to Janez Strancar (Jozef Stefan Institut, Ljubljana, Slovenia) for providing access to the EPRSIM-C software.