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Structural disorder and molecular partnership of intrinsically disordered regions of the nucleoproteins and phosphoproteins of Henipaviruses

Heads Sonia LONGHI

People involved: J. Habchi (PhD student), D. Bloquel (PhD student), A. Gruet (AI, CDD, EPHE student)

Nipah and Hendra viruses are recently emerged severe human pathogens. Their unique genome constitution and their broad host range set them apart from other Paramyxoviruses and led to their classification into the Henipavirus genus that represents the only zoonotic genus in the Paramyxoviridae family. In humans, they are responsible of encephalites with a high case fatality. Their high virulence and wide host range set them as biosafety level 4 pathogens. To date, there is no efficient therapeutic treatment against Nipah and Hendra viruses. The N-P interaction, being crucial for the replication of Henipaviruses, may constitute an efficient antiviral discovery pathway. As such, the molecular characterization of the N and P proteins is a prerequisite in view of rational drug design.

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Figure 6
Structural models of the NiV and HeV NTAIL-XD complexes, in which the NTAIL region predicted to adopt an α-helical conformation (amino acids 473–493 for NiV and 473–492 for HeV) is shown in blue with ribbon representation, while XD is shown in orange with surface representation. Hydrophobic residues are shown in yellow. The amino acid sequences of XD and of the α-MoRE are shown with the same color code. (D) Superimposition of the structural models (ribbon representation) of the NTAIL-XD complexes of HeV (pink) and NiV (orange) onto the crystal structure of the MeV chimeric construct (blue) (PDB code 1T6O) [3]. Modified from [2]

Using both computational and experimental approaches we showed that Henipaviruses N and P proteins possess large intrinsically disordered regions. By combining several disorder prediction methods, we showed that the N-terminal domain of P (PNT) and the C-terminal domain of N (NTAIL) are both mostly disordered, although they contain short order-prone segments. Using bioinformatics, we identified within NTAIL four putative molecular recognition elements (MoREs) with different structural propensities. We then cloned, expressed and purified Henipavirus PNT and NTAIL domains. By combining gel filtration, dynamic light scattering, circular dichroism and nuclear magnetic resonance, we showed that both NTAIL and PNT possess some residual structure and compactness typical of the premolten globule sub-family within the class of intrinsically disordered proteins [1]. Notably, Henipavirus NTAIL domains were also shown to be disordered in the context of full-length nucleoproteins [2]. After having purified the C-terminal X domains (PXD) of Henipavirus phosphoproteins, we showed that NTAIL and PXD form a 1:1 stoichiometric complex that is stable under NaCl concentrations as high as 1 M, and whose KD is in the µM range. Using far-UV circular dichroism and nuclear magnetic resonance, we showed that PXD triggers an increase in α–helical content of NTAIL. Fluorescence spectroscopy studies pointed out lack of PXD impact on the chemical environment of a trp residue introduced at position 527 of Henipavirus NTAIL, thus arguing for the lack of stable contacts between the C-terminus of NTAIL and PXD. Using the MeV NTAIL-PXD complex as template [3], we then proposed a tentative structural model of the NTAIL- PXD interaction where a short-order prone region of NTAIL (α-More, aa 473-493) adopts an α–helical conformation and is embedded between helices α2 and α3 of PXD, leading to a relatively small interface dominated by hydrophobic contacts (Figure 6) [2].
In a subsequent work, for each NTAIL protein, we designed four deletion constructs bearing different combinations of the predicted MoREs. Following purification of the NTAIL truncated proteins from the soluble fraction of E. coli, we characterized them in terms of their conformational, spectroscopic and binding properties. These studies provided direct experimental evidence for the structural state of the four predicted MoREs, and showed that two of them have clear α-helical propensities, with the one spanning residues 473-493 being strictly required for binding to PXD. We also showed that Henipavirus NTAIL and PXD form heterologous complexes, indicating that the PXD binding regions are functionally interchangeable between the two viruses. Finally, in those studies, we showed that the content in regular secondary structure is not a major determinant of protein compaction [4].
Altogether, these studies provide the first detailed experimental characterization of the N-P interaction in Henipaviruses. They also designate the NTAIL- PXD interaction as a valuable target for rational antiviral approaches. In collaboration with K. Alvarez and JC. Guillemot (AFMB), we are currently screening chemical libraries (see (see http://www.afmb.univ-mrs.fr/-Marsei...) for compounds able to block the NTAIL-PXD interaction.

Footnotes


[1] Habchi J, Mamelli L, Darbon H, Longhi S (2010) PLoS One 5 e11684

[2] Habchi J, Blangy S, Mamelli L, Ringkjobing Jensen M, Blackledge M, Darbon H, Oglesbee M, Shu Y, Longhi S. (2011) J Biol Chem 286 13583-13602

[3] Kingston RL, Hamel DJ, Gay LS, Dahlquist FW, Matthews BW (2004) Proc Natl Acad Sci U S A 101 8301-6

[4] Blocquel D, Habchi J, Gruet A, Blangy S and Longhi S (2012) Mol Biosyst 8 392-410
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