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Longhi’s Team Projects

Molecular partnership of the intrinsically disorder C-terminal domain of the measles virus nucleoprotein (NTAIL)

Heads Sonia LONGHI

 Unraveling the molecular mechanisms of the measles virus NTAIL-PXD interaction

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

The interaction of measles virus (MeV) NTAIL with the C-terminal X domain of the MeV phosphoprotein (PXD) has been the focus of several studies in the last years ( [1], [2], [3], [4], [5], [6], [7], [8], [9]). Altogether those studies unveiled that i) the majority of NTAIL remains disordered upon binding to PXD, ii) the Box2 region (aa 489-506 of N) undergoes alpha-helical folding upon binding to PXD, and iii) the folding coupled to binding process relies onto two non-mutually exclusive pathways, namely conformational selection and folding after binding (Figure 2).

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Figure 2.
Model for MeV NTAIL-PXD complex formation that utilizes both conformer selection and non-specific encounter complex formation (modified from (Gely et al., J Mol Recognit 2010). The helix, corresponding to the primary binding site for PXD, is partly (50%) preformed in the absence of PXD, and it encompasses residues 491-499. In the non-specific binding model, a weak encounter complex may also form between NTAIL and PXD. This encounter complex is converted to a tightly bound complex by the folding of the α-MoRE. It is unclear if this folding occurs during the lifetime of the encounter complex. In the conformer selection model, the preformed helix interacts with PXD to form a tightly bound complex. In both cases, following α-helical folding of the α-MoRE, Box3 (aa 517-525 of N) becomes more rigid. The four conformers that are contoured schematically represent the final stage in complex formation, which consists of an ensemble of conformers in which Box3 has a reduced conformational freedom that may favor the establishment of weak, non-specific contacts with PXD.
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Figure 3.
Model of the partly disordered MeV NTAIL-XD complex as a conformational ensemble. 50 best-fit structures of the 488-525 region of NTAIL in complex with PXD. The NTAIL conformers are depicted with a color gradient ranging from yellow to red with decreasing structural density, while PXD is shown in black. Modified from (Kavalenka et al., Biophys J 2010).

In collaboration with the group of J. Strancar (Jozef Stefan Institute, Ljubljana, Slovenia), we proposed a novel approach to describe the structure of partly disordered protein complexes and applied it to the 488-525 region of NTAIL in complex with PXD. This original approach relies on a combination of site-directed spin-labeling EPR spectroscopy and modeling of local rotation conformational spaces. This allowed us to model the NTAIL-PXD complex as a conformational ensemble and pointed out a considerable conformational freedom of the C-terminal region of NTAIL (Figure 3) ( [10]). In collaboration with the group of B. Guigliarelli (BIP, Marseille) the NTAIL-PXD interaction is also currently investigated using DEER EPR spectroscopy, and new spin probes endowed with different properties are currently being synthesized by the group of S. Marc (LCP, Marseille).
More recently, in collaboration with the group of M. Blackledge (IBS, Grenoble, France), an atomistic ensemble description of the free form of NTAIL could be achieved by combining RDC measurements and ensemble optimization methods. Those studies showed that NTAIL adopts a dynamic equilibrium between a completely unfolded state and four different partially helical conformations ( [11]). In those studies the disordered state of NTAIL in the context of intact nucleocapsids was also addressed. From electron microscopy studies, the mass of the nucleocapsids was estimated to be between 2 and 50 MDa, a value that would normally preclude detection of solution state NMR signals of a folded globular protein. The HSQC spectrum of the intact capsids however reveals that NTAIL remains flexible when attached to the nucleocapsid. Comparison of the HSQC spectra of the isolated NTAIL domain and intact nucleocapsids shows that the NMR resonances superimpose, demonstrating that the local conformational behavior of NTAIL, and hence its disordered state, is retained in situ. Signals for the first 50 residues of NTAIL are however absent in the spectrum of the capsid, while large variations of peak intensities indicate differential flexibility along the remainder of the chain, with the molecular recognition element (MoRE) involved in binding to PXD having particularly low intensities. From comparison with a model of MeV N that was generated by docking the structure of the related respiratory syncytial virus N protein into the electron density map of the MeV nucleocapsid [12], a model could be proposed where NTAIL escapes from the interior of the nucleocapsid helix via the interstitial space between the NCORE moieties (Figure 4). The first 50 residues of NTAIL are conformationally restricted being in sandwich between successive turns of the helical nucleocapsid, while the MoRE slowly exchanges on and off the surface of the nucleocapsids which probably locates it in the proper position for recognition by the polymerase complex [13].

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Figure 4.
The figure (courtesy of Martin Blackledge, IBS, Grenoble), shows a reconstruction of the MeV nucleocapsid from electronic microscopy data (bottom), small angles scattering (left) and high-field nuclear magnetic resonance (foreground). The red moieties of the nucleocapsid represent the disordered NTAIL domain. Modified from (Ringkjøbing Jensen et al., Proc Natl Acad Sci 2011).

Footnotes


[1] Longhi S, Receveur-Brechot V, Karlin D, Johansson K, Darbon H, Bhella D, Yeo R, Finet S, Canard B (2003) J Biol Chem 278 18638-48

[2] Johansson K, Bourhis JM, Campanacci V, Cambillau C, Canard B, Longhi S (2003) J Biol Chem 278 44567-73

[3] Bourhis JM, Johansson K, Receveur-Brechot V, Oldfield CJ, Dunker KA, Canard B, Longhi S (2004) Virus Res 99 157-167

[4] Bourhis JM, Receveur-Brechot V, Oglesbee M, Zhang X, Buccellato M, Darbon H, Canard B, Finet S, Longhi S (2005) Protein Sci 14 1975-92

[5] Morin B, Bourhis JM, Belle V, Woudstra M, Carriere F, Guigliarelli B, Fournel A, Longhi S (2006) J Phys Chem B 110 20596-608

[6] Belle V, Rouger S, Costanzo S, Liquiere E, Strancar J, Guigliarelli B, Fournel A, Longhi S (2008) Proteins 73 973-88

[7] Bernard C, Gely S, Bourhis JM, Morelli X, Longhi S, Darbon H (2009) FEBS Lett 583 (7) 1084-9

[8] Gely S, Lowry DF, Bernard C, Jensen MR, Blackledge M, Costanzo S, Bourhis JM, Darbon H, Daughdrill G, Longhi S (2010) J Mol Recognit 23 435-447

[9] Bischak CG, Longhi S, Snead DM, Costanzo S, Terrer E, Londergan CH (2010) Biophys J 99(5) 1676-83

[10] Kavalenka A, Urbancic I, Belle V, Rouger S, Costanzo S, Kure S, Fournel A, Longhi S, Guigliarelli B and Strancar J. (2010) Biophys J 98 1055-1064

[11] Ringkjobing Jensen M, Communie G, Ribeiro EA, Martinez N, Desfosses A, Salmon L, Mollica L, Gabel F, Jamin M, Longhi S, Ruigrok R and Blackeldge M (2011) Proc Natl Acad Sci 108 9839-9844

[12] Desfosses A, Goret G, Farias Estrozi L, Ruigrok RW, Gutsche I (2011) J Virol 85 1391-5

[13] Ringkjobing Jensen M, Communie G, Ribeiro EA, Martinez N, Desfosses A, Salmon L, Mollica L, Gabel F, Jamin M, Longhi S, Ruigrok R and Blackeldge M (2011) Proc Natl Acad Sci 108 9839-9844
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