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The intrinsically disordered C-terminal domain of the measles virus nucleoprotein interacts with the C-terminal domain of the phosphoprotein via two distinct sites and remains predominantly unfolded - PubMed

The intrinsically disordered C-terminal domain of the measles virus nucleoprotein interacts with the C-terminal domain of the phosphoprotein via two distinct sites and remains predominantly unfolded

Jean-Marie Bourhis et al. Protein Sci. 2005 Aug.

Abstract

Measles virus is a negative-sense, single-stranded RNA virus within the Mononegavirales order,which includes several human pathogens, including rabies, Ebola, Nipah, and Hendra viruses. The measles virus nucleoprotein consists of a structured N-terminal domain, and of an intrinsically disordered C-terminal domain, N(TAIL) (aa 401-525), which undergoes induced folding in the presence of the C-terminal domain (XD, aa 459-507) of the viral phosphoprotein. With in N(TAIL), an alpha-helical molecular recognition element (alpha-MoRE, aa 488-499) involved in binding to P and in induced folding was identified and then observed in the crystal structure of XD. Using small-angle X-ray scattering, we have derived a low-resolution structural model of the complex between XD and N(TAIL), which shows that most of N(TAIL) remains disordered in the complex despite P-induced folding within the alpha-MoRE. The model consists of an extended shape accommodating the multiple conformations adopted by the disordered N-terminal region of N(TAIL), and of a bulky globular region, corresponding to XD and to the C terminus of N(TAIL) (aa 486-525). Using surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and heteronuclear magnetic resonance, we show that N(TAIL) has an additional site (aa 517-525) involved in binding to XD but not in the unstructured-to-structured transition. This work provides evidence that intrinsically disordered domains can establish complex interactions with their partners, and can contact them through multiple sites that do not all necessarily gain regular secondary structure.

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Figures

Figure 1.
Figure 1.

Schematic representation of the modular organization of P (A) and of the NNUC–P complex (B) of measles virus. Disordered regions are represented by thin bars (A) or by lines (B). The encapsidated RNA is shown as a dotted line. PMD is represented with a dumbbell shape according to Tarbouriech et al. (2000). The tetrameric P (Rahaman et al. 2004) is shown bound to NNUC through three of its four C-terminal XD “arms,” as in the model of Curran and Kolakofsky (1999). The L protein is shown as a rectangle contacting P through PMD by analogy with SeV (Smallwood et al. 1994).

Figure 2.
Figure 2.

Low-resolution structure of the NTAIL–XD complex derived by SAXS. (A) Distance distribution function of the NTAIL–XD complex. (B) Overall shape of the NTAIL–XD complex, as obtained by GASBOR, shown in two orientations rotated by 90°, with the crystal structure of XD–NTAIL486–505 inserted in the bulky cluster. The XD molecule is shown in red, while the 486–505 region of NTAIL is shown in blue. (C) Low-resolution model of the NTAIL–XD complex provided by CREDO. Colors are as in B. The schematic organization of NTAIL, showing the location of the α-MoRE, is given below. The inset shows the experimental scattering curve (black circles) and fit (red line) obtained by CREDO with the low-resolution model.

Figure 3.
Figure 3.

(A) Schematic representation of NTAIL deletion proteins. (Top) Domain organization of N showing that it is composed of two regions, NCORE (aa 1–399) and NTAIL (aa 401–525). The epitope recognized by the anti-N mAbs, Cl 25 mAb (aa 457–476) is shaded. NTAILΔ3, NTAILΔ2,3, and NTAILΔ1 are devoid of Box3, Box2 plus Box3 and of Box1, respectively. The three NTAIL deletion proteins contain an N-terminal hexahistidine tag and a C-terminal Flag. The predicted α-helix (residues 489–504), as well as the α-MoRE (aa 488–499), i.e., the region shown to be involved in induced folding of NTAIL through binding to P (see Bourhis et al. 2004), are indicated. The position (aa 518) targeted for the Tyr → Trp substitution is highlighted by a black diamond. (B) Purification of NTAIL deletion proteins from E. coli. Coomassie blue staining of a 12% SDS-PAGE. (TF) Bacterial lysate (total fraction); (SN) clarified supernatant (soluble fraction); (IMAC) eluent from immobilized metal affinity chromatography; (GF) eluent from gel filtration.

Figure 4.
Figure 4.

Far-UV CD spectra and analysis of the α-helical propensities of NTAIL deletion proteins. Far-UV CD spectra of NTAILΔ3 (A) NTAILΔ2,3 (B) and NTAILΔ1 (C) at 0.1 mg/mL in 10 mM sodium phosphate at pH 7 in the presence of increasing concentrations of TFE (0%, 10%, 20%, and 30%) recorded at 20°C. Each spectrum is the mean of three independent acquisitions. (D) α-Helical content of NTAIL deletion proteins in the presence of increasing TFE concentrations. The α-helical content was derived from the ellipticity value at 222 nm as described in Myers et al. (1997a).

Figure 5.
Figure 5.

Induced folding on NTAIL deletion proteins in the presence of XD. Far-UV CD spectra of NTAILHNFC (A), NTAILΔ3 (B), NTAILΔ2,3 (C), and NTAILΔ1 (D) either alone (black line) or in the presence of a threefold molar excess of XD (full circles). The CD spectrum of XD alone (gray line), as well as the theoretical average curves calculated by assuming that no structural variations occur (see Materials and Methods) are also shown (open circles).

Figure 6.
Figure 6.

(A) Purification of NTAILW518 from E. coli. Coomassie blue staining of a 12% SDS-PAGE. Purified NTAILW518 protein. (B) Far-UV CD spectra of NTAILHN and NTAILW518. The spectra were recorded on a 0.1 mg/mL protein solution in 10 mM sodium phosphate (pH 7) at 20°C, and represent the mean of three independent acquisitions. (C) Fluorescence spectroscopy studies of NTAILW518. The relative fluorescence increase of NTAILW518 (1 μM in 10 mM sodium phosphate at pH 7) is plotted as a function of the XD concentration. The KDapp value (see text) results from the fitting of the data to a single exponential.

Figure 7.
Figure 7.

2D-HSQC NMR spectra. HSQC spectrum of a 0.5 mM solution of purified 15N-NTAILHN alone (black) or in the presence of a twofold molar excess of XD (red), of a 0.125-mM solution of purified 15N-NTAILΔ3 alone (green) or in the presence of a twofold molar excess of XD (blue). All proteins were in 10 mM sodium phosphate at pH 7. All spectra are recorded at 283 K. ppm quotes for resonance shifts in parts per million of the spectrophotometer frequency. Some peaks are labeled as follows: stars indicate peaks present in spectra of both complexes only, while diamonds quote for peaks present in the spectrum recorded with the 15N-NTAILHN–XD complex, but not with the 15N-NTAILΔ3–XD complex. The inset shows the purified 15N-NTAILΔ3 and 15N-NTAILHN proteins.

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