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Modulation of Measles Virus NTAIL Interactions through Fuzziness and Sequence Features of Disordered Binding Sites - PubMed

  • ️Mon Jan 01 2018

Review

Modulation of Measles Virus NTAIL Interactions through Fuzziness and Sequence Features of Disordered Binding Sites

Christophe Bignon et al. Biomolecules. 2018.

Abstract

In this paper we review our recent findings on the different interaction mechanisms of the C-terminal domain of the nucleoprotein (N) of measles virus (MeV) NTAIL, a model viral intrinsically disordered protein (IDP), with two of its known binding partners, i.e., the C-terminal X domain of the phosphoprotein of MeV XD (a globular viral protein) and the heat-shock protein 70 hsp70 (a globular cellular protein). The NTAIL binds both XD and hsp70 via a molecular recognition element (MoRE) that is flanked by two fuzzy regions. The long (85 residues) N-terminal fuzzy region is a natural dampener of the interaction with both XD and hsp70. In the case of binding to XD, the N-terminal fuzzy appendage of NTAIL reduces the rate of α-helical folding of the MoRE. The dampening effect of the fuzzy appendage on XD and hsp70 binding depends on the length and fuzziness of the N-terminal region. Despite this similarity, NTAIL binding to XD and hsp70 appears to rely on completely different requirements. Almost any mutation within the MoRE decreases XD binding, whereas many of them increase the binding to hsp70. In addition, XD binding is very sensitive to the α-helical state of the MoRE, whereas hsp70 is not. Thus, contrary to hsp70, XD binding appears to be strictly dependent on the wild-type primary and secondary structure of the MoRE.

Keywords: IDP; fuzzy interactions; kinetics; protein complementation assays; split-GFP reassembly.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Figures

Figure 1
Figure 1

(A) Schematic representation of the C-terminal domain of the nucleoprotein (N) of measles virus (MeV) NTAIL (upper panel) and cartoon representation of an NTAIL conformer generated using Flexible-Mecano [49]. (B) Ribbon representation of the crystal structure of the C-terminal X domain of the phosphoprotein of MeV XD (PDB code 1OKS). (C) The structure of the chimeric construct made of MeV XD (blue) and of the molecular recognition element MoRE of NTAIL (red) (PDB code 1T6O). (D) Cartoon representation of the crystal structure of hsp70 based on PDB codes 1HJO and 4JNF. The relative orientation of the two hsp70 domains (i.e., amino acids 3 to 382 and amino acids 389 to 610) is based on the structure of a form encompassing residues 1 to 554 (PDB code 1YUW). The three constituent domains of hsp70, i.e., nucleotide binding domain (NBD, aa 1 to 384), peptide binding domain (PBD, aa 384 to 543) and “lid” (aa 543 to 641) (see [18] and references therein cited) are highlighted.

Figure 2
Figure 2

Effect of the N-terminal fuzzy region of NTAIL on XD and hsp70 binding. (A) NTAIL deletion variants were generated as described [60]. The N-terminal residue is indicated. The N-terminal fuzzy region subjected to truncation is shown in grey and the MoRE is shown in green. (B,C) Split- green fluorescent protein (GFP) complementation assay using XD (B) and hsp70 (C). Shown are the mean values and standard deviation (SD) of an experiment performed in triplicate. Results are expressed as percentage with 100% being the fluorescence value provided by full-length NTAIL (401). For a detailed description of the procedure see Supplementary Information. (D) IUPred [64] disorder prediction of wtNTAIL (blue) and artNTAIL (red) from residue 401 to residue 480. (E) Fluorescence values obtained by split-GFP complementation assays using wild type (wt) (blue line) and art (red line) NTAIL truncation variants and XD. Shown are the mean values and SD of an experiment performed in triplicate. Results are expressed as percentage with 100% being the fluorescence value provided by full-length wtNTAIL (401). (F) Binding kinetics of XD (at a constant concentration of 2 μM) with excess concentrations of either wtNTAIL (black circles) or a peptide mimicking the MoRE (green circles) in 10 mM sodium phosphate buffer and 150 mM NaCl at pH 7.0. Under all conditions, there was an at least fivefold difference in concentration between the two proteins to ensure pseudo-first order conditions. Experiments were carried out using a PTJ-64 capacitor-discharge T-jump apparatus (Hi-Tech, Salisbury, UK). The temperature was rapidly changed with a jump size of 9 °C, from 11 °C to 20 °C. Data were taken from [60].

Figure 3
Figure 3

Effect of primary and secondary structures of the MoRE on XD and hsp70 binding. (A,B) Alanine scanning mutagenesis of NTAIL MoRE. MoRE residues (aa 486 to 504) of MeV NTAIL truncation variant 471 were individually mutated into an alanine (or a glycine when the wild-type residue was an alanine). The binding ability of each single NTAIL variant was then compared to that of wild-type NTAIL by split-GFP complementation assay using either XD (A) or hsp70 (B). Ø, negative control (fluorescence background obtained using an empty vector encoding NGFP alone); wt, positive control (i.e., wild-type truncation variant 471). Results are expressed as percentage with 100% being the fluorescence value provided by wt truncation variant 471. The horizontal dotted line indicates the binding of the positive control. (C) Far-UV circular dichroism spectra of wtMoRE, AlaMoRE, and GlyMoRE peptides. (D) Fluorescence values obtained by split-GFP complementation assays using NTAIL MoRE variants with different α-helicities. See A for details; 401, full-length wtNTAIL; wt471, 471 truncated variant with a wtMoRE; Ala471, 471 truncated variant with AlaMoRE; Gly471, 471 truncated variant with GlyMoRE. (E) Binding kinetics of MoRE peptides to XD. Data shown in panels A, B, and D are the mean values and SD of an experiment performed in triplicate. Data were taken from [66].

Figure 4
Figure 4

Binding abilities of hsb towards hsp70. (A) Amino acid sequence of wt and hsbMore. In the latter, all the residues individually shown to lead to increased NTAIL-hsp70 binding strength by the alanine scanning mutagenesis were collectively replaced with alanine, or with glycine when the wild-type residue was alanine. (B) Binding abilities of NTAIL variants as obtained by split-GFP complementation assays. Y-axis: fluorescence values of each culture divided by the optical density at 600 nm. X-axis: NTAIL variants-hsp70 pairs. Shown are the mean values and SD of an experiment performed in triplicate. The scheme of the NTAIL constructs is shown above the graph, with wt and hsb MoREs being represented in grey and black, respectively, and fuzzy regions in white. Orientation is from left (N-terminal end) to right (C-terminal end). Data were taken from [66].

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