Hydrodynamic Behavior of the Intrinsically Disordered Potyvirus Protein VPg, of the Translation Initiation Factor eIF4E and of their Binary Complex

Protein intrinsic disorder is involved in many biological processes and good experimental models are valuable to investigate its functions. The potyvirus genome-linked protein, VPg, displays many features of an intrinsically disordered protein. The virus cycle requires the formation of a complex between VPg and eIF4E, one of the host translation initiation factors. An in-depth characterization of the hydrodynamic properties of VPg, eIF4E, and of their binary complex VPg-eIF4E was carried out. Two complementary experimental approaches, size-exclusion chromatography and fluorescence anisotropy, which is more resolving and revealed especially suitable when protein concentration is the limiting factor, allowed to estimate monomers compaction upon complex formation. VPg possesses a high degree of hydration which is in agreement with its classification as a partially folded protein in between a molten and pre-molten globule. The natively disordered first 46 amino acids of eIF4E contribute to modulate the protein hydrodynamic properties. The addition of an N-ter His tag decreased the conformational entropy of this intrinsically disordered region. A comparative study between the two tagged and untagged proteins revealed the His tag contribution to proteins hydrodynamic behavior.

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Spectroscopic Investigation of the Kinetic Mechanism Involved in the Association of Potyviral VPg with the Host Plant Translation Initiation Factor eIF4E

International Journal of Molecular Sciences ◽

10.3390/ijms21165618 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5618

Author(s):

Jocelyne Walter ◽

Amandine Barra ◽

Justine Charon ◽

Geneviève Tavert-Roudet ◽

Thierry Michon

Keyword(s):

Amino Acids ◽

Translation Initiation ◽

Plant Resistance ◽

Kinetic Mechanism ◽

Single Step ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Intrinsically Disordered ◽

Resistance Breaking ◽

Central Helix

The infectious cycle of potyviruses requires the formation of a complex between the viral genome-linked protein VPg and the host eukaryotic translation initiation factor 4E, eIF4E. Mutations associated with plant resistance to potyviruses were previously mapped at the eIF4E surface, while on the virus side, mutations leading to plant resistance breaking were identified within the VPg. In the present study, fluorescence spectroscopy was used to probe the contribution of the VPg intrinsically disordered region bearing amino acids determinant of the resistance breaking, to the VPg–eIF4E binding mechanism. Synthetic peptides encompassing the VPg88–120 central region were found to tightly bind to eIF4E. Fluorescence energy transfer experiments show that, upon binding to eIF4E, the N and C termini of the VPg88–111 fragment move closer to one another, at a distance compatible with a α-helix folding. When the VPg112–120 region, which contains amino acids associated with resistance breakdown, is appended to VPg88–111, the complex formation with eIF4E switches from a single-step to a two-step kinetic model. This study revisits a recent investigation of the VPg–eIF4E complex by specifying the contribution of the VPg central helix and its appended disordered region to VPg association with eIF4E.

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Conformational ordering of intrinsically disordered peptides for targeting translation initiation

10.1101/2020.08.20.230268 ◽

2020 ◽

Author(s):

Christopher J Brown ◽

Chandra S Verma ◽

David P Lane ◽

Dilraj Lama

Keyword(s):

Translation Initiation ◽

Protein Interactions ◽

Bound State ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Structural Reorganization ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Conformational Ordering ◽

Natural Amino Acids

AbstractIntrinsically disordered regions (IDRs) in proteins can regulate their activity by facilitating protein-protein interactions (PPIs) as exemplified in the recruitment of the eukaryotic translation initiation factor 4E (eIF4E) protein by the protein eIF4G. Deregulation of this PPI module is central to a broad spectrum of cancer related malignancies and its targeted inhibition through bioactive peptides is a promising strategy for therapeutic intervention. We have employed a structure-guided approach to rationally develop peptide derivatives from the intrinsically disordered eIF4G scaffold by incorporating non-natural amino acids that facilitates disorder-to-order transition. The conformational heterogeneity of these peptides and the degree of structural reorganization required to adopt the optimum mode of interaction with eIF4E underscores their differential binding affinities. The presence of a pre-structured local helical element in the ensemble of structures was instrumental in the efficient docking of the peptides on to the protein surface. These insights were exploited to further design features into the peptide to propagate bound-state conformations in solution which resulted in the generation of a potent eIF4E binder. The study illustrates the molecular basis of eIF4E recognition by a disordered epitope from eIF4G and its modulation to generate peptides that can potentially attenuate translation initiation in oncology.

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