Submission
| Title: | Elucidating determinants of Ebola virus VP40 dimerization using computational simulations and biophysical approaches |
| Co-Authors: |
Narkhede, Yogesh, University of Notre Dame; Jacob P. Conarty; Department of Medicinal Chemistry & Molecular Pharmacology, Purdue University; Sheng Li, Department of Medicine, University of California San Diego; Olaf G. Wiest, Department of Chemistry and Biochemistry, University of Notre Dame; Robert V. Stahelin, Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University |
Abstract
Background/Significance/Rationale: The Ebola virus lipid-binding matrix protein VP40 (eVP40) is a multifunctional protein involved in viral budding as a filamentous structure and an octameric ring facilitating transcription. Binding to membranous lipids triggers conformational rearrangement of the N- and C-terminal domains of VP40 that precedes filament formation and budding. Several interdomain contacts have been identified that regulate the formation of filaments, affect dimer stability, and govern conformational dynamics. However, comprehensive dynamics and mutation information pertaining to other residues affecting eVP40 dimerization unknown. This critical information would aid in understanding eVP40 dimerization process and driving drug design targeting the viral budding process.
Methods: In the present work, computational simulations – Monte Carlo (Rosetta) and molecular dynamics (MM-GB/PBSA based alanine scanning) were used to ascertain critical interdomain residues that affect the dimerization of the VP40 monomer. Validation of computational findings performed with Hydrogen-Deuterium exchange mass spectrometry (HDX-MS), confocal microscopy and plasma membrane (PM) localization experiments.
Results/Findings: The following residues from eVP40 dimer interface affecting its dimerization revealed with aid of computational simulations – Arg52, Ile54, His61, Asp109, Ser110, Thr112, Leu117, and Arg137. HDX-MS and PM localization experiments revealed Leu51, Arg52, Ile54, Ala55, Asp56, and Asp60 to affect dimerization and impaired PM localization.
Conclusions/Discussion: Computational simulations and wet lab experiments in tandem were able to identify new residues from eVP40 dimer interface affecting its dimerization and binding to plasma membrane. Further studies are underway to study the effect of mutations on eVP40 dimerization and PM localization. The findings form the basis for structure-based design of peptide therapeutics targeting eVP40 dimerization.
Translational/Human Health Impact: Ebola has a mortality rate of ~65% and is categorized as a priority pathogen by the NIH, but there is no FDA approved treatment. Previous studies identified VP40 as a potential target. Understanding the oligomerization of VP40 will provide the basis for a targeted disruption of the essential protein-protein interactions in VP40.
