Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry

Kaveesha J Wijesinghe,Sarah Urata,Nisha Bhattarai,Edgar E Kooijman,Bernard S Gerstman,Prem P Chapagain,Sheng Li,Robert V Stahelin

doi:10.1074/jbc.m116.758300

Abstract

Marburg virus (MARV) is a lipid-enveloped virus from the Filoviridae family containing a negative sense RNA genome. One of the seven MARV genes encodes the matrix protein VP40, which forms a matrix layer beneath the plasma membrane inner leaflet to facilitate budding from the host cell. MARV VP40 (mVP40) has been shown to be a dimeric peripheral protein with a broad and flat basic surface that can associate with anionic phospholipids such as phosphatidylserine. Although a number of mVP40 cationic residues have been shown to facilitate binding to membranes containing anionic lipids, much less is known on how mVP40 assembles to form the matrix layer following membrane binding. Here we have used hydrogen/deuterium exchange (HDX) mass spectrometry to determine the solvent accessibility of mVP40 residues in the absence and presence of phosphatidylserine and phosphatidylinositol 4,5-bisphosphate. HDX analysis demonstrates that two basic loops in the mVP40 C-terminal domain make important contributions to anionic membrane binding and also reveals a potential oligomerization interface in the C-terminal domain as well as a conserved oligomerization interface in the mVP40 N-terminal domain. Lipid binding assays confirm the role of the two basic patches elucidated with HD/X measurements, whereas molecular dynamics simulations and membrane insertion measurements complement these studies to demonstrate that mVP40 does not appreciably insert into the hydrocarbon region of anionic membranes in contrast to the matrix protein from Ebola virus. Taken together, we propose a model by which association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers.

Highlights

Through hydrophobic C-terminal domain (CTD)-CTD interactions Ebola VP40 (eVP40) dimers can further assemble into hexamers that have been shown to be crucial for viral matrix assembly and budding [12]. eVP40 dimers were shown to assemble into hexamers via the CTD-CTD interface as well as a NTD interface that is somewhat buried in the dimer
The NTD of eVP40 was important in oligomerization of rearranged hexamers following eVP40 conformational changes induced by a membrane mimetic as described previously [12]. ␤2, ␤3, and ␤7 strands together form an antiparallel ␤ sheet structure and we found the concentration of two major deuteration level changes in this region is intriguing
We hypothesize that this ␤ sheet structure provides an oligomerization interface for the MARV VP40 (mVP40) NTD following association with PS containing membranes

Summary

To whom correspondence may be addressed

Association of the mVP40 dimer with the anionic plasma membrane facilitates assembly of mVP40 oligomers. A conformational change is hypothesized to occur in eVP40 monomers or dimers, which would exclude the monomers at the ends of the hexamer, displacing the CTD from the NTD revealing the oligomerization site This conformational displacement exposes a residue, which was much more buried in the dimer, Trp. The NTD-NTD dimer interface interactions are shown to be important for viral assembly and budding as mutation of threonine (Thr105) at the dimer interface prevented mVP40 assembly, budding of VLPs, and greatly reduced anionic membrane binding [11]. The key residues involved in NTD oligomerization in eVP40 hexamers were found to align in sequence and structure with mVP40, Trp and Asn148, respectively Mutation of this Trp and Asn in mVP40 greatly reduced VLP budding [11]. Molecular dynamics (MD) simulations, and membrane insertion measurements were used to complement the HDX-MS work and elucidate the mechanism by which mVP40 associates with the plasma membrane

Results

Discussion

Experimental procedures