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

View Article: PubMed Central - PubMed

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.

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mVP40 interaction with PI(4,5)P2 containing vesicles results in a similar hydrogen/deuterium exchange pattern compared with PS containing vesicles.A, space filling models of mVP40 protein after preincubation with PI(4,5)P2 vesicles (right) and PS + PI(4,5)P2 combined vesicles (left). Side view of a mVP40 monomer is also shown for both conditions. B, difference in deuteration percentage of mVP40 mapped to the sequence over the 10-s exchange period for all lipid compositions tested. Color coding: blue suggests the regions that exchange slower in the presence of liposomes; and red suggests the regions that exchange faster in the presence of liposomes.
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Figure 13: mVP40 interaction with PI(4,5)P2 containing vesicles results in a similar hydrogen/deuterium exchange pattern compared with PS containing vesicles.A, space filling models of mVP40 protein after preincubation with PI(4,5)P2 vesicles (right) and PS + PI(4,5)P2 combined vesicles (left). Side view of a mVP40 monomer is also shown for both conditions. B, difference in deuteration percentage of mVP40 mapped to the sequence over the 10-s exchange period for all lipid compositions tested. Color coding: blue suggests the regions that exchange slower in the presence of liposomes; and red suggests the regions that exchange faster in the presence of liposomes.

Mentions: We have previously shown mVP40 to be an anionic charge sensor that exclusively interacts with negatively charged lipids (17). We have hypothesized that for the structural changes in mVP40 necessary for oligomerization to occur, the mVP40 dimer must first form interactions with the phospholipid membrane. Therefore, we performed several other HDX-MS experiments where we preincubated mVP40 protein with lipid vesicles that were composed entirely of zwitterionic phospholipid/phosphatidylcholine. In the absence of an anionic membrane surface, mVP40 cannot form significant electrostatic interactions with membranes and hence does not bind zwitterionic membranes at nanomolar or micromolar concentrations (17). In support of the central hypothesis, the HDX pattern of mVP40 protein alone and mVP40 in the presence of PC vesicles looked very similar at 10- and 100-s time points, indicating the protein exhibited no significant changes in the solvent accessibility in the presence of PC liposomes (Figs. 11 and 12A). This is in stark contrast to the HDX-MS pattern observed when mVP40 was preincubated with PI(4,5)P2 or PS vesicles, which caused significant reduction in solvent accessibility (Fig. 13B). We also observed that at the 1000-s time point, mVP40 exhibited higher deuterium incorporation compared with the protein alone sample suggesting an increase in the solvent accessibility of the protein in the presence of neutral phospholipid membrane (Figs. 11B and 12B). This can be a result of nonspecific zwitterionic interactions causing the mVP40 protein to slightly deform its structure making some regions more accessible to solvent than in the absence of PC. However, further studies are necessary to understand this occurrence and its significance including more HDX-MS data at longer time points (10,000 s and 100,000 s) especially to see if the trend continues.


Detection of lipid-induced structural changes of the Marburg virus matrix protein VP40 using hydrogen/deuterium exchange-mass spectrometry
mVP40 interaction with PI(4,5)P2 containing vesicles results in a similar hydrogen/deuterium exchange pattern compared with PS containing vesicles.A, space filling models of mVP40 protein after preincubation with PI(4,5)P2 vesicles (right) and PS + PI(4,5)P2 combined vesicles (left). Side view of a mVP40 monomer is also shown for both conditions. B, difference in deuteration percentage of mVP40 mapped to the sequence over the 10-s exchange period for all lipid compositions tested. Color coding: blue suggests the regions that exchange slower in the presence of liposomes; and red suggests the regions that exchange faster in the presence of liposomes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5391744&req=5

Figure 13: mVP40 interaction with PI(4,5)P2 containing vesicles results in a similar hydrogen/deuterium exchange pattern compared with PS containing vesicles.A, space filling models of mVP40 protein after preincubation with PI(4,5)P2 vesicles (right) and PS + PI(4,5)P2 combined vesicles (left). Side view of a mVP40 monomer is also shown for both conditions. B, difference in deuteration percentage of mVP40 mapped to the sequence over the 10-s exchange period for all lipid compositions tested. Color coding: blue suggests the regions that exchange slower in the presence of liposomes; and red suggests the regions that exchange faster in the presence of liposomes.
Mentions: We have previously shown mVP40 to be an anionic charge sensor that exclusively interacts with negatively charged lipids (17). We have hypothesized that for the structural changes in mVP40 necessary for oligomerization to occur, the mVP40 dimer must first form interactions with the phospholipid membrane. Therefore, we performed several other HDX-MS experiments where we preincubated mVP40 protein with lipid vesicles that were composed entirely of zwitterionic phospholipid/phosphatidylcholine. In the absence of an anionic membrane surface, mVP40 cannot form significant electrostatic interactions with membranes and hence does not bind zwitterionic membranes at nanomolar or micromolar concentrations (17). In support of the central hypothesis, the HDX pattern of mVP40 protein alone and mVP40 in the presence of PC vesicles looked very similar at 10- and 100-s time points, indicating the protein exhibited no significant changes in the solvent accessibility in the presence of PC liposomes (Figs. 11 and 12A). This is in stark contrast to the HDX-MS pattern observed when mVP40 was preincubated with PI(4,5)P2 or PS vesicles, which caused significant reduction in solvent accessibility (Fig. 13B). We also observed that at the 1000-s time point, mVP40 exhibited higher deuterium incorporation compared with the protein alone sample suggesting an increase in the solvent accessibility of the protein in the presence of neutral phospholipid membrane (Figs. 11B and 12B). This can be a result of nonspecific zwitterionic interactions causing the mVP40 protein to slightly deform its structure making some regions more accessible to solvent than in the absence of PC. However, further studies are necessary to understand this occurrence and its significance including more HDX-MS data at longer time points (10,000 s and 100,000 s) especially to see if the trend continues.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus