Extensive Vesicle Formation by a Monotopic Membrane Glycosyltransferase

Abstract

Over-expression of the foreign monotopic glycosyltransferase MGS in Escherichia coli triggers the formation of a large amount of internal vesicles (J. Biol. Chem. 284:33904, 2009). This phenomenon seems to be directly related with the interactions between the protein and the inner membrane lipids. It has been shown that this massive vesiculation process is not induced by the enzymatic activity of the protein, but rather due to the structural and mechanical properties of MGS. In this work we study the specific interactions of MGS with the lipid membrane and we unveil the biophysical mechanisms that lead to the massive formation of internal vesicles in E. coli.We use both molecular dynamics (MD) simulations and analytical modeling to understand the molecular basis for this phenomenon and the relative importance of various potential driving forces that may induce membrane deformations, such as lipid-protein interactions and protein crowding. First we studied the specific interactions between a single MGS and the lipid membrane. By using MD simulations we show that the N-domain (of the double Rossmann fold) has a leading role in modulating the bonds with the lipid bilayer, and that the connection between MGS and lipidic membranes is modulated by both hydrophobic and electrostatic interactions. These conclusions are in perfect agreement with chemical experiments run in parallel.Furthermore, we are using coarse-grained MD simulations to study the effects of asymmetric crowding and determine how several MGS molecules bound to the same bilayer leaflet may induce membrane deformations and trigger the formation of internal vesicles. Both experiments and simulations allow us to gain a deeper insight into the mechanism of vesicle formation by MGS, and potentially by other monotopic proteins.