Study Of Insertion Of Dengue E Into Lipid Bilayers By Neutron Reflectivity And Molecular Dynamics Simulations

The envelope (E) protein of Dengue virus rearranges to a trimeric hairpin to mediate fusion of the viral and host membranes. Insertion of E into host membranes is essential to the process, serving to anchor E into the target membrane and possibly also to disrupt local order within the membrane. Both aspects are likely to be affected by the depth of insertion, the orientation of the trimer with respect to the membrane normal, and the interactions that form between the trimer and the membrane. In the present work, we resolved the depth of insertion, the tilt angle, and the fundamental interactions for the soluble portion of Dengue E trimers (sE) associated with planar lipid bilayer membranes of various combinations of POPC with POPG, POPE, and cholesterol by neutron reflectivity (NR) and by molecular dynamics (MD) simulations. The tip of E containing the fusion loop (FL) is located at the interface of the headgroups and acyl chains of the outer leaflet of the lipid bilayers, in good agreement with prior predictions. The NR measurements and the MD simulations both indicate that E tilts with respect to the membrane normal upon insertion, promoted by either the anionic lipid POPG or cholesterol. The simulations show that tilting of the protein correlates with hydrogen bond formation between lysines located on the sides of the trimer close to the tip (K246 and K247) and nearby lipid headgroups. These hydrogen bonds provide the majority of the interaction energy whereas interactions involving the FL are a minor contribution. POPG promotes formation of these hydrogen bonds through direct interactions with K246, K247, and other polar residues whereas cholesterol indirectly facilitates formation of these hydrogen bonds as a result of a greater hydrated volume in the headgroups. Simulations in which the protein was held in a vertical orientation with respect to the membrane show that these strong hydrogen bonding interactions of K246 and K247 with lipid headgroups causes significant local membrane deformation of a 70:30 POPC:POPG bilayer as the lipids wrap around the periphery of the E trimer. We propose that these interactions play the dominant role in membrane anchoring and may also play a role in initiating mixing of the outer leaflets during the fusion process.