Insertion of Dengue E into lipid bilayers studied by neutron reflectivity and molecular dynamics simulations

Abstract

The envelope (E) protein of Dengue virus rearranges to a trimeric hairpin to mediate fusion of the viral and target membranes, which is essential for infectivity. Insertion of E into the target membrane serves to anchor E and possibly also to disrupt local order within the membrane. Both aspects are likely to be affected by the depth of insertion, orientation of the trimer with respect to the membrane normal, and the interactions that form between trimer and 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 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and cholesterol (CHOL) by neutron reflectivity (NR) and by molecular dynamics (MD) simulations. The results show that 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 results also indicate that E tilts with respect to the membrane normal upon insertion, promoted by either the anionic lipid POPG or CHOL. The simulations show that tilting of the protein correlates with hydrogen bond formation between lysines and arginines located on the sides of the trimer close to the tip (K246, K247, and R73) and nearby lipid headgroups. These hydrogen bonds provide a major contribution to the membrane anchoring and may help to destabilize the target membrane.

Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering

“…To determine the orientation of the NEC on the membrane and how deeply it inserts into the lipid bilayer, we turned to neutron reflectometry (NR) ( 43 ), which allows low-resolution structural characterization of the membrane and any associated protein. A tethered lipid bilayer composed of POPC/POPS/POPA in a 3:1:1 molar ratio was prepared in a flow cell, and the reflectivity and scattering length densities of the bilayer interface to a collimated neutron beam, incident at various angles, was measured before and after incubation with NEC220 at 100 nM or 500 nM ( Fig.…”

Highly basic clusters in the HSV-1 nuclear egress complex drive membrane budding by inducing lipid ordering

“…To determine the orientation of the NEC on the membrane and how deeply it inserts into the lipid bilayer, we turned to neutron reflectometry (NR) (Vanegas et al, 2018) Within the NEC coats formed in vitro and in vivo, the NEC molecules are oriented perpendicularly to the plane of the membrane, with the protein density extending ~110 Å from the membrane, in accordance with the cryo-EM measurements (Bigalke et al, 2014;Hagen et al, 2015). However, the NEC220 density profile obtained from the NR measurements only extended to ~90 Å, and an orientation probability plot showed significant tilt of the NEC220 from a vertical orientation (Fig 3C).…”

Computational modelling of flavivirus dynamics: The ins and outs

“…For example, several all-atom simulation studies investigated viral envelope conformational changes occurring upon exposure to low pH, both during the fusion and maturation processes [26][27][28][29][30][31][32][33][34]. The capacity for individual E/M proteins or their complexes to initiate membrane fusion [35][36][37][38][39][40] and host membrane bending [41] has also been extensively studied via simulation. Nevertheless, it is important to consider the dynamics of the ~50 nm diameter flavivirus envelope in its entirety [42,43].…”