Molecular insight into the efficient & robust design of vesiculated protein nano-cages

Recently, recapitulation of viromimetic function in non-viral protein nanocages (PNCs) has emerged as a strategy to successfully encapsulate them in membrane vesicles. This method successfully evaded immune system detection. The mechanism responsible for triggering membrane budding and vesiculation remains elusive. This is primarily because the membrane initially interacts with flat protein arrangements from nanocages (whether pyramidal, dodecahedral, or icosahedral), and it is unclear how these seemingly flat arrangements can overcome the inherent mechanical resistance of the lipid bilayer to induce curvature. In this study, we considered a trimeric interface of a dodecahedron nanocage and explored the energetic and molecular role of its viromimetic module in protein nanocage packaging. Using a combination of all-atom and MARTINI coarse-graining molecular dynamics, we show that a stronger highly basic region (HBR) promotes electrostatic sequestration of PIP2 lipids, known for their larger headgroups, to accumulate around trimer binding sites, forming a PIP2 depletion zone in the central region of the trimer interface. Such lipid-sorting events resulted in membrane thickness distributions with taller lipids accumulating toward the margins and shorter ones at the center of the trimer and inducing curvature to the lipid bilayer due to stretching and contraction in different regions of the lipid interface. Our findings give molecular-level mechanistic insights into curvature generation and propagation in membranes induced by engineered PNC interactions, as well as a generic molecular design approach for clathrin-independent nanoparticle exocytosis.