Molecular insight toward 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, primarily because the membrane initially interacts with a flat arrangement of proteins from nanocages (whether their shape is pyramidal, dodecahedron or icosahedron) and it is unclear how these seemingly flat protein 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 on protein nanocage packaging. Using a combination of all-atom and Martini coarse-graining molecular dynamics, we show that stronger highly basic region (HBR) promotes electrostatic sequestration of PIP2 lipids, known for their larger headgroups, around trimer binding sites, forming a PIP2 depletion zone in the central region of the trimer interface. Such lipid-sorting event resulted in membrane-thickness distribution with taller lipids accumulating toward the margins and shorter at the center of the trimer and inducing curvature to the lipid bilayer due to stretching and contraction events at different lipid interfaces. 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 clathrinindependent nanoparticle exocytosis.