Lipopolysaccharide chain length and coat assembly keep guanylate-binding proteins upright on bacterial membranes

Abstract

Guanylate-binding proteins (GBPs) defend host cells by assembling on intracellular pathogen membranes. For human GBP1, this requires a switch from a compact, closed conformation to an extended, open one, whereas for murine orthologues the open state is not known. How the open state behaves at a membrane has remained inaccessible to experiment. Using all-atom and coarse-grained molecular dynamics simulations covering over 0.5 ms, we followed human GBP1 and murine GBP2 as monomers, dimers, and higher-order assemblies on phosphatidylcholine membranes, Shigella flexneri lipopolysaccharide (LPS), and phosphoinositide-containing bilayers. Closed GBPs dock through their prenyl anchor with minimal lipid selectivity. Open GBPs, by contrast, are intrinsically unstable: monomers fold back into membrane-bound resting states, and dimers tip over unless supported, either by long LPS O-antigen chains or by neighboring molecules within a coat. The open state thus appears to be maintained not by the nucleotide alone but by the protein's molecular environment.