Structural basis of membrane targeting and coatomer assembly by human GBP1

Abstract

Guanylate-Binding Proteins (GBPs) are interferon-inducible guanosine triphosphate hydrolases (GTPases) that mediate immune effector functions against intracellular pathogens. A key step for the antimicrobial activity of GBPs is the formation of homo- and heterooligomeric complexes on the membrane of pathogen-associated compartments or cytosol-invasive bacteria. Similar to other large GTPases of the dynamin family, oligomerisation and membrane association of GBPs depend on their GTPase activity. How nucleotide binding and hydrolysis prime GBPs for membrane targeting and coatomer formation remains unclear. Here, we report the cryo-EM structure of the full-length human GBP1 dimer in its guanine nucleotide-bound state and resolve the molecular ultrastructure of GBP1 coatomer assemblies on liposomes and bacterial lipopolysaccharide membranes. We show how nucleotide binding promotes large-scale conformational changes of the middle and GTPase effector domains that expose the isoprenylated carboxyl-terminus for association with lipid membranes. Our structure reveals how the alpha-helical stalks of the middle domain form a parallel arrangement firmly held in a unique cross-over arrangement by intermolecular contacts between adjacent monomers. This conformation is critical for GBP1 dimers to assemble into densely packed coatomers on target membranes. The extended alpha-helix of the effector domain is flexible and permits intercalation into the dense lipopolysaccharide layer on the outer membrane of gram-negative bacterial pathogens. We show that nucleotide-dependent oligomerisation and GTP hydrolysis yield GBP1 membrane scaffolds with contractile abilities that promote the formation of tubular membrane protrusions and membrane fragmentation. Collectively, our data provide a structural and mechanistic framework for interrogating the molecular basis for GBP1 effector functions in intracellular immunity.

Integrative modeling of guanylate binding protein dimers

“…Thus far, only three partial dimer structures of GBPs are available (Cui et al, 2021 ; Ghosh et al, 2006 ). The structure by Ghosh et al (Ghosh et al, 2006 ) is a dimer only formed by the G domains of hGBP1, while in the partial hGBP1 and hGBP5 dimer structures reported by others the G and M domains (the E domain is missing) interact lengthwise (Cui et al, 2021 ; Kuhm et al, 2023 ; Weismehl et al, 2023 ). However, in either case the protein–protein interaction is mainly mediated via the same G domain interaction motif.…”