Kuhm, T. I. | bioRxiv Science

bioRxiv2026

State-selective nanobodies to probe Guanylate-binding protein coat assembly and function

Cell-autonomous immunity against gram-negative bacterial pathogens relies on Guanylate-binding proteins (GBPs). GBPs are interferon-inducible guanosine triphosphate hydrolases that coat cytosolic bacteria and promote non-canonical inflammasome activation. GBP coat assembly is initiated by GBP1, which binds bacterial lipopolysaccharide and recruits further paralogs in a hierarchical and nucleotide-dependent manner. Dissecting the contributions of individual GBPs has remained challenging because the seven human paralogs are structurally similar and undergo nucleotide-driven conformational changes that are difficult to resolve in cellular and biochemical experiments. Here, we developed a nanobody toolbox for the state-selective probing of human GBP coat assembly. We identify eight nanobodies with distinct paralog, domain, and conformational specificities and define their binding profiles and epitopes using complementary biophysical assays and cryogenic electron microscopy. We further demonstrate the utility of selected nanobodies by demonstrating their functionality in membrane reconstitution assays and in infected cells. A dimer-disrupting nanobody blocks progression of GBP-coat assembly from soluble GBP1 assemblies to LPS-associated coats, suggesting a stage-specific structural transition during coat formation. In contrast, a dimer selective nanobody binds assembled coats without measurably perturbing their organisation and is robustly recruited to GBP1-positive bacteria in infected cells, indicating that this dimer interface remains intact and accessible in coat-associated GBP1. Together, these nanobodies establish a set of state-selective reagents to distinguish, perturb, and probe GBP assemblies in structural and cellular studies.

biophysics↗

bioRxiv2023

Structural basis of membrane targeting and coatomer assembly by human GBP1

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 cytosolinvasive 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 -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 -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.

biophysics↗