Abstract
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, biochemical, and cellular studies