Year: 2026
Type: preprint
Abstract: Guanylate-binding proteins (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, nucleotide-dependent manner. Dissecting individual GBP functions remains challenging because the seven human paralogs are structurally similar and undergo nucleotide-driven conformational changes. Here, we developed a nanobody toolbox for 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 biophysical assays and cryogenic electron microscopy. Selected nanobodies were functionally validated in membrane reconstitution assays and infected cells. An inhibitory nanobody blocks progression 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 recruited to GBP1-positive bacteria, indicating that this dimer interface remains intact and accessible in coat-associated GBP1. Together, these nanobodies provide state-selective reagents to distinguish, perturb, and probe GBP assemblies in structural and cellular studies.
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W7219836234