Structure-function analysis of the cyclic β-1,2-glucan synthase from Agrobacterium tumefaciens

The synthesis of complex sugars is a key aspect of microbial biology. Cyclic β-1,2-glucan (CβG) is a circular polysaccharide critical for host interactions of many bacteria, including major pathogens of humans (Brucella) and plants (Agrobacterium). CβG is produced by the cyclic glucan synthase (Cgs), a multi-domain membrane protein. So far, its structure as well as the mechanism underlining the synthesis have not been clarified. Here we use cryo-electron microscopy (cryo-EM) and functional approaches to study Cgs from A. tumefaciens. We determine the structure of this complex protein machinery and clarify key aspects of CβG synthesis, revealing a distinct mechanism that uses a tyrosine-linked oligosaccharide intermediate in cycles of polymerization and processing of the glucan chain. Our research opens possibilities for combating pathogens that rely on polysaccharide virulence factors and may lead to synthetic biology approaches for producing complex cyclic sugars.


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Christoph DehioJan 16, 2024 SerialEM 3; 3; MotionCor2; Focus 11; Digital Micrograph 3; 3; EPU; No No custom code was developed for this study CryoSparc 2; 2; CTFFIND4; UCSF Chimera 1.16; UCSF ChimeraX 1.7; Coot 0.9.4;Phenix 1.18; Molprobity; Origin 9.7; GraphPad Prism 9; 9; Geneious Prime 2023; No No custom code was developed for this study All data needed to to evaluate the conclusions in in the paper are present in in the paper and/or the Supplementary Materials.PDB models generated in in this study have been deposited at at the Protein Data Bank with accession numbers of of 8RF0 (CgsWT), 8RF9 (CgsiGP1), 8RFE (CgsiGP2), 8RFG (CgsiGP3).Cryo-EM maps determined in in this study have been deposited at at the Electron Microscopy Data Bank with accession numbers of ofEMD-19114 (CgsWT),EMD-19116 (CgsiGP1),EMD-19118  (CgsiGH2),EMD-19119 (CgsiGP3),EMD-19123 (CgsiGT).Raw data files and FragPipe output files related to to the mass spectrometry analysis of of Cgs glycosylation haveField-specific reportingFor a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdfLifesciencesstudy designAll studies must disclose on these points even when the disclosure is negative.