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Acetylation of surface carbohydrates in bacterial pathogens requires coordinated action of a two-domain membrane-bound acyltransferase

DOI: 10.1128/mBio.01364-20 DOI Help

Authors: Caroline R. Pearson (University of York) , Sarah N. Tindall (University of York) , Reyme Herman (University of York) , Huw Jenkins (University of York) , Alex Bateman (European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI)) , Gavin H. Thomas (University of York) , Jennifer R. Potts (University of York) , Marjan W. Van Der Woude (University of York)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Mbio , VOL 11

State: Published (Approved)
Published: August 2020
Diamond Proposal Number(s): 13587

Open Access Open Access

Abstract: Membrane bound acyltransferase-3 (AT3) domain-containing proteins are implicated in a wide range of carbohydrate O-acyl modifications, but their mechanism of action is largely unknown. O-antigen acetylation by AT3 domain-containing acetyltransferases of Salmonella spp. can generate a specific immune response upon infection and can influence bacteriophage interactions. This study integrates in situ and in vitro functional analyses of two of these proteins, OafA and OafB (formerly F2GtrC), which display an “AT3-SGNH fused” domain architecture, where an integral membrane AT3 domain is fused to an extracytoplasmic SGNH domain. An in silico-inspired mutagenesis approach of the AT3 domain identified seven residues which are fundamental for the mechanism of action of OafA, with a particularly conserved motif in TMH1 indicating a potential acyl donor interaction site. Genetic and in vitro evidence demonstrate that the SGNH domain is both necessary and sufficient for lipopolysaccharide acetylation. The structure of the periplasmic SGNH domain of OafB identified features not previously reported for SGNH proteins. In particular, the periplasmic portion of the interdomain linking region is structured. Significantly, this region constrains acceptor substrate specificity, apparently by limiting access to the active site. Coevolution analysis of the two domains suggests possible interdomain interactions. Combining these data, we propose a refined model of the AT3-SGNH proteins, with structurally constrained orientations of the two domains. These findings enhance our understanding of how cells can transfer acyl groups from the cytoplasm to specific extracellular carbohydrates.

Journal Keywords: O-antigen; SGNH superfamily; Salmonella; acetylation; acyltransferase-3 family; Gram-negative bacteria; lipopolysaccharide; membrane proteins; structure-activity relationship; surface antigens

Subject Areas: Biology and Bio-materials

Instruments: I04-1-Macromolecular Crystallography (fixed wavelength)