Structural analysis of acyltransferases involved in O-antigen modification

Acyltransferase-3 (AT3) domain-containing proteins are involved in acylation of a diverse range of carbohydrates across all domains of life. In bacteria they are essential in processes including symbiosis, resistance to antimicrobials, and biosynthesis of antibiotics. AT3 domain containing proteins from Salmonella spp. are responsible for acetylation of the O-antigen of lipopolysaccharides (LPS), this can generate a specific immune response upon infection and influence bacteriophage interactions. Despite this, the mechanism of action of these AT3 proteins is largely unknown. Here we analysed two AT3 proteins, OafA and OafB, which acetylate O-antigen of Salmonella serovars. Both contain an integral membrane AT3 domain fused to a periplasmic SGNH domain, essential residues from each domain suggest both domains are required for substrate acetylation. The crystal structure of the SGNH domain from OafA was determined and found to form a dimer with each subunit similar to the previously solved structure of the SGNH domain from OafB. However, striking differences are seen in surface loops surrounding the active site. The region linking the AT3 and SGNH domains was previously found to be structured in OafB and form an extension of the SGNH domain (SGNH-ext). Removal of this region in multiple AT3-SGNH proteins suggests that the SGNH-ext is important for stability of some SGNH domains. The structure of the SGNH-extension suggests the SGNH domain is located in close proximity to the acyltransferase domain and the domains may interact. Co-evolution analysis, used to make predictions about the structure of both domains, suggests that inter-domain interactions are likely. This co-evolution analysis was used further to predict the structure of OafB and suggests AT3-SGNH proteins are likely to form a novel fold. Combining these data, we propose a refined model of the AT3-SGNH proteins which enhances our understanding of the mechanism and structure of the AT3 protein family required for modification of cell-surface carbohydrates.