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Structure and Mechanism of Staphylococcus Aureus Tarm, The Structure and mechanism of Staphylococcus aureus PNAS PLUS TarM, the wall teichoic acid α-glycosyltransferase Solmaz Sobhanifara,1, Liam James Worralla,1, Robert J. Gruningera, Gregory A. Wasneya, Markus Blaukopfb, Lars Baumannb, Emilie Lameignerea, Matthew Solomonsona, Eric D. Brownc, Stephen G. Withersb, and Natalie C. J. Strynadkaa,2 aDepartment of Biochemistry and Center for Blood Research, University of British Columbia, Vancouver, BC, Canada V6T 1Z3; bDepartment of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1; and cDepartment of Chemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada L8S 4K1 Edited by Scott J. Hultgren, Washington University School of Medicine, St. Louis, MO, and approved December 30, 2014 (received for review September 19, 2014) Unique to Gram-positive bacteria, wall teichoic acids are anionic conditions, but that its ability to colonize and infect is greatly glycopolymers cross-stitched to a thick layer of peptidoglycan. The compromised (5, 6). Deletion of LTA leads to temperature-sen- polyol phosphate subunits of these glycopolymers are decorated sitive strains only viable at temperatures lower than 30 °C, and, with GlcNAc sugars that are involved in phage binding, genetic interestingly, strains with deletions of both WTA and LTA are exchange, host antibody response, resistance, and virulence. The nonviable, suggesting some degree of redundancy in their re- Staphylo- search for the enzymes responsible for GlcNAcylation in spective roles (7). TAs have furthermore been implicated in re- coccus aureus has recently identified TarM and TarS with respective sistance to antimicrobial molecules (8–11), resistance to lysozyme α β – - and -(1 4) glycosyltransferase activities. The stereochemistry of (11), coping with environmental stresses (7, 12), mediating in- the GlcNAc attachment is important in balancing biological pro- teractions with receptors and biomaterials (6, 13), induction of cesses, such that the interplay of TarM and TarS is likely important inflammation (14–16), phage binding (17, 18), and biofilm for- for bacterial pathogenicity and survival. Here we present the crys- tal structure of TarM in an unusual ternary-like complex consisting mation (19). Faced with so many functions in an uncertain envi- ronment, TAs must remain highly adaptive, a large part of which of a polymeric acceptor substrate analog, UDP from a hydrolyzed MICROBIOLOGY donor, and an α-glyceryl-GlcNAc product formed in situ. These is achieved by the D-alanylation and glycosylation of polyol hy- structures support an internal nucleophilic substitution-like mech- droxyl groups. These modifications have a significant effect on the anism, lend new mechanistic insight into the glycosylation of gly- physical and interactive properties of the cell wall. copolymers, and reveal a trimerization domain with a likely role in In S. aureus, C4 hydroxyls of polyRboP are heavily substituted acceptor substrate scaffolding. with GlcNAc. The configuration of the glycosidic linkage varies, with certain strains of S. aureus containing WTA with almost glycosyltransferase | wall teichoic acid | cell wall | crystal structure | exclusively α-orβ-O-linked GlcNAc, and others displaying bacterial pathogenicity a mixture of anomers (20, 21). An important role of WTA sugar modification in S. aureus is that of a receptor for bacteriophage hallmark difference between Gram-negative and Gram- binding. This role was discovered early on, when phage-resistant Apositive bacteria relates to the composition of their cell wall. mutants were observed to lack GlcNAc in their WTA (18). WTA Gram-negative bacteria possess an inner and an outer mem- brane, the latter of which is lined by a thin layer of peptidoglycan. Significance Gram-positive bacteria, in contrast, lack this outer membrane, instead possessing a thick layer of peptidoglycan complemented This paper describes the structure of Staphylococcus aureus by anionic glycopolymers known as teichoic acids (TAs) that can TarM, an enzyme responsible for the glycosylation of wall comprise an astonishing 60% of the dry weight of the cell wall (1). teichoic acid that is important in pathological processes such as Charged with this thick cross-stitched protective matrix, Gram- host immunity, phage binding, and antibiotic resistance in positive bacteria are able to inhabit severe environments such strains such as Methicillin-resistant S. aureus. The TarM struc- as the gastrointestinal tract, similar to their Gram-negative ture is presented in an unusual ternary-like complex that fea- counterparts. tures a polymeric acceptor substrate analogue and a trapped TAs are constitutively manufactured and covalently incor- product of enzyme action, lending novel structural and mech- porated into the cell walls of Gram-positive bacteria by attachment anistic insight into the glycosylation of glycopolymers. More to peptidoglycan in the case of wall TAs (WTAs) or to the plasma generally, the positioning of this product in the active site as membrane as with lipoteichoic acids (LTAs). TA composition well as the distorted conformation of its pyranose ring provide varies widely depending on bacterial strain and subtype (reviewed direct structural evidence for an internal substitution-like cat- in ref. 2). In most Staphylococcus aureus strains, WTA consists of alytic mechanism for retaining GT-B class enzymes. polyribitol phosphate (polyRboP) chains of 40–60 repeats, at- tached via a disaccharide linkage unit to the C6 hydroxyl group Author contributions: S.S., L.J.W., R.J.G., and N.C.J.S. designed research; S.S., L.J.W., R.J.G., of occasional N-acetylmuramic acid residues of peptidoglycan (3). G.A.W., M.B., L.B., E.L., and M.S. performed research; S.S., L.J.W., R.J.G., G.A.W., M.B., L.B., E.L., M.S., E.D.B., S.G.W., and N.C.J.S. analyzed data; and S.S., L.J.W., and N.C.J.S. wrote LTA composition tends to be less diverse than WTA, and in the paper. S. aureus most often contains polyglycerol phosphate attached to The authors declare no conflict of interest. the plasma membrane via a glycolipid anchor (reviewed in ref. 4). This article is a PNAS Direct Submission. Although our current understanding of the exact functions of Data deposition: The atomic coordinates and structure factors have been deposited in the TAs is incomplete, it is clear that bacteria invest an impressive Protein Data Bank, www.pdb.org (PDB ID codes 4X6L, 4X7M, 4X7P, and 4X7R). amount of energy and resources in their construction and main- 1S.S. and L.J.W. contributed equally to this work. tenance. Indeed, efforts to study these extraordinary polymers 2To whom correspondence should be addressed. Email: [email protected]. have yielded some important findings. For instance, it is known This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. that, in the absence of WTA, S. aureus is viable under laboratory 1073/pnas.1418084112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1418084112 PNAS Early Edition | 1of10 Downloaded by guest on October 1, 2021 GlcNAc residues have also long been recognized as important tals (2.1–2.4 Å) were obtained that were found to contain UDP antigens in the host-antibody response (22–24). and UDP-GlcNAc bound monomers in the asymmetric unit. A Recently, Xia et al. identified a phage-resistant S. aureus RN4220 complex with fondaparinux, a pentameric, polyanionic synthetic transposon mutant, whose disrupted gene was designated as heparin used clinically as an anticoagulant (trade name Arixtra; TarM, a soluble cytoplasmic glycosyltransferase (GT) enzyme GlaxoSmithKline), was also obtained. This compound was ini- (25). According to the Carbohydrate-Active Enzymes database, tially tested as a ligand on the basis of the high affinity of TarM for which classifies GTs into 94 families based on sequence identity, heparin Sepharose that was exploited during purification. Binding TarM belongs to the GT4 family that includes various anomeric of fondaparinux was confirmed by affinity measurements de- stereochemistry-retaining enzymes (26). On verification, recom- scribed later, and its defined length and similar physicochemical α binant TarM was shown to -O-glycosylate WTA in vitro in a properties to those of polyRboP (both are polysaccharides with – UDP-GlcNAc dependent manner. Soon after, a second S. aureus negatively charged functional groups) made it a suitable acceptor β inverting GT responsible for the -O-GlcNAcylation of WTA was analog candidate for cocrystallization. In addition to fonda- identified and designated as TarS (27). The differences in the parinux, the aforementioned structure also contained product UDP stereochemistries of these GlcNAc glycosidic linkages appear to as well as an α-glyceryl-GlcNAc glycoside product. Phases for directly influence the biology and pathogenicity of Gram-positive the native trimeric UDP bound structure were solved by using bacteria, often on a strain-specific level (24, 27, 28). selenomethionine single-wavelength anomalous dispersion (SAD) Here, to our knowledge, we present the first structure of TarM methods. All other structures were subsequently solved with mo- in a ternary-like complex with a polyRboP acceptor substrate lecular replacement by using the native structure as a search model. analog, fondaparinux, as well as a cleaved donor UDP-GlcNAc. In addition, the surprising TarM-catalyzed formation of α-glyceryl- Refinement statistics of the final structural models are presented GlcNAc and its positioning in the active
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