Structure and Mechanism of Staphylococcus aureus TarS, the Wall Teichoic Acid β-glycosyltransferase Involved in Methicillin Resistance

In recent years, there has been a growing interest in teichoic acids as targets for antibiotic drug design against major clinical pathogens such as Staphylococcus aureus, reflecting the disquieting increase in antibiotic resistance and the historical success of bacterial cell wall components as drug targets. It is now becoming clear that β-O-GlcNAcylation of S. aureus wall teichoic acids plays a major role in both pathogenicity and antibiotic resistance. Here we present the first structure of S. aureus TarS, the enzyme responsible for polyribitol phosphate β-O-GlcNAcylation. Using a divide and conquer strategy, we obtained crystal structures of various TarS constructs, mapping high resolution overlapping N-terminal and C-terminal structures onto a lower resolution full-length structure that resulted in a high resolution view of the entire enzyme. Using the N-terminal structure that encapsulates the catalytic domain, we furthermore captured several snapshots of TarS, including the native structure, the UDP-GlcNAc donor complex, and the UDP product complex. These structures along with structure-guided mutants allowed us to elucidate various catalytic features and identify key active site residues and catalytic loop rearrangements that provide a valuable platform for anti-MRSA drug design. We furthermore observed for the first time the presence of a trimerization domain composed of stacked carbohydrate binding modules, commonly observed in starch active enzymes, but adapted here for a poly sugar-phosphate glycosyltransferase.

近年来，磷壁酸（teichoic acids）作为抗临床主要病原菌（如金黄色葡萄球菌（Staphylococcus aureus））的抗生素研发靶点受到日益广泛的关注，这一趋势既反映出抗生素耐药性问题的严峻增长态势，也印证了细菌细胞壁组分作为药物靶点的历史成功经验。目前已有明确研究表明，金黄色葡萄球菌细胞壁磷壁酸的β-O-乙酰葡糖胺修饰（β-O-GlcNAcylation）在其致病性与抗生素耐药性中均发挥关键作用。本研究首次解析了负责聚核糖醇磷酸（polyribitol phosphate）β-O-乙酰葡糖胺修饰的TarS酶（TarS）的三维结构。本研究采用分而治之的策略，解析了多种TarS重组构建体的晶体结构，并将高分辨率的N端与C端重叠结构拟合至低分辨率的全长结构中，最终获得了完整酶的高分辨率三维视图。借助包含催化结构域的N端结构，我们还捕获了TarS的多个瞬时结构快照，包括天然态结构、尿苷二磷酸-N-乙酰葡糖胺（UDP-GlcNAc）供体复合物结构以及尿苷二磷酸（UDP）产物复合物结构。这些结构结合结构导向突变体研究，帮助我们阐明了TarS的多种催化特性，鉴定了关键的活性位点残基以及催化环的构象重排现象，为抗耐甲氧西林金黄色葡萄球菌（MRSA）药物研发提供了极具价值的研究平台。此外，我们首次观测到由堆叠的碳水化合物结合模块（carbohydrate binding modules）构成的三聚化结构域：这类结构域通常见于淀粉活性酶（starch active enzymes）中，但本研究中其功能适配于多糖磷酸糖基转移酶（poly sugar-phosphate glycosyltransferase）。