Acetylation of Surface Carbohydrates in Bacterial Pathogens Requires Coordinated Action of a Two-Domain Membrane-Bound Acyltransferase
Acetylation of Surface Carbohydrates in Bacterial Pathogens Requires Coordinated Action of a Two-Domain Membrane-Bound Acyltransferase
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DOI：
10.1128/mbio.01364-20
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发表时间：
2020-07-01
期刊：
影响因子：
6.4
通讯作者：
Van der Woude, Marjan W.
中科院分区：
文献类型：
--
作者：
Pearson, Caroline R.;Tindall, Sarah N.;Van der Woude, Marjan W.
膜结合酰基转移酶-3（AT 3）结构域的蛋白质涉及广泛的碳水化合物0-酰基修饰，但其作用机制在很大程度上是未知的。0-沙门氏菌属的含AT 3结构域的乙酰转移酶的抗原乙酰化可以在感染时产生特异性免疫应答，并且可以影响噬菌体相互作用。本研究整合了两个这些蛋白质，OafA和OafB（以前的F2 GtrC），显示了一个“AT 3-SGNH融合”的结构域架构，其中一个完整的膜AT 3域融合到一个胞外SGNH域的原位和体外功能分析。一个在电脑启发的诱变方法的AT 3域确定了7个残基，这是基本的OafA的作用机制，特别是保守的基序在TMH 1表明一个潜在的酰基供体相互作用位点。遗传和体外证据表明，SGNH结构域是必要的和足够的脂多糖乙酰化。OafB的周质SGNH结构域的结构鉴定了先前未报道的SGNH蛋白的特征。特别地，结构域间连接区的周质部分是结构化的。值得注意的是，该区域限制受体底物特异性，显然是通过限制进入活性位点。这两个领域的协同进化分析表明可能的域间相互作用。结合这些数据，我们提出了一个改进的模型的AT 3-SGNH蛋白，结构上限制的方向的两个域。这些发现增强了我们对细胞如何将酰基从细胞质转移到特定胞外碳水化合物的理解。重要信息酰基转移酶-3（AT 3）结构域包含的膜蛋白参与各种碳水化合物的O-乙酰化，这些碳水化合物跨越生命的所有领域。在细菌中，它们在包括共生、抗微生物剂抗性和抗生素生物合成在内的过程中是必不可少的。然而，其作用机制的特点很差。我们分析了两个乙酰转移酶作为模型，这个重要的家庭的膜蛋白，修改碳水化合物的表面上的病原体沙门氏菌肠道，影响免疫原性，毒力和噬菌体耐药性。我们表明，当这些AT 3域融合到一个周质伴侣域，这两个域都需要底物乙酰化。数据显示了AT 3结构域中的保守元件和周质结构域的独特结构特征。我们的数据提供了一个工作模型来探测广泛分布的AT 3蛋白家族的不同和重要成员的机制和功能，这些成员是细胞表面碳水化合物进行生物学显著修饰所必需的。
Membrane bound acyltransferase-3 (AT3) domain-containing proteins are implicated in a wide range of carbohydrate 0-acyl modifications, but their mechanism of action is largely unknown. 0-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.IMPORTANCE Acyltransferase-3 (AT3) domain-containing membrane proteins are involved in 0-acetylation of a diverse range of carbohydrates across all domains of life. In bacteria they are essential in processes including symbiosis, resistance to anti-microbials, and biosynthesis of antibiotics. Their mechanism of action, however, is poorly characterized. We analyzed two acetyltransferases as models for this important family of membrane proteins, which modify carbohydrates on the surface of the pathogen Salmonella enterica, affecting immunogenicity, virulence, and bacteriophage resistance. We show that when these AT3 domains are fused to a periplasmic partner domain, both domains are required for substrate acetylation. The data show conserved elements in the AT3 domain and unique structural features of the periplasmic domain. Our data provide a working model to probe the mechanism and function of the diverse and important members of the widespread AT3 protein family, which are required for biologically significant modifications of cell-surface carbohydrates.