Structural Studies of Protein-Carbohydrate Interactions

该提案的重点是（1）探索抗体（Ab）对寡糖抗原的反应和（2）使用结构-功能方法（主要是X射线晶体学）合成和结合二级细胞壁聚合物（SCWP）。 首先，与蛋白质和肽不同，碳水化合物免疫原通常不能诱导亲和力成熟和类别转换，这导致Ab种系经历直接识别碳水化合物的巨大进化压力。已经表明，抗原决定簇的多样性远远超过了Ab种系的组合潜力，这需要至少一些种系Ab来显示多特异性。 我们正在研究几组腹肌。在一个强有力的证明多特异性，我们正在探索抗独特型抗体，模仿原来的碳水化合物抗原。在另一个，我们正在探索的能力，两个近种系抗体，通过多特异性抗原识别可能会通过构象选择，通过采用多种稳定的构象。在第三个，我们正在研究有趣的交叉反应潜力的抗体特异性的糖肽。 第二，自组装蛋白质表面层（S层）是涉及关键生理过程的常见原核细胞壁特征，所述关键生理过程包括营养物分解、细胞壁组装以及在感染的情况下的宿主细胞粘附。在革兰氏阳性细菌中，S层蛋白通过与特定肽聚糖连接的SCWP连接的表面层同源性（SLH）结构域锚定在细胞壁上。在几种革兰氏阳性细菌菌株中，这种相互作用依赖于乙酰基化的SCWP。值得注意的是，尽管乙酰化SCWP普遍存在，但令人惊讶的是，在分子水平上对它们的生物合成、识别或伴随的对这种理解带来的治疗抑制的见解知之甚少。 来自模式生物蜂房类芽孢杆菌CCM 2051 T的SCWP由乙酰化GlcNAc-ManNAc二糖重复序列组成。 SCWP延伸涉及的两个步骤是TagA催化的N-乙酰甘露糖胺基从UDP-ManNAc转移到末端GlcNAc，以及CsaB催化的N-乙酰甘露糖基从磷酸烯醇丙酮酸转移到末端ManNAc-GlcNAc受体。 我们已经在TagA（属于CAZy家族26，只有一个结构表征的例子）和CsaB（目前还没有合适的模型）上取得了重大进展，旨在确定它们的结构unliganded和与合成供体和受体类似物的复合物。 最后，我们正在扩展SLH结构域SpaA的结构研究。我们最近在Nature Commun上发表了SpaA与末端SCWP单糖复合的结构。揭示了一个意想不到的构象转变的蛋白质，我们证明有生物学意义。我们正朝着使用更长和更生物相关的合成SCWP片段表征SpaA SLH相互作用的方向发展。

This proposal is focused (1) on the exploring antibody (Ab) response to oligosaccharide antigens and (2) on the synthesis and binding of secondary cell wall polymers (SCWPs) using a structure-function approach, primarily X-ray crystallography. First, unlike proteins and peptides, a carbohydrate immunogen cannot generally induce affinity maturation and class switching, which results in the Ab germline experiencing great evolutionary pressure to recognize carbohydrates directly. It has been shown that the diversity of antigenic determinants far exceeds the combinatorial potential of the Ab germline, which requires at least some germline Abs to display polyspecificity. We are studying several groups of Abs. In one, in a powerful demonstration of polyspecificity, we are exploring anti-idiotypic Abs that mimic the original carbohydrate antigens. In another, we are exploring the ability of two near-germline Abs to adopt multiple stable conformations through which polyspecific antigen recognition may proceed via conformational selection. In a third, we are studying the interesting cross-reactive potential of an Ab specific for a glycopeptide. Second, self-assembling protein surface layers (S-layers) are common prokaryotic cell wall features involved in key physiological processes including nutrient breakdown, cell wall assembly and, in the case of infection, host cell adhesion. In Gram-positive bacteria, S-layer proteins are anchored to the cell wall by surface layer homology (SLH) domains linked to specific peptidoglycan-linked SCWPs. In several Gram-positive bacterial strains this interaction relies on pyruvylated SCWPs. Remarkably, despite the ubiquity of pyruvylated SCWPs, surprisingly little is known at the molecular level about their biosynthesis, recognition, or the concomitant insights into therapeutic inhibition such understanding would bring. The SCWP's from model organism Paenibacillus alvei CCM 2051T consists of pyruvylated GlcNAc-ManNAc disaccharide repeats. Two steps involved in SCWP extension are TagA-catalyzed transfer of a N-acetylmannosaminyl group from UDP-ManNAc to the terminal GlcNAc, and CsaB-catalyzed transfer of a pyruvyl group from phosphoenolpyruvate to the terminal ManNAc-GlcNAc acceptor. We have made significant progress on TagA (which belongs to CAZy family 26 with only one structurally characterized example) and CsaB (for which there is currently no suitable model), and aim to determine their structures unliganded and in complex with synthetic donor and acceptor analogues. Finally, we are extending structural studies of the SLH domain SpaA. Our recent publication of the structure of SpaA in complex with the terminal SCWP monosaccharide reported in Nature Commun. revealed an unexpected conformational shift in the protein which we demonstrated had biological significance. We are moving toward characterizing SpaA SLH interactions using longer and more biologically relevant synthetic SCWP fragments.