Structural Studies of Protein-Carbohydrate Interactions

蛋白质-碳水化合物相互作用的结构研究

• 批准号: RGPIN-2020-06021
• 负责人: Evans, Stephen
• 金额: $ 4.23万
• 依托单位: University of Victoria
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=740979
• 关键词: Structural; Studies; Protein; Carbohydrate; Interactions

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.

这项建议的重点是(1)探索抗体(Ab)对寡糖抗原的反应，以及(2)利用结构-功能方法，主要是X射线结晶学，合成和结合次级细胞壁聚合物(SCWPs)。首先，与蛋白质和多肽不同，碳水化合物免疫原通常不能诱导亲和力成熟和类别转换，这导致抗体种系经历了巨大的进化压力，需要直接识别碳水化合物。已有研究表明，抗原决定簇的多样性远远超过了抗体种系的组合潜力，这就要求至少有一些种系抗体表现出多特异性。我们正在研究几组抗体。首先，在多重特异性的有力证明中，我们正在探索模仿原始碳水化合物抗原的抗独特型抗体。在另一个方面，我们正在探索两个近胚系抗体采用多个稳定构象的能力，通过这些构象可以通过构象选择进行多特异性抗原识别。在第三项研究中，我们正在研究糖肽特异性抗体的有趣的交叉反应潜力。其次，自组装蛋白表层(S层)是原核生物常见的细胞壁特征，参与关键生理过程，包括营养分解、细胞壁组装，在感染的情况下，还涉及宿主细胞黏附。在革兰氏阳性细菌中，S层蛋白通过与特定的肽聚糖连接的SCWP的表层同源结构域固定在细胞壁上。在几个革兰氏阳性细菌菌株中，这种相互作用依赖于丙酮化的SCWP。值得注意的是，尽管丙酮化的SCWP随处可见，但令人惊讶的是，人们在分子水平上对它们的生物合成、识别或伴随而来的治疗抑制的洞察知之甚少。来自模式生物Paenibacillusalvei CCM 2051T的SCWP由丙酮化的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-乙酰甘露糖基从UDP-ManNAc到末端GlcNAc的转移，以及CSAB催化的丙酮基从磷酸烯醇式丙酮酸到末端ManNAc-GlcNAc受体的转移。我们在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)方面取得了重大进展，目标是确定它们的结构，并与合成的给体和受体类似物形成络合物。最后，我们正在扩大对SLH结构域SPAA的结构研究。我们最近在《自然通讯》上发表了SPAA与末端SCWP单糖的络合物的结构。揭示了蛋白质中意想不到的构象变化，我们证明了这一点具有生物学意义。我们正朝着使用更长和更具生物相关性的合成SCWP片段来表征SPAA SLH相互作用的方向发展。