Department of Biochemistry & Microbiology, University of Victoria, Victoria, British Columbia, Canada.
Department of NanoBiotechnology, NanoGlycobiology Unit, Universität für Bodenkultur Wien, Vienna, Austria.
J Biol Chem. 2022 Apr;298(4):101745. doi: 10.1016/j.jbc.2022.101745. Epub 2022 Feb 18.
Self-assembling (glyco)protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and other processes, which makes them appealing targets for therapeutics and biotechnological applications as biosensors or drug delivery systems. However, unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment. S-layers of Gram-positive bacteria often are attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Cocrystal structures of the SLH domain trimer from the Paenibacillus alvei S-layer protein SpaA (SpaA) with synthetic, terminal SCWP disaccharide and trisaccharide analogs, together with isothermal titration calorimetry binding analyses, reveal that while SpaA accommodates longer biologically relevant SCWP ligands within both its primary (G2) and secondary (G1) binding sites, the terminal pyruvylated ManNAc moiety serves as the nearly exclusive SCWP anchoring point. Binding is accompanied by displacement of a flexible loop adjacent to the receptor site that enhances the complementarity between protein and ligand, including electrostatic complementarity with the terminal pyruvate moiety. Remarkably, binding of the pyruvylated monosaccharide SCWP fragment alone is sufficient to cause rearrangement of the receptor-binding sites in a manner necessary to accommodate longer SCWP fragments. The observation of multiple conformations in longer oligosaccharides bound to the protein, together with the demonstrated functionality of two of the three SCWP receptor-binding sites, reveals how the SpaA-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division.
自组装（糖）蛋白表面层（S-层）是普遍存在于原核细胞表面的结构，参与结构维持、营养扩散、宿主黏附、毒力等过程，这使得它们成为治疗和生物技术应用的有吸引力的靶点，如生物传感器或药物输送系统。然而，要挖掘这种潜力，就需要扩大我们对 S-层特性的理解，尤其是对表面附着细节的理解。革兰氏阳性细菌的 S-层通常通过 S-层同源（SLH）结构域三聚体与肽聚糖连接的次级细胞壁聚合物（SCWP）的相互作用来附着。来自巴氏芽胞杆菌 S-层蛋白 SpaA（SpaA）的 SLH 结构域三聚体与合成的、末端 SCWP 二糖和三糖类似物的共晶结构，以及等温滴定量热法结合分析，揭示了虽然 SpaA 在其主要（G2）和次要（G1）结合位点内容纳了更长的生物相关 SCWP 配体，但末端丙酮酸化 ManNAc 部分是几乎唯一的 SCWP 锚固点。结合伴随着与受体结合位点相邻的柔性环的位移，这增强了蛋白质和配体之间的互补性，包括与末端丙酮酸部分的静电互补性。值得注意的是，仅结合末端吡喃酮化单糖 SCWP 片段就足以引起受体结合位点的重排，从而适应更长的 SCWP 片段。在与蛋白质结合的较长寡糖中观察到多种构象，以及三个 SCWP 受体结合位点中的两个表现出的功能，揭示了 SpaA-SCWP 相互作用是如何进化以适应更长的 SCWP 配体，并减轻细菌 S-层附着在生长和分裂过程中固有的应变。
