Structural and functional characterization of glycosyltransferases in the Campylobacter concisus N-linked glycoconjugate biosynthetic pathway

弯曲杆菌 N 连接糖复合物生物合成途径中糖基转移酶的结构和功能表征

• 批准号: 10607139
• 负责人: Nemanja Vuksanovic
• 金额: $ 6.95万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10607139
• 关键词: Active Sites; Address; Affinity; Anabolism; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Antibiotic Resistance; Binding; Biochemical; Bioinformatics; Biological Assay; Buffers; Campylobacter; Campylobacter jejuni; Complex; Crystallization; Data; Deltaproteobacteria; Detergents; Development; Engineering; Enzymes; Epitopes; Epsilonproteobacteria; Evolution; Exhibits; Future; Glycoconjugates; Glycoproteins; Goals; Human; Investigation; Kinetics; Knowledge; Label; Laboratories; Life; Link; Lipids; Mediating; Mediation; Membrane; Membrane Proteins; Methods; Network-based; Nucleotides; Organism; Orthologous Gene; Pathogenicity; Pathway interactions; Patients; Play; Polysaccharides; Population; Prokaryotic Cells; Proteins; Rapid screening; Research Personnel; Research Proposals; Resolution; Resources; Roentgen Rays; Role; Sampling; Scanning; Site-Directed Mutagenesis; Specificity; Structure; Substrate Specificity; Testing; Therapeutic; Transferase; Uridine Diphosphate Sugars; Variant; Virulence; Work; X-Ray Crystallography; analog; biophysical techniques; clinically relevant; clinically significant; effective therapy; experimental study; glycosyltransferase; human pathogen; inhibitor; inorganic phosphate; insight; luminescence; macromolecule; member; nano; novel; pathogenic bacteria; preference; protein protein interaction; screening; structural determinants; sugar; sugar nucleotide

Bacterial glycoconjugates are a diverse group of macromolecules that play a key role in bacterial survival and pathogenicity by mediating bacteria-host interactions. Despite their structural diversity, many glycoconjugates are made by prokaryotes utilizing a common mechanism, which includes transferring glycans to a Pren-PP- linked carrier at the membrane interface. The striking diversity of glycoconjugates is achieved through sequential addition of glycans by glycosyltransferases (GTs), which transfer sugars from soluble nucleotide-activated donors (NDP-sugars) to the lipid-based carrier molecule. Despite being ubiquitous across all kingdoms of life, GTs still represent an excellent target for antibiotics, due to the remarkable degree of selectivity they exhibit for structurally very similar sugars. Understanding the structural features that influence GT substrate selectivity and protein-protein interactions in pathogenic bacteria like Campylobacter species is critical for antibiotic development, and this research proposal strives to address that knowledge gap. To date, the substrates for C. jejuni GTs have been confirmed in the Campylobacter genus, but structural information for PglA, PglJ, PglH1, and PglH2 of C. concisus, another clinically significant human pathogen, is still unavailable. Additionally, characterizing GTs from the same organism's glycoconjugate biosynthetic pathway will offer the structural information needed for future protein-protein interaction studies and will pave the way for analysis to clarify how these proteins interact to modulate pathway flux. Aim 1 of this proposal will identify preferred GT substrates using nano-differential scanning fluorimetry (nanoDSF) and determine steady-state kinetic parameters using luminescence-based GT activity assays. Aim 2 will be to discover the detergent and buffer conditions required for crystallization of purified GTs, as well as to optimize protein crystals and collect X-ray crystal data to obtain high-resolution crystal structures. Aim 3 will be to characterize a number of orthologs from a GT-B sequence similarity network (SSN) in order to gain insight into GT evolution and to pinpoint the primary sequence variables that account for their various substrate specificities. Interprotein covariance between GT residues will be investigated via the GREMLIN method in order to identify interactions responsible for the hypothesized formation of supramolecular complexes between these enzymes. The resulting findings will reveal binding determinants controlling specificity for glycans prevalent in pathogenic prokaryotes and demonstrate crucial structure-function links and protein-protein interactions.

细菌性糖缀合物是一组多样的大分子，在细菌存活中起关键作用 通过介导细菌宿主相互作用的致病性。尽管它们的结构多样性，但许多糖缀合物 是通过利用通用机制的原核生物制成的，其中包括将聚糖转移到pren-pp- 膜接口处的链接载体。糖缀合物的惊人多样性是通过顺序实现的 通过糖基转移酶（GTS）添加糖，从而从可溶性核苷酸激活中转移糖 基于脂质的载体分子的供体（NDP-糖）。尽管在生活的所有王国中无处不在，但 由于它们表现出显着的选择性，GTS仍然代表了抗生素的绝佳靶标 结构上非常相似的糖。了解影响GT底物选择性的结构特征和 病原细菌（如弯曲杆菌）中的蛋白质 - 蛋白质相互作用对于抗生素至关重要 发展，这项研究建议致力于解决这一知识差距。迄今为止，C。 空肠GTS已在弯曲杆菌属中得到证实，但PGLA，PGLJ，PGLH1， C. cocisus的PGLH2是另一种临床上显着的人类病原体，仍然不可用。此外， 表征来自同一生物体的糖缀合物生物合成途径的GTS将提供结构性 将来蛋白质 - 蛋白质相互作用研究所需的信息，并将为分析铺平道路，以阐明如何 这些蛋白质相互作用以调节途径通量。该提案的目标1将确定首选的GT基板 使用纳米差异扫描荧光法（Nanodsf），并确定使用稳态动力学参数 基于发光的GT活性测定。目标2将是发现所需的洗涤剂和缓冲条件 纯化的GTs以及优化蛋白质晶体并收集X射线晶体数据以获得的结晶 高分辨率晶体结构。 AIM 3将是表征来自GT-B序列的许多直系同源物 相似性网络（SSN），以洞悉GT演化并查明主要序列变量 该解释其各种底物特异性。 GT残留物之间的解释蛋白协方差将是 通过Gremlin方法进行了研究，以确定负责假设形成的相互作用 这些酶之间的超分子复合物。最终的发现将揭示结合决定因素 控制在致病性原核生物中普遍存在的聚糖的特异性，并证明了至关重要的结构功能 链接和蛋白质 - 蛋白质相互作用。