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.

细菌糖结合物是一组不同的大分子，在细菌存活和 通过调节细菌与宿主的相互作用而致病。尽管它们的结构多样化，但许多糖共轭化合物 是由原核生物利用一种共同的机制制造的，这包括将多糖转移到Pren-PP- 膜界面上的连接载体。糖偶联物的惊人多样性是通过连续的 糖基转移酶(GTS)通过糖基转移酶(GTS)将糖从可溶性核苷酸激活转移到糖链上 脂质载体分子的供体(NDP-糖)。尽管在所有的生命王国中无处不在， GTS仍然是抗生素的极好靶点，因为它们对抗生素表现出非凡的选择性 在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 弯曲杆菌等致病细菌中的蛋白质-蛋白质相互作用是抗生素的关键 这项研究提案致力于解决这一知识鸿沟。迄今为止，C. 空肠弯曲菌GT在弯曲杆菌属中已被证实，但PglA、PglJ、PglH1、 而另一种具有临床意义的人类病原体--致密梭菌的PglH2仍然无法获得。另外， 从同一生物体的糖共轭生物合成途径中鉴定GT将提供结构 未来蛋白质-蛋白质相互作用研究所需的信息，并将为分析澄清 这些蛋白质相互作用，调节途径的流量。该提案的目标1将确定首选的GT基材 使用纳米差示扫描荧光法(NanDSF)，并使用 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 用于纯化GT的结晶，以及优化蛋白质晶体和收集X射线晶体数据以获得 高分辨率的晶体结构。目标3将描述来自GT-B序列的一些同源基因 相似性网络(SSN)，以深入了解成组进化并精确定位主要序列变量 这解释了它们不同的底物特性。GT残基之间的蛋白质间协方差将是 通过Gremlin方法进行调查，以确定对假设的形成负责的相互作用 这些酶之间的超分子复合体。由此产生的发现将揭示具有约束力的决定因素。 控制致病原核生物中普遍存在的糖链的特异性并展示关键的结构功能 链接和蛋白质-蛋白质相互作用。