Metal Binding to the Bacterial Cell Wall

金属与细菌细胞壁的结合

• 批准号: 8463215
• 负责人: Charles V Rice
• 金额: $ 24.03万
• 依托单位: UNIVERSITY OF OKLAHOMA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8463215
• 关键词: Abbreviations; Acetylglucosamine; Address; Adsorption; Alanine; Amides; Amino Acids; Antibiotic Therapy; Antibiotics; Architecture; Area; Bacillus (bacterium); Bacillus anthracis; Bacteria; Binding; Biochemical; Biochemistry; Biology; Cadmium; Cell Wall; Cells; Chemicals; Chemistry; Complex; Coupled; Cytolysis; Data; Development; Dialysis procedure; Divalent Cations; Drug Design; Environment; Equilibrium; Faculty; Foundations; Genetic; Genus staphylococcus; Goals; Gram-Positive Bacteria; Health; Homeostasis; Human; Ions; Kinetics; Knowledge; Label; Lipids; Measurement; Measures; Mechanics; Mediating; Membrane; Metal Ion Binding; Metals; Methods; Microbiology; Modeling; Molecular Models; Molecular Structure; Mono-S; NMR Spectroscopy; Outcome; Peptidoglycan; Physiological; Polyamines; Polymers; Research; Resolution; Resources; Rice; Role; Sampling; Series; Site; Solvents; Spectrum Analysis; Staphylococcus aureus; Structure; System; Technology; Teichoic Acids; Training; Translating; Vertebral column; Virulence; Water; Work; absorption; antimicrobial; base; chelation; computational chemistry; crosslink; drug development; extracellular; functional group; innovation; inorganic phosphate; insight; member; molecular dynamics; molecular modeling; mutant; novel; pathogen; pathogenic bacteria; polyglycerol; polyribitol phosphate; programs; public health relevance; quantum; research study; solid state nuclear magnetic resonance; structural biology; uptake

DESCRIPTION (provided by applicant): Binding of metals to the bacterial cell wall is essential for peptidoglycan integrity and metal ion homeostasis. Although a potential antibiotic target, drug development suffers from insufficient understanding of how metal binding occurs. The peptidoglycan (PG) and teichoic acids (TA) work in harmony to form the metal binding pocket. Our long-term goal is to provide chemistry and biochemistry explanations of TA function in its different physiological roles. The objective of this application is to determine these rules with regard to metal ion binding in cell walls composed of TA and A13 or A31 PG. These form the sacculus for pathogens B. anthracis and S. aureus, respectively. The central hypothesis of this application is that the metal binding mechanism is mediated through solvent-separated ion-pairs with anionic groups and/or amide carbonyls of PG and the TA polymer. This hypothesis arises from Preliminary NMR Data used to measure the 111Cd2+ to TA distance, which is long enough to allow water molecules to separate these species. Likewise, NMR data show that the phosphate to D-Ala distance is 4.5 ¿ and increases to 5.4 ¿ when Mg2+ is present. Molecular modeling of this distance constraint yields a solvent-separated zwitterion pair. This result contradicts the current paradigm of TA in metal binding, where D-Ala and phosphate supposedly form a contact ion pair and inhibit the chelation of mono and divalent cations. Additional NMR data show that metal binding brings the TA polymer closer the D-Ala group of the PG. This is the first measurement of the TA/PG architecture. Preliminary data guide the development of two specific aims: 1) Identify Changes in TA Structure Upon Metal Chelation; and 2) Characterize the Cell Wall (TA and PG) Structure Before and After Metal Adsorption. The approach uses equilibrium dialysis of Cd2+, Mg2+, Ca2+, K+, and Na+ with cell wall (PG+TA), PG only, and TA only. The concentration of free ions is measured with atomic absorption spectroscopy, providing kinetic data for the equilibrium binding constants. This functional data provides mechanistic insight to the structural data collected with REDOR NMR spectroscopy. Here, 13C and 15N isotopic labeling of the TA and PG components enables REDOR NMR to measure the internuclear distances. Molecular models of localized structure are created with ab-initio calculations with the NMR-based distance constraints. Molecular dynamics simulations using the TA/PG interactions generate models of the cell wall architecture. The innovation of this work arises because it capitalizes on advances in NMR spectroscopy, genetic mutants, and isotopic labeling to solve a complex biochemical problem. Metal binding in the cell wall is an under-investigated, complex, and biologically important system where solid-state NMR experiments could make a truly high impact and yield high-resolution structural information. The proposed research is significant because solid-state NMR methods are coupled with quantum mechanical calculations to elucidate the interactions between teichoic acid, metals, and peptidoglycan. If successful, these studies could potentially guide the development of novel antibiotics.

描述(由申请人提供)： 金属与细菌细胞壁的结合对于肽聚糖的完整性和金属离子的动态平衡是必不可少的。尽管药物开发是一个潜在的抗生素靶点，但对金属结合是如何发生的了解不足。肽聚糖(PG)和磷壁酸(TA)协调工作，形成金属结合口袋。我们的长期目标是为TA在不同生理作用下的功能提供化学和生物化学解释。本申请的目的是确定关于TA和A13或A31 PG组成的细胞壁中的金属离子结合的这些规则。它们分别形成了炭疽杆菌和金黄色葡萄球菌的球囊。这一应用的中心假设是，金属结合机制是通过与PG和TA聚合物的阴离子基团和/或酰胺基团的溶剂分离离子对来调节的。这一假设源于初步的核磁共振数据，该数据用于测量111Cd2+到TA的距离，该距离足以使水分子区分这些物种。同样，核磁共振数据表明，磷酸到D-丙氨酸的距离为4.5？，当有镁离子存在时，距离增加到5.4？这种距离限制的分子模拟产生了溶剂分离的两性离子对。这一结果与目前TA在金属结合中的范式相矛盾，在金属结合中，D-丙氨酸和磷酸盐被认为形成接触离子对，并抑制一价和二价阳离子的螯合。另外的核磁共振数据表明，金属结合使TA聚合物更接近PG的D-Ala基团。这是对TA/PG架构的第一次测量。初步数据指导制定了两个具体目标：1)确定金属螯合后TA结构的变化；2)表征金属吸附前后的细胞壁(TA和PG)结构。该方法利用细胞壁(PG+TA)平衡透析Cd~(2+)、Mg~(2+)、Ca~(2+)、K~+和Na~+，仅PG和TA。用原子吸收光谱测量了自由离子的浓度，提供了平衡结合常数的动力学数据。这些功能数据提供了对用REDOR核磁共振光谱学收集的结构数据的机械洞察力。在这里，TA和PG组分的13C和15N同位素标记使Redor核磁共振能够测量核间距离。局域结构的分子模型是用基于核磁共振的距离约束的从头计算建立的。使用TA/PG相互作用的分子动力学模拟产生了细胞壁结构的模型。这项工作的创新之处在于，它利用了核磁共振光谱学、基因突变和同位素标记的进展来解决一个复杂的生化问题。细胞壁中的金属结合是一个未被研究的、复杂的、具有生物学意义的系统，在其中，固态核磁共振实验可以产生真正的高影响并产生高分辨率的结构信息。这项拟议的研究意义重大，因为固体核磁共振方法与量子力学计算相结合，以阐明磷壁酸、金属和肽聚糖之间的相互作用。如果成功，这些研究可能会指导新型抗生素的开发。