Metal Binding to the Bacterial Cell Wall

金属与细菌细胞壁的结合

• 批准号: 8071135
• 负责人: Charles V Rice
• 金额: $ 24.92万
• 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/8071135
• 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; Public Health; 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. PUBLIC HEALTH RELEVANCE: Alleviating harmful effects of bacteria on human health requires precise knowledge of bacterial biochemistry. Solid-state NMR methods are ideally suited to address the metal binding mechanism. Our data can explain metal binding differences between B. anthracis and S. aureus pathogens. After the biochemical mechanisms are known, metal chelation can be blocked with antibiotic therapies.

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稳态至关重要。尽管是潜在的抗生素靶点，但药物开发却因对金属结合如何发生的了解不足而受到困扰。肽聚糖 (PG) 和磷壁酸 (TA) 协同作用形成金属结合袋。我们的长期目标是提供 TA 不同生理作用的化学和生物化学解释。本申请的目的是确定关于由 TA 和 A13 或 A31 PG 组成的细胞壁中金属离子结合的这些规则。它们分别形成病原体炭疽芽孢杆菌和金黄色葡萄球菌的球囊。该申请的中心假设是金属结合机制是通过溶剂分离的离子对与PG和TA聚合物的阴离子基团和/或酰胺羰基介导的。这一假设源自用于测量 111Cd2+ 到 TA 距离的初步 NMR 数据，该距离足够长，足以让水分子分离这些物质。同样，NMR 数据显示磷酸盐与 D-Ala 的距离为 4.5 ¿，当存在 Mg2+ 时，该距离增加至 5.4 ¿这种距离约束的分子建模产生了溶剂分离的两性离子对。这一结果与金属结合中 TA 的当前范例相矛盾，其中 D-Ala 和磷酸盐据推测形成接触离子对并抑制单价和二价阳离子的螯合。其他 NMR 数据表明，金属结合使 TA 聚合物更接近 PG 的 D-Ala 基团。这是TA/PG架构的第一次测量。初步数据指导两个具体目标的制定：1）识别金属螯合时 TA 结构的变化； 2) 表征金属吸附前后的细胞壁（TA 和 PG）结构。 该方法使用 Cd2+、Mg2+、Ca2+、K+ 和 Na+ 与细胞壁 (PG+TA)、仅 PG 和仅 TA 的平衡透析。使用原子吸收光谱测量自由离子的浓度，提供平衡结合常数的动力学数据。这些功能数据为通过 REDOR NMR 波谱收集的结构数据提供了机制洞察。此处，TA 和 PG 组分的 13C 和 15N 同位素标记使 REDOR NMR 能够测量核间距离。局域结构的分子模型是通过基于 NMR 的距离约束的从头算来创建的。使用 TA/PG 相互作用的分子动力学模拟生成细胞壁结构的模型。这项工作的创新之处在于它利用核磁共振波谱、基因突变和同位素标记的进步来解决复杂的生化问题。细胞壁中的金属结合是一个尚未充分研究的复杂且具有生物学重要性的系统，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。这项研究意义重大，因为固态核磁共振方法与量子力学计算相结合，阐明了磷壁酸、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。 公共卫生相关性： 减轻细菌对人类健康的有害影响需要对细菌生物化学的精确了解。固态核磁共振方法非常适合解决金属结合机制。我们的数据可以解释炭疽芽孢杆菌和金黄色葡萄球菌病原体之间的金属结合差异。了解生化机制后，可以用抗生素疗法阻断金属螯合。