Metal Binding to the Bacterial Cell Wall

金属与细菌细胞壁的结合

• 批准号: 8270487
• 负责人: Charles V Rice
• 金额: $ 24.91万
• 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/8270487
• 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; 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的稳态至关重要。虽然是潜在的抗生素靶点，但药物开发对金属结合如何发生的理解不足。肽聚糖（PG）和磷壁酸（TA）协调工作以形成金属结合口袋。我们的长期目标是提供化学和生物化学解释TA功能在其不同的生理作用。本申请的目的是确定关于由TA和A13或A31 PG组成的细胞壁中的金属离子结合的这些规则。这些形成病原体B的球囊。anthracis和S.金黄色葡萄球菌。本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。这一假设来自于用于测量111Cd2+到TA距离的初步NMR数据，该距离足够长以允许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+时增加到5.4。这种距离约束的分子建模产生了溶剂分离的两性对。这一结果与TA在金属结合中的当前范例相矛盾，其中D-Ala和磷酸根据称形成接触离子对并抑制一价和二价阳离子的螯合。额外的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结构的第一次测量。初步数据指导了两个具体目标的开发：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。 该方法使用Cd2+、Mg2+、Ca2+、K+和Na+与细胞壁（PG + TA）、仅PG和仅TA的平衡透析。用原子吸收光谱法测量游离离子的浓度，为平衡结合常数提供动力学数据。该功能数据为用REDOR NMR光谱收集的结构数据提供了机理见解。在这里，TA和PG组分的13 C和15 N同位素标记使得REDOR NMR能够测量核间距离。局域化结构的分子模型创建与从头计算的NMR为基础的距离约束。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了NMR光谱学、遗传突变体和同位素标记的进步来解决复杂的生物化学问题。细胞壁中的金属结合是一个研究不足的、复杂的和生物学上重要的系统，固态NMR实验可以产生真正高的影响并产生高分辨率的结构信息。该研究是重要的，因为固态NMR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。 公共卫生关系： 减轻细菌对人类健康的有害影响需要精确的细菌生物化学知识。固态NMR方法非常适合于解决金属结合机制。我们的数据可以解释B之间的金属结合差异。anthracis和S.金黄色葡萄球菌在生物化学机制已知后，金属螯合作用可以用抗生素治疗来阻断。