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稳态至关重要。尽管潜在的抗生素靶标，但药物发育却不足以了解金属结合的发生方式。肽聚糖（PG）和Teichoic Acid（TA）和谐起作用，形成金属结合袋。我们的长期目标是在其不同的身体角色中提供对TA功能的化学和生物化学解释。该应用的目的是确定由TA和A13或A31 Pg组成的细胞壁中的金属离子结合。这些分别形成了病原体的囊。该应用的中心假设是金属结合机理是通过溶剂分离的离子对介导的，该离子对与PG和TA聚合物的阴离子基和/或酰胺羰基介导。该假设来自用于测量111CD2+ TA距离的初步NMR数据，该数据足够长，可以使水分子分离这些物种。同样，NMR数据表明，在存在Mg2+时，磷酸盐至D-ALA的距离为4.5€，并增加到5.4？。该距离约束的分子建模产生溶剂分离的zwitterion对。该结果与金属结合中TA的当前范式相矛盾，在金属结合中，D-ALA和磷酸盐预期形成接触离子对，并抑制单声道和二价阳离子的螯合作用。其他NMR数据表明，金属结合使TA聚合物更接近Pg的D-ALA组。这是TA/PG体系结构的第一个测量。初步数据指南的发展两个特定目的：1）在金属螯合时确定TA结构的变化； 2）表征金属吸附之前和之后的细胞壁（TA和PG）结构。该方法将CD2+，Mg2+，Ca2+，K+和Na+的等效透析与细胞壁（PG+TA），仅PG和TA一起使用。通过原子吸收光谱法测量游离离子的浓度，为等效结合常数提供了动力学数据。该功能数据为使用重新NMR光谱收集的结构数据提供了机械洞察力。在这里，TA和PG组件的13C和15N同位素标记使REDOR NMR能够测量核切间距离。通过基于NMR的距离约束，创建了局部结构的分子模型。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。这项工作的创新之所以出现，是因为它利用了NMR光谱，基因突变体和同位素标记的进步，以解决复杂的生化问题。细胞壁中的金属结合是一种不足的，复杂且在生物学上重要的系统，固态NMR实验可以产生真正的高影响并产生高分辨率的结构信息。提出的研究之所以重要，是因为固态NMR方法与量子机械计算相结合，以阐明Teichoic Acid，Metals和Pepperidoglycan之间的相互作用。如果成功，这些研究可以潜在地指导新型抗生素的发展。