Structure and Mechanism of Metalloregulatory Proteins

金属调节蛋白的结构和机制

• 批准号: 8370344
• 负责人: DAVID P. GIEDROC
• 金额: $ 33.44万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8370344
• 关键词: ATP phosphohydrolase; Affinity; Animal Model; Antibiotics; Bacillus (bacterium); Bacteria; Bacterial Adhesins; Bacterial Infections; Binding; Biological; Biological Assay; Biological Availability; Buffers; Catalysis; Cells; Communication; Companions; Competence; Complex; Copper; Cytoplasm; DNA; DNA Binding; Disease; Enzymes; Fluorescence Resonance Energy Transfer; Foundations; Free Will; Freezing; Genetic Transcription; Goals; Homeostasis; Human; Image; Immunity; Invaded; Ions; Lead; Life; Link; Lipid Bilayers; Manganese; Measures; Membrane; Metal Binding Site; Metals; Modeling; Molecular; Molecular Chaperones; Mycobacterium tuberculosis; N-terminal; Nasopharynx; Nutritional; Operon; Physiology; Play; Pneumococcal vaccine; Process; Protein Family; Proteins; Regulation; Resistance; Roentgen Rays; Role; Shapes; Specificity; Staphylococcus aureus; Streptococcus pneumoniae; Structure; System; Testing; Therapeutic; Thermodynamics; Transition Elements; Virulence; Zinc; antimicrobial drug; base; cupredoxin; dimer; ferryl iron; insight; killings; metal complex; metal poisoning; metalloregulatory protein; mutant; next generation; novel; pathogen; protein transport; research study; respiratory; response; sensor; stoichiometry; tool; trafficking; uptake

DESCRIPTION (provided by applicant): Transition metal ions play highly specialized roles in biological catalysis and all cells encode regulatory machinery that controls their intracellular availability so that mis-metallation and metal toxicity is avoided. In bacteria, this process is transcriptionally regulated by a panel of "metal sensor" proteins in response to changes in metal bioavailability. The proposed experiments strongly integrate structural and cell biological approaches to elucidate determinants of metal homeostasis and cross-talk in the respiratory pathogen Streptococcus pneumoniae, focusing on zinc, manganese and copper, three metals whose concentrations change dramatically during the course of invasive disease. In the next project period, our specific aims are to 1) Obtain a structural and physicochemical characterization of the novel zinc uptake regulator S. pneumoniae AdcR (adhesin competence repressor). Here we propose NMR, structural and thermodynamic studies of wild-type and mutant AdcRs in an effort to understand allosteric activation of DNA operator binding; 2) Perform a direct test of the thermodynamic "set-point" model for zinc homeostasis in S. pneumoniae, which posits that "free" zinc is buffered in a concentration range dictated by the zinc affinities of the uptake (AdcR) and efflux (SczA, streptococcal czcD activator) regulators. Since intracellular metallation status is dictated by free or chelatable zinc, not total zinc, we propose to measure and manipulate free Zn in the cytoplasm using a FRET-based imaging assay; 3) Obtain structural and mechanistic insights into copper resistance and trafficking in S. pneumoniae, a process characterized by features not previously observed; and 4) Carry out structural studies of the CuI sensor CsoR and CsoR-related proteins in Mycobacterium tuberculosis and other bacilli. Small angle X-ray scattering, NMR and crystallographic studies are proposed. Since an emerging aspect of nutritional immunity to infection by bacterial human pathogens is the host control of metal availability, these studies lay the foundation for our long-term goal of disrupting metal availability or inducing metal toxicity by developing new antimicrobial agents that selectively target key components of transition metal homeostasis systems. PUBLIC HEALTH RELEVANCE: Bacterial pathogens require metal ions to perform processes related to both normal physiology and life- threatening invasive disease. As such, host control of metal ion availability is a critical component of the host-pathogen interface. Our studies of this process in the important respiratory pathogen Streptococcus pneumoniae may lead to the identification of new antibiotic targets that play key roles in this process.

描述（由申请人提供）：过渡金属离子在生物催化中扮演高度专业的角色，并且所有细胞编码控制其细胞内可用性的调节机制，从而避免了错误的金属化和金属毒性。在细菌中，该过程在转录下由一组“金属传感器”蛋白的调节，以应对金属生物利用度的变化。提出的实验强烈整合了结构和细胞生物学方法，以阐明金属稳态的决定因素和呼吸道病原体肺炎链球菌的跨对词的决定因素，重点介绍了锌，锰和铜，三种金属在侵入性疾病过程中浓度急剧变化。在下一个项目时期，我们的具体目的是1）获得新型锌摄取调节剂S.肺炎ADCR（粘附素能力抑制剂）的结构和物理化学表征。在这里，我们提出了野生型和突变体ADCR的NMR，结构和热力学研究，以了解DNA算子结合的变构激活； 2）对肺炎链球菌中锌稳态​​的热力学“设定点”模型进行直接测试，该模型认为“游离”锌在摄取锌亲和力（ADCR）和Efflux（SCZA，SCZA，链球菌，链球菌CZCD Activator）调节器中的浓度范围内进行了缓冲。由于细胞内金属状态由游离或螯合锌决定，而不是总锌，因此我们建议使用基于FRET的成像测定法测量和操纵细胞质中的游离锌。 3）在肺炎链球菌中获得对铜的耐药性和运输的结构和机械洞察，这一过程为特征以前未观察到的特征； 4）在结核分枝杆菌和其他杆菌中进行CUI传感器CSOR和与CSOR相关的蛋白进行结构研究。提出了小角度X射线散射，NMR和晶体学研究。由于通过细菌性人类病原体感染营养免疫的新兴方面是金属可用性的宿主控制，因此，我们的长期目标是破坏金属可用性或诱导金属毒性的长期目标，从而选择性地靶向过渡金属稳态系统的关键成分，从而诱导金属毒性。 公共卫生相关性：细菌病原体要求金属离子执行与正常生理和威胁生命的侵入性疾病有关的过程。因此，金属离子可用性的宿主控制是宿主 - 病原体界面的关键组成部分。我们在重要的呼吸病原体肺炎链球菌中对这一过程的研究可能导致鉴定出在此过程中起关键作用的新抗生素靶标。