Parameters Involved in Metal Recognition

描述(由申请人提供)： 保持适当的细胞内金属离子水平是所有生物体生存的关键。细菌的生存能力，包括人类病原体，一直与获得或竞争过渡金属的能力有关。几种人类疾病被证明是由于细胞金属贩运的崩溃造成的。就过渡金属而言，保持适当的金属浓度涉及控制拥有功能酶等所需的最佳水平与金属有毒浓度之间的微妙平衡。为了实现这一控制，生物体已经开发出机制，以确保获得生长所需的特定金属，排除有毒金属，并控制它们的细胞浓度。这些金属运输系统依赖于能够区分通常具有相似大小和电荷的金属离子的蛋白质。涉及的金属蛋白统称为金属转运蛋白，控制金属的摄取和外排(金属转运体)，目标是将金属输送到特定的酶(金属离子转运体)，并调节其他蛋白的表达，以响应金属离子浓度(金属调节剂)。虽然许多蛋白质对不同的金属离子实现这些功能的例子已经被表征，但允许金属识别和金属特异的生物反应的机制还不是很清楚。拟议研究的总体目标是了解使运输蛋白质能够区分金属的结构参数，以及驱动特定金属生物反应的相关蛋白质结构变化。在许多运输蛋白质中，金属识别是必不可少的，以便产生针对一种或一小群金属的生物反应，并避免与其他金属离子的串扰。为了实现这一目标，我们计划在大肠杆菌和幽门螺杆菌中检测与金属识别有关的结构参数，包括金属转运蛋白(NICA)、金属配位酮(HYPA)和金属调节剂(NikR和RcnR)。该方法包括克隆和表达运输蛋白，表征它们的金属离子亲和力，阐明金属中心的结构，使用X射线吸收光谱(XAS)作为金属中心结构的探针，并使用结晶学和核磁共振来检测蛋白质结构。通过定点突变可以引起金属结合位点和其他关键蛋白质特征的改变，并解决对金属位点结构和/或蛋白质结构的影响。包括体外和体内试验的策略被用来评估对突变运输蛋白功能的影响。除了对基本生物化学的了解外，对金属转移所涉及的分子机制的详细了解可能会导致开发治疗金属过载(包括中毒)或由于金属代谢缺陷而导致的患者的治疗方法，以及设计干扰细菌金属代谢的新抗生素。与公共健康相关：拟议的研究试图提供对细胞金属贩运所涉及的分子机制的详细了解。这些信息将有助于开发治疗金属过载(包括中毒)或由于金属代谢缺陷而导致的患者的治疗方法，以及设计干扰细菌金属代谢的新抗生素。

DESCRIPTION (provided by applicant): Maintaining proper cellular levels of metal ions is key to the survival of all organisms. The viability of bacteria, including human pathogens, has been linked to the ability to acquire or compete for transition metals. Several human diseases have been shown to result from a breakdown in cellular metal trafficking. In the case of transition metals, maintaining proper metal concentrations involves controlling a delicate balance between optimal levels required to have functional enzymes, etc., and the concentration at which the metals become toxic. To achieve this control, organisms have developed mechanisms to ensure the acquisition of specific metals necessary for growth, exclude toxic metals, and control their cellular concentration. These metal trafficking systems rely on proteins that have the ability to distinguish between metal ions that often have similar sizes and charges. The metalloproteins involved, collectively known as metal trafficking proteins, control uptake and efflux (metallotransporters), target the delivery of metals to specific enzymes (metallochaperones) and regulate the expression of the other proteins in response to metal ion concentration (metalloregulators). Although many examples of proteins that achieve these functions for various metal ions have been characterized, the mechanisms that allow for metal recognition and metal-specific biological responses are not well known. The overall objective of the proposed research is to understand the structural parameters that that allow trafficking proteins to distinguish between metals, and the related protein structural changes that drive metal specific biological responses. Metal recognition is essential in many trafficking proteins in order to generate biological responses geared to one, or a small group of, metals and avoid crosstalk with other metal ions. Toward this goal, we plan to examine structural parameters that are involved in metal-recognition in a metallotransporter (NikA), a metallochaperone (HypA) and metalloregulators (NikR and RcnR) in E. coli and Helicobacter pylori. The approach involves cloning and expressing trafficking proteins, characterizing their metal ion affinities, and elucidating the structure of the metal sites, employing X-ray absorption spectroscopy (XAS) as a probe of metal site structure and crystallography and NMR to examine protein structure. Alterations in the metal binding site and other critical protein features can be produced by site-directed mutagenesis, and the effect on metal site structure and/or protein structure addressed. Strategies involving both in vitro and in vivo assays are employed to assess the effect on function in mutant trafficking proteins. In addition to the understanding of the basic biochemistry, a detailed understanding of the molecular mechanisms involved in metal trafficking may lead to the development of therapies for the treatment of patients with metal overloads (including poisoning) or deficiencies resulting from defects in metal metabolism, to the design of new antibiotics that interfere with bacterial metal metabolism. PUBLIC HEALTH RELEVANCE: The research proposed seeks to provide a detailed understanding of the molecular mechanisms involved in cellular metal trafficking. This information will be useful in the development of therapies for the treatment of patients with metal overloads (including poisoning) or deficiencies resulting from defects in metal metabolism, and in the design of new antibiotics that interfere with bacterial metal metabolism.