Structural and Mechanistic Characterization of the ZIP Metal Transporters

项目摘要 过渡金属锌是人体内第二丰富的微量元素，一直以来 在生物系统中广泛用于酶催化、结构稳定性和信号 转导。为了精确控制这种潜在有毒金属的水平，指定运输 系统已经进化了。锌的转运主要由ZNT家族进行外排。 和zrt-/irt-like蛋白(ZIP)家族，用于细胞外或细胞内的内流 细胞器。人类的14个拉链广泛地参与了各种生理和 病理过程，但对其结构和转运机制知之甚少 拉链，这阻碍了新疗法的发展。我们最近在解决第一个问题上的进展 原核生物ZIP的晶体结构为该项目的最终目标铺平了道路- 对拉链的金属输送机制有透彻的了解。为了实现这一目标， 我们将重点研究一种典型的细菌ZIP和人类蛋白ZIP4，这是 专门负责从饮食食物中摄取锌，并参与遗传疾病和 癌症，在以下三个方面。在Aim1中，我们将从结构上描述外部- 面向拉链构象的结构、计算、生化相结合的方法 以及细胞生物学方法。此结构信息与以前的 特征是向内的构象，将使我们能够建立一种交替的通道 金属运输中的机制。在AIM2中，我们将阐明双核金属中心的作用， 它出人意料地被鉴定在运输途径的中间，在锌结合和锌 结合促进了运输周期中的构象转变。在中获得的知识 AIM1和AIM2将帮助我们描绘一个完整的运输周期。在Aim3中，我们将识别分子 人体拉链底物专一性的决定因素。我们将对ZIP4进行比较 和ZIP8，其生理底物分别为锌和锰，并鉴定了 决定底物选择性的关键残基/元件通过系统诱变和活性 测量。总体而言，通过这些努力获得的结果将大大增加。 对拉链独特的金属传输机制的知识，这将填补知识空白 在锌信号和锌稳态中，扩大结构和机制的多样性 膜转运蛋白，重要的是，促进了新的治疗方法的发展 人类疾病，包括几种癌症。

Project Summary Transition metal zinc is the second most abundant trace element in human body and it has been extensively exploited in biological systems for enzymatic catalysis, structural stability and signal transduction. To precisely control the levels of this potential toxic metal, designated transport systems have been evolved. Zinc transport is primarily conducted by the ZnT family for efflux and the Zrt-/Irt-like protein (ZIP) family for influx from either extracellular space or intracellular organelles. The fourteen human ZIPs are broadly involved in a variety of physiological and pathological processes, but little is known about the structure and the transport mechanism of the ZIPs, which hinders the development of novel therapies. Our recent progress of solving the first crystal structure of a prokaryotic ZIP has paved a way towards the ultimate goal of this project - a thorough understanding of the metal transport mechanism of the ZIPs. To achieve this goal, we will focus our research on a prototypical bacterial ZIP and the human protein ZIP4, which is exclusively responsible for zinc uptake from dietary food and involved in genetic disease and cancers, in the following three aspects. In Aim1, we will structurally characterize the outward- facing conformation of the ZIPs by using a combination of structural, computational, biochemical and cell biological approaches. This structural information, together with the previously characterized inward-facing conformation, will enable us to establish an alternating access mechanism in metal transport. In Aim2, we will clarify the roles of the binuclear metal center, which was unexpectedly identified in the middle of the transport pathway, in zinc binding and zinc binding facilitated conformational transition during a transport cycle. The knowledge obtained in Aim1 and Aim2 will help us to depict a full transport cycle. In Aim3, we will identify the molecular determinants of substrate specificity in the human ZIPs. We will make comparison between ZIP4 and ZIP8, whose physiological substrates are zinc and manganese, respectively, and identify the key residues/elements dictating the substrate preference by systematic mutagenesis and activity measurement. Overall, the findings obtained through these efforts will greatly increase knowledge of the unique metal transport mechanism of the ZIPs, which will fill the knowledge gap in zinc signaling and zinc homeostasis, expand the structural and mechanistic diversity of membrane transporters, and importantly, facilitate the development of new therapeutics against human diseases, including several types of cancers.