MIT家族二价阳离子转运蛋白金属传感机制的阐明

金属离子转运蛋白（MIT）超家族蛋白是从原核到真核生物普遍存在的二价阳离子转运蛋白,感知细胞中的二价阳离子浓度以维持金属内稳态，广泛参与生物学进程。. ZntB是细菌MIT家族的主要成员之一。然而，ZntB的确切功能和转运机制仍存在争议。此外，尚未报道ZntB与二价阳离子复合体的全长结构。. MRS2是MIT超家族蛋白的真核成员，广泛保守于哺乳动物和植物中，并且参与神经元变性以及植物的叶绿素合成。到目前为止尚未有任何MRS2结构报道，因此其感知金属离子的机制还不清楚。. 申请人前期针对多个二价阳离子转运蛋白的研究已经分别以通讯作者发表于Nature Communications。我们拟在前期研究基础上，重点针对细菌ZntB和真核MRS2进行冷冻电镜结构解析，以揭示二价阳离子识别机制和与金属结合相关的结构变化。进一步在生化和遗传学等层面验证这些MIT蛋白传感二价阳离子来调控金属转运的机制。

The Metal Ion Transporter (MIT) superfamily proteins are divalent cation transporters ubiquitously conserved from bacteria to eukaryotes. They sense cellular divalent cation concentrations to maintain metal homeostasis, and are implicated in a wide range of biological process such as metal tolerance in prokaryotes, cancer and neuronal degeneration in human, and chlorophyll synthesis in plants. However, despite the physiological importance of the MIT superfamily, only one of the MIT superfamily proteins, a bacterial CorA Mg2+ channel, has been functionally and structurally well-characterized so far, and thus the structures and transport mechanisms of other MIT superfamily members have been poorly understood yet.. In our proposal, we focus the structural and functional characterization of two members of the MIT superfamily proteins, a bacterial ZntB and an eukaryotic MRS2. . ZntB is one of the major bacterial MIT members, and was originally identified as a transporter implicated in Zn2+ efflux. However, the exact function and transport mechanism of ZntB still remain controversial Furthermore, the full-length structure of ZntB in complex with divalent cations has not been reported yet, obscuring the metal sensing mechanism of ZntB for metal ion transport regulation. . MRS2 is an eukaryotic member of the MIT superfamily proteins, widely distributed in yeast, mammals and plants. MRS2 is also implicated in sensing metal ions to regulate its metal transport, as in the case of its prokaryotic counterpart, CorA. However, MRS2 proteins have very weak sequence identity to their prokaryotic homologues, and the metal binding sites in the CorA cytoplasmic domain structure are not conserved in MRS2. Furthermore, none of the MRS2 structures has been reported so far. Accordingly, the metal ion sensing mechanisms by MRS2 proteins also has been unclear.. In our proposal, we aim to understand how ZntB and MRS2 sense divalent cations to regulate metal transport by a combination of Cryo-EM techniques, biochemical and genetics analyses. We will perform Cryo-EM of ZntB and MRS2 in the presence and absence of divalent cations to reveal divalent cation recognition mechanism and structural changes associated with metal binding. The further structure-based biochemical and genetics analysis will verify the metal transport regulation mechanism by divalent cation sensing by these MIT proteins. Overall, our work will provide insights into the functional and mechanistic diversities of the MIT superfamily.