江涛研究员《Nature》子刊解析肉碱膜转运蛋白三维结构

　　

生物通报道，中国科学院生物物理研究所江涛研究员领衔的科研小组在肉碱膜转运蛋白CaiT三维结构研究方面取得最新的进展，相关成果文章Crystal structure of the carnitine transporter and insights into the antiport mechanism刊登在3月28的《Nature Structural & Molecular Biology》上。

 

肉碱（carnitine）在生物体内广泛存在，它在哺乳动物长链脂肪酸的β-氧化中发挥着重要作用。一些细菌在缺氧环境下，还可以利用肉碱作为电子受体，并将其转化为γ-三甲氨基丁内盐（γ-butyrobetaine）。大肠杆菌中的膜转运蛋白CaiT是一个具有12次跨膜螺旋的膜蛋白，它负责双向转运肉碱以及肉碱的代谢产物γ-butyrobetaine。关于转运机制，过去的研究认为，膜转运蛋白采用一种alternating-access的底物转运机制，即转运蛋白有inward-facing和outward-facing两种主要构象，通过在二者间的变换来转运底物。然而，由于缺乏足够的蛋白-底物复合物结构和功能实验证据，具体的作用细节还很不清楚。

 

经过长期努力，江涛课题组解析了膜转运蛋白CaiT与其底物L-肉碱复合物3.15埃分辨率的晶体结构。该晶体结构中，CaiT以三聚体形式存在，每个单体表现为介于inward-facing和outward-facing构象中间态的一种构象。每个CaiT分子结合了4个L-肉碱分子。这4个肉碱分子在CaiT的中心部位沿跨膜方向依次分布，勾勒出一条底物的转运通道。通过对关键氨基酸残基进行突变体转运实验，研究结果提示位于CaiT中心位置的主要结合位点和位于胞内通道底部的次要结合位点在转运过程中起重要作用。该项研究工作使得人们对于具有双向运输功能的膜转运蛋白的作用机制有了更加深入的认识。

 

图注：膜转运蛋白CaiT与底物复合物的结构示意图（1-12表示12段跨膜螺旋）

 

a 为平行于膜的视角，b为垂直于膜的视角

（生物通 小茜）

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Crystal structure of the carnitine transporter and insights into the antiport mechanism

Lin Tang,Lin Bai,Wen-hua Wang& Tao Jiang

Affiliations Contributions Corresponding author Journal name:

Nature Structural & Molecular Biology

Year published:

(2010)

DOI:

doi:10.1038/nsmb.1788

Received 16 October 2009 Accepted 17 February 2010 Published online 28 March 2010

Abstract

CaiT is a membrane antiporter that catalyzes the exchange of l-carnitine with γ-butyrobetaine across the Escherichia coli membrane. To obtain structural insights into the antiport mechanism, we solved the crystal structure of CaiT at a resolution of 3.15 Å. We crystallized CaiT as a homotrimer complex, in which each protomer contained 12 transmembrane helices and 4 l-carnitine molecules outlining the transport pathway across the membrane. Mutagenesis studies revealed a primary binding site at the center of the protein and a secondary substrate-binding site at the bottom of the intracellular vestibule. These results, together with the insights obtained from structural comparison with structurally homologous transporters, provide mechanistic insights into the association between substrate translocation and the conformational changes of CaiT.

 

江涛研究员

 

简历 & 研究组工作摘要：

1998年毕业于中科院生物物理研究所，获博士学位

1999－2005年 中科院生物物理研究所副研究员        

2004年至今   中科院生物物理研究所研究员。

研究方向为利用蛋白质晶体学手段研究生物大分子的三维结构与功能关系。

目前主要的研究对象分为两个方面:       

1. 细胞表面受体复合物的结构研究。        

神经营养因子对于神经系统的发育具有重要的意义。研究发现，p75NTR与神经营养因子的直接相互作用与神经系统退行性疾病关系密切。但是二者间的相互作用方式一直存在争议。我们最近完成了神经营养因子NT-3与p75NTR 复合物晶体结构解析，揭示了神经营养因子与其受体P75NTR天然的结合模式。      

2. 结构基因组研究计划。

 

Biography & Introduction

1998 Ph.D degree from Institute of Biophysics, CAS;

1999 Associate professor of Structural Biology, Institute of Biophysics, CAS.

2004，Professor of Structural Biology, Institute of Biophysics, CAS.

 

Our research interest  is in the relationship between the structure and function of biomacromolecules. The main technique employed is x-ray protein crystallography. The current focus is on:

1.Structual studies of cell surface receptor conplexes   

NTs are key factors for the development and maintenance of the mammalian nervous system. The binding of NTs to the p75NTR receptor induces either cell survival or apoptosis. p75NTR also plays a very important role in Alzheimer's disease, and tumor development and metastasis. In spite of this knowledge, the fundamental mechanism of interaction between p75 and NTs is not clear. Recently，we solved the crystal structure of a 2:2 symmetrical complex of NT-3 and p75NTR. This structure revealed the detailed interaction mode between p75 with NT-3. This structure provides a basis to understand diverse p75NTR functions and facilitate the therapeutic utility of neurotrophins as clinical agents.

2. Structural genomics project.

 

Selected Publications：

Gong Y, Cao P, Yu HJ, Jiang T*. Crystal structure of the neurotrophin-3 and p75(NTR) symmetrical complex. NATURE, 2008, [Epub ahead of print]

Liu SQ,  Wang F,  Tang L,  Gui WJ ,  Cao P , Liu XQ ,  Poon WS , Shaw PC* ,  Jiang T*. Crystal structure of mastoparan from Polistes jadwagae at 1.2 angstrom resolution. J STRU BIOL, 2007, 160(1): 28-34.    

Wang F, Li H, Liu MN,  Song H,  Han HM,  Wang CC, Yin YC, Zhou, Z,  Qi Z, Shu Y, Lin ZJ,  Jiang T*. Structural and functional analysis of natrin, a venom protein that targets various ion channels. BIOCHEM BIOPHY RES COMMUN, 2006, 351(2): 443-448

Wang F, Liu XQ, Li H, Liang KN, Miner JN, Hong M, Kallel EA, Van Oeveren A, Zhi L, Jiang T*. Structure of the ligand-binding domain (LBD) of human androgen receptor in complex with a selective modulator LGD2226. ACTA CRYSTALLOGR SECT F STRUCT BIOL CRYST COMMUN, 2006, 62: 1067-1071

Wang F, Liu XQ, Li H, Lang XJ, Peng H, Liu SQ, Jiang T*. Crystallization and preliminary X-ray diffraction analysis of three mastoparans. PROTEIN PEPT LETT. 2006;13(6):629-31

Cao P, Gong Y,  Tang L, Leung YC,  Jiang T*. Crystal structure of human pyridoxal kinase, J STRUCT BIOL, 2006, 154(3): 327-332.

Tang L, Li MH, Cao P, Wang F, Chang WR, Bach S, Reinhardt J, Ferandin Y, Galons H, Wan YQ, Gray N, Meijer L, Jiang T*, Liang DC. Crystal structure of pyridoxal kinase in complex with roscovitine and derivatives. J Biol Chem 280(35), 31220–31229(2005).

Bach S, Knockaert M, Lozach O, Reinhard J , Baratte B, Schmitt S, Coburn SP, Tang L, Jiang T, Liang DC, Meijer L. Roscovitine Targets, Protein Kinases and Pyridoxal Kinase. J Biol Chem 280(35), 31208–31219(2005).

Li MH, Kwok F, Chang WR, Liu SQ, Lo SC, Zhang JP, Jiang T*, Liang DC. Conformational changes in the reaction of pyridoxal kinase. J Biol Chem 279 (17), 17459-65 (2004).

Li MH, Kowk F, Chang WR, Lau CK, Zhang JP, Lo SC, Jiang T*, Liang DC*. Crystal structure of brain pyridoxal kinase, a novel member of the ribokinase superfamily. J Biol Chem 277 (48), 46385-90 (2002).

Li MH, Kwok F, An XM, Chang WR, Lau CK, Zhang JP, Liu SQ, Leung YC, Jiang T*, Liang DC*. Crystallization and preliminary crystallographic studies of pyridoxal kinase from sheep brain. Acta Crystallogr D Biol Crystallogr 58(Pt 9),1479-81 (2002).

Jiang T, Zhang JP, Chang WR, Liang DC. Crystal structure of R-phycocyanin and possible energy transfer pathways in the phycobilisome. Biophys J 81(2), 1171-9 (2001).

Jiang T, Zhang J, Liang D. Structure and function of chromophores in R-Phycoerythrin at 1.9 A resolution. Proteins 34(2), 224-31(1999).

Liu, J. Y., Jiang, T., Zhang, J. P., Liang, D. C., Crystal structure of allophycocyanin from red algae Porphyra yezoensis at 2.2 A resolution. J.Biol.Chem 274(24), 16945-16952 (1999).

Chang, W. R., Jiang, T., Wan, Z. L., Zhang, J. P., Yang, Z. X., Liang, D. C., Crystal structure of R-phycoerythrin from Polysiphonia unceolata at 2.8 A resolution. J. Mol. Biol. 262, 721-731 (1996).