Substituted cysteine accessibility method (SCAM) analysis of the transport domain of human concentrative nucleoside transporter 3 (hCNT3) and other family members reveals features of structural and functional importance.
Substituted cysteine accessibility method (SCAM) analysis of the transport domain of human concentrative nucleoside transporter 3 (hCNT3) and other family members reveals features of structural and functional importance.
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DOI：
10.1074/jbc.m116.743997
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发表时间：
2017-06-09
期刊：
影响因子：
--
通讯作者：
Young JD
中科院分区：
文献类型：
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作者：
Mulinta R;Yao SYM;Ng AML;Cass CE;Young JD
人SLC28家族集中核苷转运蛋白(CNT)有三个成员：hCNT1、hCNT2和hCNT3。Na+偶联的hCNT1和hCNT2分别转运嘧啶和嘌呤核苷，而hCNT3利用Na+和/或H+电化学梯度同时转运嘧啶和嘌呤核苷。大肠杆菌CNT家族成员NupC在选择性上与hCNT1相似，但是H+偶联的。利用非洲爪哇卵母细胞异源表达和hCNT3(C−)基因工程蛋白的半胱氨酸可及性方法，用膜非平均硫醇反应试剂对氯汞苯磺酸盐对该蛋白的转运域(界面螺旋2、发夹1、可能的跨膜区(TM)7和TM8)和TM9进行半胱氨酸可及性分析。对hCNT3(C−)整个C末端的系统扫描，以及对野生型hCNT1的转运域和相应的无半胱氨酸型NupC(C−)的TMS的平行研究，验证了新开发的人CNT膜结构的结构同源性模型，揭示了转运域TMS中延长的构象可移动区，确定了具有重要功能的孔衬残基，并为一种新的升降型转运蛋白功能机制提供了证据。
The human SLC28 family of concentrative nucleoside transporter (CNT) proteins has three members: hCNT1, hCNT2, and hCNT3. Na+-coupled hCNT1 and hCNT2 transport pyrimidine and purine nucleosides, respectively, whereas hCNT3 transports both pyrimidine and purine nucleosides utilizing Na+ and/or H+ electrochemical gradients. Escherichia coli CNT family member NupC resembles hCNT1 in permeant selectivity but is H+-coupled. Using heterologous expression in Xenopus oocytes and the engineered cysteine-less hCNT3 protein hCNT3(C−), substituted cysteine accessibility method analysis with the membrane-impermeant thiol reactive reagent p-chloromercuribenzene sulfonate was performed on the transport domain (interfacial helix 2, hairpin 1, putative transmembrane domain (TM) 7, and TM8), as well as TM9 of the scaffold domain of the protein. This systematic scan of the entire C-terminal half of hCNT3(C−) together with parallel studies of the transport domain of wild-type hCNT1 and the corresponding TMs of cysteine-less NupC(C−) yielded results that validate the newly developed structural homology model of CNT membrane architecture for human CNTs, revealed extended conformationally mobile regions within transport-domain TMs, identified pore-lining residues of functional importance, and provided evidence of an emerging novel elevator-type mechanism of transporter function.