Human Concentrative Nucleoside Transporter 3 (hCNT3, SLC28A3) Forms a Cyclic Homotrimer.
Human Concentrative Nucleoside Transporter 3 (hCNT3, SLC28A3) Forms a Cyclic Homotrimer.
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人类浓缩核苷转运蛋白 3 (hCNT3、SLC28A3) 形成环状同源三聚体。
DOI：
10.1021/acs.biochem.7b00339
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发表时间：
2017
期刊：
影响因子：
2.9
通讯作者：
Sali,Andrej
中科院分区：
文献类型：
--
作者：
Stecula,Adrian;Schlessinger,Avner;Giacomini,KathleenM;Sali,Andrej
许多抗癌和抗病毒药物是嘌呤或嘧啶的类似物，它们利用膜转运蛋白穿越细胞膜。浓缩型核苷转运体(CNTs)通过将配体的转运偶联到钠的梯度来介导核苷的挽救和治疗性核苷类似物的跨膜转运。在人类CNT家族的三个成员中，CNT3具有最广泛的选择性和最广泛的表达谱。然而，转运蛋白的分子机制，包括它如何与结构不同的核苷和核苷类似物相互作用和移位，目前尚不清楚。最近，vcCNT的晶体结构表明，CNT3的原核同源物形成了一个同源三聚体。在本研究中，我们成功地表达和纯化了野生型人类同源物hCNT3，并通过大小排除图谱和戊二醛交联证明了同源三聚体的存在。此外，通过在比较结构模型的指导下在高度保守的位置创建一系列半胱氨酸突变体，我们在基于细胞的实验中将hCNT3原基交联，从而表明hCNT3同源三聚体在人类细胞中的存在。TM9上特定位置的交联链的存在和不存在告诉我们vcCNT和hCNT3之间的重要结构差异。三聚结构域的比较建模和序列协同进化分析都表明，齐聚对hCNT3的稳定性和功能至关重要。特别是，三聚化似乎缩短了核苷穿过质膜的转位路径，并可能允许通过变构来调节转运功能。
Many anticancer and antiviral drugs are purine or pyrimidine analogues, which use membrane transporters to cross cellular membranes. Concentrative nucleoside transporters (CNTs) mediate the salvage of nucleosides and the transport of therapeutic nucleoside analogues across plasma membranes by coupling the transport of ligands to the sodium gradient. Of the three members of the human CNT family, CNT3 has the broadest selectivity and the widest expression profile. However, the molecular mechanisms of the transporter, including how it interacts with and translocates structurally diverse nucleosides and nucleoside analogues, are unclear. Recently, the crystal structure of vcCNT showed that the prokaryotic homologue of CNT3 forms a homotrimer. In this study, we successfully expressed and purified the wild type human homologue, hCNT3, demonstrating the homotrimer by size exclusion profiles and glutaraldehyde cross-linking. Further, by creating a series of cysteine mutants at highly conserved positions guided by comparative structure models, we cross-linked hCNT3 protomers in a cell-based assay, thus showing the existence of hCNT3 homotrimers in human cells. The presence and absence of cross-links at specific locations along TM9 informs us of important structural differences between vcCNT and hCNT3. Comparative modeling of the trimerization domain and sequence coevolution analysis both indicate that oligomerization is critical to the stability and function of hCNT3. In particular, trimerization appears to shorten the translocation path for nucleosides across the plasma membrane and may allow modulation of the transport function via allostery.
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DOI：
10.1074/jbc.m116.743997
发表时间：
2017-06-09
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Mulinta R;Yao SYM;Ng AML;Cass CE;Young JD
通讯作者：
Young JD
影响因子：
5.8
作者：
Vangone, Anna;Spinelli, Raffaele;Oliva, Romina
通讯作者：
Oliva, Romina
取代半胱氨酸可及性方法 (SCAM)
影响因子：
4.8
作者：
G. Liapakis;J. Javitch
通讯作者：
J. Javitch
影响因子：
3.4
作者：
CAREAGA, CL;FALKE, JJ
通讯作者：
FALKE, JJ
人浓缩 Na/核苷协同转运蛋白 hCNT1 跨膜螺旋 8 的特定突变影响渗透选择性和阳离子偶联。
影响因子：
2.9
作者：
Melissa D. Slugoski;Shauna Loewen;A. Ng;Kyla M. Smith;S. Yao;E. Karpinski;C. Cass;S. Baldwin;J. Young
通讯作者：
J. Young