Novel variants provide differential stabilisation of human equilibrative nucleoside transporter 1 states.
Novel variants provide differential stabilisation of human equilibrative nucleoside transporter 1 states.
复制标题
新的变体提供人平衡核苷转运蛋白1状态的差异稳定。
DOI：
10.3389/fmolb.2022.970391
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发表时间：
2022
影响因子：
5
通讯作者：
中科院分区：
文献类型：
--
作者：
人平衡型核苷转运蛋白是心脏病、癌症和病毒治疗的主要药物靶点。了解转运的分子基础对于通过基于结构的药物设计开发改进的治疗方法至关重要。已经提出ENT利用交替进入作用机制，类似于主要易化剂超家族。然而，ENT缺乏该超家族的功能基本特征，这表明它们可能使用不同的运输机制。了解其运输的分子基础需要深入了解不同的构象状态。中间状态之间的差异可能是离散的，并通过微妙的门控相互作用（例如作为盐桥）来介导。我们在大细胞内环（ICL 6）和跨膜螺旋7（TM 7）处鉴定了人平衡型核苷转运蛋白亚型1（hENT 1）的四种变体，这些变体稳定了脱辅基状态（Δ NTm 0.7-1.5°C）。此外，我们发现变体K263 A（ICL 6）和I282 V（TM 7）特异性地稳定hENT 1的受体结合状态（受体T m 5.0 ± 1.7°C和3.0 ± 1.8°C），支持ICL 6在hENT 1门控中的作用。最后，我们发现，与野生型相比，变体T336 A被硝基苄基硫代肌苷（nitrobenzylthiosine）（Nitrobenzylthioinosine）去稳定化（Nitrobenzylthioinosine Tm-4.7 ± 1.1°C），并且与它的结合差七倍。这种残留物可能有助于确定抑制剂和底物的敏感性。残基K263不存在于解析结构中，突出表明需要包括环区域的进一步结构数据。
Human equilibrative nucleoside transporters represent a major pharmaceutical target for cardiac, cancer and viral therapies. Understanding the molecular basis for transport is crucial for the development of improved therapeutics through structure-based drug design. ENTs have been proposed to utilise an alternating access mechanism of action, similar to that of the major facilitator superfamily. However, ENTs lack functionally-essential features of that superfamily, suggesting that they may use a different transport mechanism. Understanding the molecular basis of their transport requires insight into diverse conformational states. Differences between intermediate states may be discrete and mediated by subtle gating interactions, such as salt bridges. We identified four variants of human equilibrative nucleoside transporter isoform 1 (hENT1) at the large intracellular loop (ICL6) and transmembrane helix 7 (TM7) that stabilise the apo-state (∆T m 0.7–1.5°C). Furthermore, we showed that variants K263A (ICL6) and I282V (TM7) specifically stabilise the inhibitor-bound state of hENT1 (∆∆T m 5.0 ± 1.7°C and 3.0 ± 1.8°C), supporting the role of ICL6 in hENT1 gating. Finally, we showed that, in comparison with wild type, variant T336A is destabilised by nitrobenzylthioinosine (∆∆T m -4.7 ± 1.1°C) and binds it seven times worse. This residue may help determine inhibitor and substrate sensitivity. Residue K263 is not present in the solved structures, highlighting the need for further structural data that include the loop regions.
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影响因子：
64.8
作者：
Jumper J;Evans R;Pritzel A;Green T;Figurnov M;Ronneberger O;Tunyasuvunakool K;Bates R;Žídek A;Potapenko A;Bridgland A;Meyer C;Kohl SAA;Ballard AJ;Cowie A;Romera-Paredes B;Nikolov S;Jain R;Adler J;Back T;Petersen S;Reiman D;Clancy E;Zielinski M;Steinegger M;Pacholska M;Berghammer T;Bodenstein S;Silver D;Vinyals O;Senior AW;Kavukcuoglu K;Kohli P;Hassabis D
通讯作者：
Hassabis D
影响因子：
64.8
作者：
Nomura N;Verdon G;Kang HJ;Shimamura T;Nomura Y;Sonoda Y;Hussien SA;Qureshi AA;Coincon M;Sato Y;Abe H;Nakada-Nakura Y;Hino T;Arakawa T;Kusano-Arai O;Iwanari H;Murata T;Kobayashi T;Hamakubo T;Kasahara M;Iwata S;Drew D
通讯作者：
Drew D
影响因子：
21.1
作者：
Huang W;Zeng X;Shi Y;Liu M
通讯作者：
Liu M
影响因子：
5.7
作者：
Fowler, Philip W.;Orwick-Rydmark, Marcella;Radestock, Sebastian;Solcan, Nicolae;Dijkman, Patricia M.;Lyons, Joseph A.;Kwok, Jane;Caffrey, Martin;Watts, Anthony;Forrest, Lucy R.;Newstead, Simon
通讯作者：
Newstead, Simon
影响因子：
4.8
作者：
Baldwin, SA;Yao, SYM;Young, JD
通讯作者：
Young, JD