Ho Su-Chin, Goh Sui-Sin, Li Sun, Khoo Daphne Hsu-Chin, Paterson Malcolm
SingHealth Research, Singapore Health Service, Singapore, Republic of Singapore.
Thyroid. 2008 Dec;18(12):1313-9. doi: 10.1089/thy.2008.0240.
Cysteine (Cys) residues pair to form disulfide bonds that are important in maintaining structure and function of the thyrotropin receptor (TSHR). There are 11 Cys residues in the ectodomain (ECD). Cys 41 at the N-terminus and Cys 283 at the SHCC motif have been identified as important for ligand binding. The present study evaluated the effects of mutating Cys distal to the S281HCC motif at the C-terminus of the ECD on the functional characteristics of TSHR.
We introduced (i) individual Cys and (ii) consecutive cumulative Cys mutations into the starting template SHCS-TSHR, a truncated TSHR-ECD moiety previously shown to behave like the wild-type TSHR. Each mutant receptor was evaluated for relative specific binding (RSB), calculated as a measure of TSH-binding ability after normalization with receptor surface expression.
In the first approach, RSB was severely affected when Cys 390 and Cys 398 were individually switched to serine. Failed receptor trafficking occurred with Cys 408 mutation. These findings were likely results of altered receptor conformation due to illegitimate disulfide bridge formation. Only SHCS-301 TSHR bound TSH in a specific manner, and it formed the base for sequential Cys mutations. Through this second approach, both Cys 301 and 390 could be removed simultaneously without hindering TSH binding significantly. Cys 398, however, was shown to be critical. Its absence resulted in huge loss of TSH binding. Leaving Cys 283 and 398 as the only Cys pair in the C-terminus alone could support 40% of the total ligand-binding capacity.
From these data, we proposed Cys 398 as a stable disulfide bond partner of Cys 283, corroborating with a model based on evolutionary history of TSHR across species. This pairing of Cys 283 and Cys 398 also provides an objective alternative to conventional hypotheses on Cys coupling based on other predictive models.
半胱氨酸（Cys）残基配对形成二硫键，这对维持促甲状腺激素受体（TSHR）的结构和功能很重要。胞外域（ECD）中有11个半胱氨酸残基。N端的Cys 41和SHCC基序中的Cys 283已被确定对配体结合很重要。本研究评估了在ECD C端S281HCC基序远端的Cys发生突变对TSHR功能特性的影响。
我们将（i）单个Cys和（ii）连续累积的Cys突变引入起始模板SHCS - TSHR，这是一种截短的TSHR - ECD部分，先前已证明其行为类似于野生型TSHR。评估每个突变受体的相对特异性结合（RSB），计算方法是在用受体表面表达进行归一化后作为TSH结合能力的度量。
在第一种方法中，当Cys 390和Cys 398分别转换为丝氨酸时，RSB受到严重影响。Cys 408突变导致受体转运失败。这些发现可能是由于非法二硫键形成导致受体构象改变的结果。只有SHCS - 301 TSHR以特异性方式结合TSH，并且它构成了连续Cys突变的基础。通过第二种方法，可以同时去除Cys 301和390而不会显著妨碍TSH结合。然而，Cys 398被证明是至关重要的。它的缺失导致TSH结合能力大幅丧失。仅将Cys 283和398作为C端唯一的Cys对就可以支持总配体结合能力的40％。
根据这些数据，我们提出Cys 398是Cys 283的稳定二硫键伙伴，这与基于跨物种TSHR进化史的模型一致。Cys 283和Cys 398的这种配对也为基于其他预测模型的关于Cys偶联的传统假设提供了一个客观的替代方案。
