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首页> 外文期刊>Journal of Molecular Biology >Thermostabilisation of the serotonin transporter in a cocaine-bound conformation
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Thermostabilisation of the serotonin transporter in a cocaine-bound conformation

机译：血清素转运蛋白在可卡因结合构象中的热稳定作用

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Structure determination of mammalian integral membrane proteins is challenging due to their instability upon detergent solubilisation and purification. Recent successes in the structure determination of G-protein-coupled receptors (GPCRs) resulted from the development of GPCR-specific protein engineering strategies. One of these, conformational thermostabilisation, could in theory facilitate structure determination of other membrane proteins by improving their tolerance to detergents and locking them in a specific conformation. We have therefore used this approach on the cocaine-sensitive rat serotonin transporter (SERT). Out of a panel of 554 point mutants throughout SERT, 10 were found to improve its thermostability. The most stabilising mutations were combined to make the thermostabilised mutants SAH6 (L99A + G278A + A505L) and SAH7 (L405A + P499A + A505L) that were more stable than SERT by 18 C and 16 C, respectively. Inhibitor binding assays showed that both of the thermostabilised SERT mutants bound [125I]RTI55 (β-CIT) with affinity similar to that of the wild-type transporter, although cocaine bound with increased affinity (17- to 56-fold) whilst ibogaine, imipramine and paroxetine all bound with lower affinity (up to 90-fold). Neither SAH6 nor SAH7 was capable of transporting [3H]serotonin into HEK293 cell lines stably expressing the mutants, although serotonin bound to them with an apparent Ki of 155 μM or 82 μM, respectively. These data combined suggest that SAH6 and SAH7 are thermostabilised in a specific cocaine-bound conformation, making them promising candidates for crystallisation. Conformational thermostabilisation is thus equally applicable to membrane proteins that are transporters in addition to those that are GPCRs.
机译：哺乳动物整合膜蛋白的结构确定是有挑战性的，因为它们在去污剂增溶和纯化时不稳定。 G蛋白偶联受体（GPCR）的结构确定方面的最新成功来自于GPCR特异性蛋白工程策略的发展。其中之一，构象热稳定理论上可以通过提高其他膜蛋白对去污剂的耐受性并将它们锁定在特定构象中，来促进其他膜蛋白的结构确定。因此，我们在可卡因敏感的大鼠血清素转运蛋白（SERT）上使用了这种方法。在整个SERT的554个点突变体中，发现10个可以改善其热稳定性。结合最稳定的突变，使热稳定的突变体SAH6（L99A + G278A + A505L）和SAH7（L405A + P499A + A505L）分别比SERT稳定18 C和16C。抑制剂结合实验表明，两个可热稳定的SERT突变体都以与野生型转运蛋白相似的亲和力与[125I] RTI55（β-CIT）结合，尽管可卡因与依巴加因的亲和力增加了（17至56倍），丙咪嗪和帕罗西汀均具有较低的亲和力（最多90倍）结合。 SAH6和SAH7均不能将[3H] 5-羟色胺转运到稳定表达该突变体的HEK293细胞系中，尽管5-羟色胺与它们结合的表观Ki分别为155μM或82μM。这些数据加起来表明SAH6和SAH7在特定的可卡因结合构象中是热稳定的，使其成为有希望的结晶候选物。因此，构象热稳定化同样适用于作为GPCR的转运蛋白的膜蛋白。

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