Department of Pharmacology, Toxicology and Therapeutics, The University of Kansas Medical Center, Kansas City, KS 66160, USA.
Program in Molecular Therapeutics, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA.
Int J Mol Sci. 2022 Mar 16;23(6):3211. doi: 10.3390/ijms23063211.
In the Na/taurocholate cotransporting polypeptide (NTCP), the clinically relevant S267F polymorphism occurs at a "rheostat position". That is, amino acid substitutions at this position ("S267X") lead to a wide range of functional outcomes. This result was particularly striking because molecular models predicted the S267X side chains are buried, and thus, usually expected to be less tolerant of substitutions. To assess whether structural tolerance to buried substitutions is widespread in NTCP, here we used Rosetta to model all 19 potential substitutions at another 13 buried positions. Again, only subtle changes in the calculated stabilities and structures were predicted. Calculations were experimentally validated for 19 variants at codon 271 ("N271X"). Results showed near wildtype expression and rheostatic modulation of substrate transport, implicating N271 as a rheostat position. Notably, each N271X substitution showed a similar effect on the transport of three different substrates and thus did alter substrate specificity. This differs from S267X, which altered transport kinetics and specificity. As both transport and specificity may change during protein evolution, the recognition of such rheostat positions may be important for evolutionary studies. We further propose that the presence of rheostat positions is facilitated by local plasticity within the protein structure. Finally, we note that identifying rheostat positions may advance efforts to predict new biomedically relevant missense variants in NTCP and other membrane transport proteins.
在牛磺胆酸钠共转运多肽（NTCP）中，临床相关的 S267F 多态性发生在“变阻器位置”。也就是说，该位置的氨基酸取代（“S267X”）导致了广泛的功能结果。这一结果尤其引人注目，因为分子模型预测 S267X 侧链被掩埋，因此通常预期对取代的容忍度较低。为了评估 NTCP 中对埋藏取代的结构耐受性是否广泛，我们在这里使用 Rosetta 对另外 13 个埋藏位置的所有 19 个潜在取代进行建模。同样，仅预测了计算稳定性和结构的微小变化。在密码子 271（“N271X”）处对 19 个变体进行了实验验证。结果表明，近野生型表达和底物转运的变阻器调节，暗示 N271 是变阻器位置。值得注意的是，每个 N271X 取代对三种不同底物的转运都有类似的影响，因此并没有改变底物特异性。这与 S267X 不同，S267X 改变了转运动力学和特异性。由于在蛋白质进化过程中运输和特异性都可能发生变化，因此识别这种变阻器位置对于进化研究可能很重要。我们进一步提出，变阻器位置的存在是由蛋白质结构中的局部可塑性促成的。最后，我们注意到，识别变阻器位置可能会推动预测 NTCP 和其他膜转运蛋白中新型生物医学相关错义变异的努力。
