Division of Chemistry & Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
J Am Chem Soc. 2012 Sep 12;134(36):14890-6. doi: 10.1021/ja304560x. Epub 2012 Aug 31.
The recently published crystal structure of the D3 dopamine receptor shows a tightly packed region of aromatic residues on helices 5 and 6 in the space bridging the binding site and what is thought to be the origin of intracellular helical motion. This highly conserved region also makes contacts with residues on helix 3, and here we use double mutant cycle analysis and unnatural amino acid mutagenesis to probe the functional role of several residues in this region of the closely related D2 dopamine receptor. Of the eight mutant pairs examined, all show significant functional coupling (Ω > 2), with the largest coupling coefficients observed between residues on different helices, C3.36/W6.48, T3.37/S5.46, and F5.47/F6.52. Additionally, three aromatic residues examined, F5.47, Y5.48, and F5.51, show consistent trends upon progressive fluorination of the aromatic side chain. These trends are indicative of a functionally important electrostatic interaction with the face of the aromatic residue examined, which is likely attributed to aromatic-aromatic interactions between residues in this microdomain. We also propose that the previously determined fluorination trend at W6.48 is likely due to a sulfur-π interaction with the side chain of C3.36. We conclude that these residues form a tightly packed structural microdomain that connects helices 3, 5, and 6, thus forming a barrier that prevents dopamine from binding further toward the intracellular surface. Upon activation, these residues likely do not change their relative conformation, but rather act to translate agonist binding at the extracellular surface into the large intracellular movements that characterize receptor activation.
最近发表的 D3 多巴胺受体的晶体结构显示，在结合部位和被认为是细胞内螺旋运动起源的部位之间的空间中，5 号和 6 号螺旋上有一个紧密堆积的芳香族残基区域。这个高度保守的区域也与 3 号螺旋上的残基接触，在这里，我们使用双突变体循环分析和非天然氨基酸诱变来探测这个密切相关的 D2 多巴胺受体中这个区域的几个残基的功能作用。在所检查的八个突变对中，所有都显示出显著的功能偶联（Ω>2），最大的偶联系数观察到在不同的螺旋上的残基之间，C3.36/W6.48、T3.37/S5.46 和 F5.47/F6.52。此外，检查的三个芳香族残基，F5.47、Y5.48 和 F5.51，在芳香族侧链的逐步氟化过程中表现出一致的趋势。这些趋势表明与所检查的芳香族残基的表面存在功能上重要的静电相互作用，这可能归因于该微域中残基之间的芳香族-芳香族相互作用。我们还提出，以前在 W6.48 上确定的氟化趋势可能是由于与 C3.36 的侧链的硫-π相互作用。我们得出结论，这些残基形成一个紧密堆积的结构微域，连接 3 号、5 号和 6 号螺旋，从而形成一个阻止多巴胺进一步结合到细胞内表面的屏障。在激活时，这些残基可能不会改变它们的相对构象，而是作用于将激动剂结合在细胞外表面转化为受体激活的特征性的大的细胞内运动。
