Department of Psychiatry, and Biochemistry and Molecular Pharmacology, Alexandria Center for Life Sciences, New York University School of Medicine, 450 E 29th Street, New York, NY 10016, USA.
Neurochem Res. 2013 Jul;38(7):1301-2. doi: 10.1007/s11064-013-1025-0. Epub 2013 Mar 27.
The study reported by Wenge and Bönisch in this issue provides critical structural information regarding extracellular loop 2 (EL2) of the human norepinephrine transporter (NET). A systematic search among all 10 cysteine and 13 histidine residues in NET led to His222 in EL2 as the target for N-ethylmaleimide: its alkylation interferes with [(3)H]nisoxetine binding, indicating the part of EL2 containing His 222 reaches back into the protein interior where it prevents access by nisoxetine to its binding site. Thus, EL2 in human NET does much more than conformationally assisting substrate translocation. The present study underscores the importance of site-directed mutagenesis approaches to elucidate structural features that cannot be deduced from crystals of homolog proteins. In the case of NET, the closest crystal structure is that of the homolog LeuT, but EL2 is difficult to align with 22 less loop residues in LeuT than in NET. The present results could only be achieved by the systematic mutagenesis study of all cysteines and all histidines in NET.
文本中提到的温格和博尼施的这项研究提供了有关人去甲肾上腺素转运体（NET）细胞外环 2（EL2）的关键结构信息。对 NET 中所有 10 个半胱氨酸和 13 个组氨酸残基进行系统搜索，结果表明 EL2 中的组氨酸 222 是 N-乙基马来酰亚胺的靶标：其烷基化会干扰[3H]去甲替林结合，表明包含组氨酸 222 的 EL2 部分向后延伸到蛋白质内部，从而阻止去甲替林进入其结合位点。因此，人 NET 中的 EL2 不仅仅是协助底物转运的构象。本研究强调了通过定点突变方法阐明不能从同源蛋白晶体中推断出的结构特征的重要性。在 NET 的情况下，最接近的晶体结构是同源的 LeuT，但与 LeuT 相比，EL2 与 NET 中少 22 个环残基的对齐困难。只有通过对 NET 中的所有半胱氨酸和组氨酸进行系统的突变研究，才能得到本研究的结果。
