Photoaffinity labeling of mutant neurokinin-1 receptors reveals additional structural features of the substance P/NK-1 receptor complex.
Photoaffinity labeling of mutant neurokinin-1 receptors reveals additional structural features of the substance P/NK-1 receptor complex.
复制标题
突变型神经激肽-1 受体的光亲和标记揭示了 P/NK-1 受体复合物物质的其他结构特征。
作者：
Macdonald,D;Mierke,DF;Li,H;Pellegrini,M;Sachais,B;Krause,JE;Leeman,SE;Boyd,ND
结合光亲和标记、受体定点突变和高分辨核磁共振波谱，进一步确定了P物质(SP)与大鼠神经激肽-1(NK-1)受体结合的分子细节。突变的NK-1受体是通过用丙氨酸取代Met174和/或Met181： 残基构建而成的，这些残基是分别在第4位和第8位含有对苯甲酰-L-苯丙氨酸的放射性碘标记的SP的光反应衍生物的共价结合位点。用放射性碘标记的Bpa8-SP对M181a突变体进行光亲和标记，与野生型受体相比，光掺入效率显著降低。相反，使用放射性碘标记的Bpa4-SP对M174A突变体进行光亲和标记得到了意想不到的结果，即与野生型受体相比，光掺入效率提高了。对光标记受体突变体进行的酶和化学碎片分析表明，共价结合位点不是被取代的丙氨酸，而是第二个胞外(E2)环序列上的另一个蛋氨酸，这不是野生型受体的主要附着位点。这些结果表明，NK-1受体上的Met174和Met181之间存在密切的空间关系。为了评估这种结构配置，对具有与整个E2环和相邻跨膜螺旋片段相对应的序列的合成肽进行了核磁共振分析，以将该肽锚定在用于模拟膜的脂类中。E2环的结构特征包括从Pro175延伸到Glu183的中心位置的α-螺旋，以及与跨膜区相对应的末端较小的α-螺旋。这两个甲硫氨酸残基位于中心α-螺旋的同一面上，彼此之间的距离约为11，因此与光亲和标记结果的结论一致。
Photoaffinity labeling, receptor site-directed mutagenesis, and high-resolution NMR spectroscopy have been combined to further define the molecular details of the binding of substance P (SP) to the rat neurokinin-1 (NK-1) receptor. Mutant NK-1 receptors were constructed by substituting Ala for Met174 and/or Met181: residues previously identified as the sites of covalent attachment of radioiodinated, photoreactive derivatives of SP containingp-benzoyl-l-phenylalanine (Bpa) in positions 4 and 8, respectively. Photoaffinity labeling of the M181A mutant using radioiodinated Bpa8-SP resulted in a marked reduction in photoincorporation efficiency compared to the wild-type receptor. In contrast, photoaffinity labeling of the M174A mutant using radioiodinated Bpa4-SP gave the unexpected result of an increase in the efficiency of photoincorporation compared to the wild-type receptor. Enzymatic and chemical fragmentation analysis of the photolabeled receptor mutants established that the sites of covalent attachment were not the substituted alanine, but rather the other methionine on the second extracellular (E2) loop sequence, that is not the primary site of attachment in the wild-type receptor. The results thus suggest a close spatial relationship between Met174 and Met181 on the NK-1 receptor. To evaluate this structural disposition, NMR analyses were performed on a synthetic peptide with a sequence corresponding to the entire E2 loop and segments of the adjoining transmembrane helices to anchor the peptide in the lipids used to mimic a membrane. The structural features of the E2 loop include a centrally located α-helix, extending from Pro175 to Glu183, as well as smaller α-helices at the termini, corresponding to the transmembrane regions. The two methionine residues are located on the same face of the central α-helix, approximately 11 Å apart from each other, and are therefore consistent with the conclusions of the photoaffinity labeling results.