Engineering a "steric doorstop" in rhodopsin: converting an inverse agonist to an agonist.
Engineering a "steric doorstop" in rhodopsin: converting an inverse agonist to an agonist.
复制标题
在视紫红质中设计“空间门挡”：将反向激动剂转化为激动剂。
DOI：
10.1021/bi701003b
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发表时间：
2007
期刊：
影响因子：
2.9
通讯作者：
Kono,Masahiro
中科院分区：
文献类型：
--
作者：
McKee,TimothyD;Lewis,MargaretR;Kono,Masahiro
视紫红质的晶体结构描绘了视紫红质在黑暗中的非活性构象。11-顺式维甲酸发色团，反向激动剂保持视紫红质失活，是很好的解决。螺旋3中的Thr 118是紧邻发色团的9-甲基的最接近的氨基酸残基。视黄醛的9-甲基促进从无活性的后视紫红质I转变为活性的后视紫红质II中间体。在这项研究中，一个位点特异性突变的Thr 118的体积较大的色氨酸的想法，以诱导蛋白质的活性构象。这些数据表明，这种突变确实会产生一种依赖于配体（特别是9-甲基）存在的活性蛋白。由于这种突变，11-顺式视黄醛已转化为激动剂。这种突变体的脱辅基蛋白形式并不比野生型脱辅基蛋白更有活性。然而，与野生型视紫红质不同，配体的共价键在黑暗中可以被羟胺攻击。Thr 118 Trp突变和发色团的9-甲基基团的组合表现为将蛋白质保持在开放和活性构象的“空间门挡”。
The crystal structures of rhodopsin depict the inactive conformation of rhodopsin in the dark. The 11-cis retinoid chromophore, the inverse agonist holding rhodopsin inactive, is well-resolved. Thr118 in helix 3 is the closest amino acid residue next to the 9-methyl group of the chromophore. The 9-methyl group of retinal facilitates the transition from an inactive metarhodopsin I to the active metarhodopsin II intermediate. In this study, a site-specific mutation of Thr118 to the bulkier Trp was made with the idea to induce an active conformation of the protein. The data indicate that such a mutation does indeed result in an active protein that depends on the presence of the ligand, specifically the 9-methyl group. As a result of this mutation, 11-cis retinal has been converted to an agonist. The apoprotein form of this mutant is no more active than the wild-type apoprotein. However, unlike wild-type rhodopsin, the covalent linkage of the ligand can be attacked by hydroxylamine in the dark. The combination of the Thr118Trp mutation and the 9-methyl group of the chromophore behaves as a “steric doorstop” holding the protein in an open and active conformation.