DIFFERENTIAL DNA-BINDING SPECIFICITY OF THE ENGRAILED HOMEODOMAIN - THE ROLE OF RESIDUE-50
DIFFERENTIAL DNA-BINDING SPECIFICITY OF THE ENGRAILED HOMEODOMAIN - THE ROLE OF RESIDUE-50
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DOI：
10.1021/bi00197a022
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发表时间：
1994-08-09
期刊：
影响因子：
2.9
通讯作者：
SAUER, RT
中科院分区：
文献类型：
--
作者：
ADES, SE;SAUER, RT
为了评估残基50在决定果蝇增强转录因子同源结构域结合特异性中的重要性，研究了含有谷氨酰胺(野生型)、丙氨酸和赖氨酸的分离同源结构域的DNA结合特性。在使用野生型增强的同源结构域进行的结合位点选择实验中，TAATTA被鉴定为一个高亲和力、共识的结合位点。当50位的谷氨酰胺被赖氨酸(QK50)取代时，结合位点的偏好变为TAATCC。与野生型蛋白与TAATTA位点之间的半衰期和亲和力相比，QK50蛋白与含有TAATCC的DNA位点之间的复合体的半衰期和亲和力显著增加。这表明Lys50与TAATCC DNA的相互作用比Gln50与TAATTA位点的作用更有利。事实上，野生型Gln50侧链(它与共晶结构中TAATTA位点的最后A：T碱基对形成疏水相互作用[Kisinger，C.R.，Liu，B.，Martin-Blanco，E.，Kornberg，T.B.，&Pabo，C.C.(1990)Cell 63,579-590])似乎只在决定TAATTA位点的结合亲和力和特异性方面起到很小的作用，因为QA50突变体与TAATTA位点的亲和力只降低了2倍，并且区分了TAATTA和TAATCC位点以及野生型蛋白。因此，除了Gln50以外的决定因素必须参与建立增强的同源结构域的不同结合特异性。
To assess the importance of residue 50 in determining the binding specificity of the homeodomain from the engrailed transcription factor of Drosophila, the DNA-binding properties of isolated homeodomains containing glutamine (wild type), alanine, and lysine at this position have been studied. In binding site selection experiments using the wild-type engrailed homeodomain, TAATTA was identified as a high-affinity, consensus binding site. When the glutamine at position 50 was replaced by a lysine (QK50), the binding site preference changed to TAATCC. The half-life and affinity of the complex between the QK50 protein and a DNA site containing TAATCC were increased significantly compared to the half-life and affinity of the complex between the wild-type protein and a TAATTA site. This suggests that Lys50 forms a more favorable interaction with the TAATCC DNA than Gln50 does with the TAATTA site. In fact, the wild-type Gln50 side chain (which forms a hydrophobic interaction with the last A:T base pair of the TAATTA site in the cocrystal structure [Kissinger, C. R., Liu, B., Martin-Blanco, E., Kornberg, T. B., & Pabo, C. O. (1990) Cell 63, 579-590]) appears to play only a small role in determining binding affinity and specificity for the TAATTA site, as the QA50 mutant has only a 2-fold reduced affinity for the TAATTA site and discriminates between the TAATTA and TAATCC sites as well as the wild-type protein. As a result, determinants in addition to Gln50 must be involved in establishing the differential binding specificity of the engrailed homeodomain.