Consensus and variant cAMP-regulated enhancers have distinct CREB-binding properties
Consensus and variant cAMP-regulated enhancers have distinct CREB-binding properties
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DOI：
10.1074/jbc.m010263200
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发表时间：
2001-04-13
影响因子：
4.8
通讯作者：
Goodman, RH
中科院分区：
文献类型：
--
作者：
Craig, JC;Schumacher, MA;Goodman, RH
最近对cAMP反应元件结合蛋白（CREB）碱性亮氨酸拉链（bZIP）共有CRE晶体结构的测定揭示了CREB/CREM/ATF-1转录因子家族成员中保守的关键二聚化和DNA结合特征。二聚化似乎是由Tyr（307）-Glu（312）螺旋间氢键和Glu（319）- Arg（314）静电相互作用介导的。一个意想不到的六水合Mg 2+离子的中心以上的CRE在二聚体腔。在本研究中，我们将这些特征与CREB二聚化和DNA结合相关。一个Y307 F取代减少二聚体的稳定性和DNA结合亲和力，而Y307 R突变产生稳定的效果。Glu（319）突变为Ala或Lys减弱二聚化和DNA结合。Mg 2+离子使野生型CREB与回文CRE的结合亲和力增强了约20倍，但对于不同的克雷斯则没有。类似地，介导CREB与水合Mg 2+相互作用的Lys（304）的突变阻断了CREB与回文CRE序列的结合，但不阻断CREB与变体CRE序列的结合。K304 A突变体与共有和变异CRE序列的不同结合特征表明CREB与这些元件的结合受Mg ~（2+）离子的差异调节。我们认为CREB通过根本不同的机制与共有和变异CRE序列结合。
Recent determination of the cAMP response element-binding protein (CREB) basic leucine zipper (bZIP) consensus CRE crystal structure revealed key dimerization and DNA binding features that are conserved among members of the CREB/CREM/ATF-1 family of transcription factors. Dimerization appeared to be mediated by a Tyr(307)-Glu(312) interhelical hydrogen bond and a Glu(319)- Arg(314) electrostatic interaction. An unexpected hexahydrated Mg2+ ion was centered above the CRE in the dimer cavity. In the present study, we related these features to CREB dimerization and DNA binding. A Y307F substitution reduced dimer stability and DNA binding affinity, whereas a Y307R mutation produced a stabilizing effect. Mutation of Glu(319) to Ala or Lys attenuated dimerization and DNA binding. Mg2+ ions enhanced the binding affinity of wild-type CREB to the palindromic CRE by similar to 20-fold but did not do so for divergent CREs, Similarly, mutation of Lys(304), which mediates the CREB interaction with the hydrated Mg2+, blocked CREB binding to the palindromic but not the variant CRE sequences. The distinct binding characteristics of the K304A mutants to the consensus and variant CRE sequences indicate that CREB binding to these elements is differentially regulated by Mg2+ ions, We suggest that CREB binds the consensus and variant CRE sequences through fundamentally distinct mechanisms.