Mg2+ and Ca2+ differentially regulate DNA binding and dimerization of DREAM
Mg2+ and Ca2+ differentially regulate DNA binding and dimerization of DREAM
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DOI：
10.1074/jbc.m500338200
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发表时间：
2005-05-06
影响因子：
4.8
通讯作者：
Ames, JB
中科院分区：
文献类型：
--
作者：
Osawa, M;Dace, A;Ames, JB
DREAM（calsenilin/KChIP 3）是一种EF-手型钙结合蛋白，抑制强啡肽原和c-fos基因的转录。在这里，我们提出了结构和结合的DREAM单位点突变体的研究，旨在禁用Ca 2+结合到每个功能EF-手（EF-2：D150 N; EF-3：E186 Q;和EF-4：E234 Q）。对Ca 2+与各种突变体结合的等温滴定量热法（ITC）分析显示，在不存在Mg 2+的情况下，Ca 2+独立地且顺序地与EF-3（Δ H =-2.4 kcal/mol）、EF-4（Δ H = +5.2 kcal/mol）和EF-2（Δ H =+1 kcal/mol）结合。相比之下，只有两个Ca 2+结合DREAM在生理水平的Mg 2+的存在下，野生型和D150 N，这表明EF-2组成型结合Mg 2+。ITC测量表明，一个Mg 2+以高亲和力（Kd = 13 μ M和Δ H =-0.79千卡/摩尔）亲合力结合，两个或更多个Mg 2+在毫摩尔范围内熵结合。尺寸排阻色谱研究表明，镁稳定DREAM作为一个单体，而钙离子诱导蛋白质二聚化。电泳迁移率变动分析表明，Mg 2+是必不可少的序列特异性结合的DREAM的DNA反应元件（DREs）在前强啡肽和c-fos基因。EF-手突变体特异性结合DRE，表明它们在功能上是完整的。EF-手突变体结合DRE在饱和Ca 2+水平，表明在EF-3或EF-4的一个单一的Ca 2+的结合足以驱动构象变化，废除DNA结合。NMR结构分析表明，无金属的DREAM采用折叠而灵活的熔融球状结构。Ca ~（2+）和Mg ~（2+）均能引起DREAM的构象变化，使DREAM的三级结构稳定。我们建议，镁离子结合EF-2可能在结构上桥DREAM的DNA目标和钙离子诱导的蛋白质二聚破坏DNA结合。
DREAM (calsenilin/KChIP3) is an EF-hand calcium-binding protein that represses transcription of prodynorphin and c-fos genes. Here we present structural and binding studies on single-site mutants of DREAM designed to disable Ca2+ binding to each of the functional EF-hands (EF-2: D150N; EF-3: E186Q; and EF-4: E234Q). Isothermal titration calorimetry (ITC) analysis of Ca2+ binding to the various mutants revealed that, in the absence of Mg2+, Ca2+ binds independently and sequentially to EF-3 (Delta H = - 2.4 kcal/mol), EF-4 (Delta H = +5.2 kcal/mol), and EF-2 (Delta H = + 1 kcal/mol). By contrast, only two Ca2+ bind to DREAM in the presence of physiological levels of Mg2+ for both wild-type and D150N, suggesting that EF-2 binds constitutively to Mg2+. ITC measurements demonstrate that one Mg2+ binds enthalpically with high affinity (K-d = 13 mu M and Delta H = - 0.79 kcal/mol) and two or more Mg2+ bind entropically in the millimolar range. Size-exclusion chromatography studies revealed that Mg2+ stabilizes DREAM as a monomer, whereas Ca2+ induces protein dimerization. Electrophoretic mobility shift assays indicated that Mg2+ is essential for sequence-specific binding of DREAM to DNA response elements (DREs) in prodynorphin and c-fos genes. The EF-hand mutants bind specifically to DRE, suggesting they are functionally intact. None of the EF-hand mutants bind DRE at saturating Ca2+ levels, suggesting that binding of a single Ca2+ at either EF-3 or EF-4 is sufficient to drive conformational changes that abolish DNA binding. NMR structural analysis indicates that metal-free DREAM adopts a folded yet flexible molten globule-like structure. Both Ca2+ and Mg2+ induce distinct conformational changes, which stabilize tertiary structure of DREAM. We propose that Mg2+ binding at EF-2 may structurally bridge DREAM to DNA targets and that Ca2+-induced protein dimerization disrupts DNA binding.