Lusin Jacqueline D, Vanarotti Murugendra, Li Congmin, Valiveti Aswani, Ames James B
Department of Chemistry, University of California, Davis, California 95616, USA.
Biochemistry. 2008 Feb 26;47(8):2252-64. doi: 10.1021/bi7017267. Epub 2008 Jan 18.
DREAM (calsenilin/KChIP3) is an EF-hand calcium-binding protein that binds to specific DNA sequences and regulates Ca2+-induced transcription of prodynorphin and c-fos genes. Here, we present the atomic-resolution structure of Ca2+-bound DREAM in solution determined by nuclear magnetic resonance (NMR) spectroscopy. Pulsed-field gradient NMR diffusion experiments and 15N NMR relaxation analysis indicate that Ca2+-bound DREAM forms a stable dimer in solution. The structure of the first 77 residues from the N-terminus could not be determined by our NMR analysis. The C-terminal DREAM structure (residues 78-256) contains four EF-hand motifs arranged in a tandem linear array, similar to that seen in KChIP1, recoverin, and other structures of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily. Mg2+ is bound at the second EF-hand, whereas Ca2+ is bound functionally at the third and fourth sites. The first and second EF-hands form an exposed hydrophobic groove on the protein surface lined by side-chain atoms of L96, F100, F114, I117, Y118, F121, F122, Y151, L155, L158, and L159 that are highly conserved in all NCS proteins. An exposed leucine near the C-terminus (L251) is suggested to form intermolecular contacts with leucine residues in the hydrophobic groove (L155, L158, and L159). Positively charged side chains of Arg and Lys (Lys87, Lys90, Lys91, Arg98, Lys101, Arg160, and Lys166) are clustered on one side of the protein surface and may mediate electrostatic contacts with DNA targets. We propose that Ca2+-induced dimerization of DREAM may partially block the putative DNA-binding site, which may suggest as to how Ca2+ abolishes DREAM binding to DNA to activate the transcription of prodynorphin and other downstream genes in pain control.
DREAM（钙调神经磷酸酶结合蛋白/KChIP3）是一种EF手型钙结合蛋白，它能与特定DNA序列结合，并调节钙离子诱导的前强啡肽原和c-fos基因的转录。在此，我们展示了通过核磁共振（NMR）光谱法测定的溶液中钙离子结合的DREAM的原子分辨率结构。脉冲场梯度NMR扩散实验和15N NMR弛豫分析表明，钙离子结合的DREAM在溶液中形成稳定的二聚体。我们的NMR分析无法确定N端前77个残基的结构。C端DREAM结构（残基78 - 256）包含四个以串联线性阵列排列的EF手基序，类似于在KChIP1、恢复蛋白以及钙调蛋白超家族的神经元钙传感器（NCS）分支的其他结构中所见。镁离子结合在第二个EF手基序上，而钙离子在功能上结合在第三和第四位点。第一和第二个EF手基序在蛋白质表面形成一个暴露的疏水凹槽，由L96、F100、F114、I117、Y118、F121、F122、Y151、L155、L158和L159的侧链原子排列而成，这些在所有NCS蛋白中高度保守。C端附近一个暴露的亮氨酸（L251）被认为与疏水凹槽中的亮氨酸残基（L155、L158和L159）形成分子间接触。精氨酸和赖氨酸的带正电侧链（Lys87、Lys90、Lys91、Arg98、Lys101、Arg160和Lys166）聚集在蛋白质表面的一侧，可能介导与DNA靶点的静电接触。我们提出，钙离子诱导的DREAM二聚化可能部分阻断假定的DNA结合位点，这可能提示了钙离子如何消除DREAM与DNA的结合以激活前强啡肽原和其他下游基因在疼痛控制中的转录。
