A novel target recognition revealed by calmodulin in complex with the basic helix-loop-helix transcription factor SEF2-1/E2-2
A novel target recognition revealed by calmodulin in complex with the basic helix-loop-helix transcription factor SEF2-1/E2-2
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DOI：
10.1110/ps.28401
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发表时间：
2001-01-01
期刊：
影响因子：
8
通讯作者：
Wijmenga, SS
中科院分区：
文献类型：
--
作者：
Larsson, G;Schleucher, J;Wijmenga, SS
钙调蛋白是细胞内主要的钙信号受体，参与多种细胞过程的调节。它可以通过一种新型的直接相互作用来抑制碱性螺旋-环-螺旋转录因子的DNA结合。为了从结构上表征这种新的钙调蛋白-靶相互作用，我们决定利用核磁共振技术研究钙调蛋白与二聚体碱性螺旋-环-螺旋转录因子SEF2-1(SEF2-1MP)的DNA结合结构域的二聚肽的复合体。在这里，我们报告了钙调蛋白：SEF2-1MP复合体的化学计量比是一个二聚肽与两个钙调蛋白分子结合。我们还报道了核磁共振大分子(类似于38kD)复合体的1H，13C和15N共振归属和钙调蛋白的二级结构，以及SEF2-1MP的1H归属和二级结构。此外，我们还测定了钙调蛋白的酰胺质子交换率，并测定了分子间钙调蛋白：SEF2-1MP和钙调蛋白：钙调蛋白NOE的接触。同位素过滤实验表明，SEF2-1MP与CaM NOE形成了紧密的复合体，分子间的CaM：CaM NOE接触证实了这一点。二级结构和酰胺质子交换数据表明，结合不是通过经典的包络结合模式进行的。相反，数据表明钙调蛋白以更开放的构象与SEF2-1MP相互作用，尽管N-末端和C-末端结构域的疏水表面仍然形成主要的相互作用部位。涉及带电残基的相互作用也与已知的结合对离子强度的相对较高的敏感性一致。最后，在经典的环合结合模式中，该多肽不形成或螺旋。
Calmodulin is the predominant intracellular receptor for Ca2+ signals, mediating the regulation of numerous cellular processes. It can inhibit the DNA binding of basic helix-loop-helix transcription factors by a direct interaction of a novel type. To structurally characterize this novel calmodulin-target interaction, we decided to study the complex of calmodulin with a dimeric peptide corresponding to the DNA-binding domains of the dimeric basic helix-loop-helix transcription factor SEF2-1 (SEF2-1mp) using NMR. Here, we report that the stoichiometry of the calmodulin:SEF2-1mp complex is one dimeric peptide binding two calmodulin molecules. We also report the 1H, 13C, and 15N resonance assignments and the secondary structure of calmodulin in this for NMR large (similar to 38 kD) complex, as well as the 1H assignments and secondary structure of SEF2-1mp. In addition, we determined the amide proton exchange rates of calmodulin and measured intermolecular calmodulin:SEF2-1mp and calmodulin:calmodulin NOE contacts. The isotope-filtered experiments show a large number of SEF2-1mp to calmodulin NOE contacts indicating that a tight complex is formed, which is confirmed by an intermolecular calmodulin:calmodulin NOE contact. The secondary structure and amide proton exchange data show that the binding does not occur via the classical wraparound binding mode. Instead, the data indicate that calmodulin interacts with SEF2-1mp in a more open conformation, although the hydrophobic surfaces of the N- and C-terminal domains still form the main interaction sites. Interactions involving charged residues are also identified in agreement with the known relatively high sensitivity of the binding to ionic strength. Finally, the peptide does not form an or-helix as in the classical wraparound binding mode.