Structural basis for the regulation of L-type voltage-gated calcium channels: interactions between the N-terminal cytoplasmic domain and Ca2+-calmodulin
Structural basis for the regulation of L-type voltage-gated calcium channels: interactions between the N-terminal cytoplasmic domain and Ca2+-calmodulin
复制标题
DOI：
10.3389/fnmol.2012.00038
复制
发表时间：
2012-01-01
影响因子：
4.8
通讯作者：
Vogel, Hans J.
中科院分区：
文献类型：
--
作者：
Liu, Zhihong;Vogel, Hans J.
众所周知，l型电压门控钙通道的打开可由钙调蛋白（calmodulin, CaM）调控。其中一个主要的调节机制是钙依赖性失活（CDI），其中载脂蛋白cam与通道的细胞质c端结构域结合可以有效地感知局部钙离子浓度的增加。钙结合CaM可以结合到c端区域的iq基序区域并阻断钙通道，从而提供一种负反馈机制，防止细胞钙浓度超过生理极限的上升。最近，在Ca(v)1.2和Ca(v)1.3的氨基端细胞质区发现了一个额外的Ca2+/ cam结合基元（NSCaTE， n端空间Ca2+转化元件）。该基元仅存在于Ca(v)1.2和Ca(v)1.3通道中，并且在Ca(v)1.3通道中发现了明显的N-lobe (Ca2+/CaM) CDI效应。为了了解这种相互作用的分子基础，研究了Ca2+/CaM与生物合成的n端区域（残基1-68）和NSCaTE肽（残基48-68）的配合物。我们发现n端细胞质区域的NSCaTE基序采用u-螺旋构象，很可能是由于其高丙氨酸含量。此外，当与Ca2+-CaM结合时，该复合物表现出不寻常的1:2蛋白：肽化学计量，并且CaM的n叶比c叶对肽具有更强的亲和力。通过核磁共振波谱和数据驱动蛋白对接方法，对Ca2+/CaM分离的N-叶和c -叶以及NSCaTE肽的复杂结构进行了测定。此外，我们还证明了钙结合蛋白1，与CaM竞争结合到c端细胞质域，仅弱结合到NSCaTE区域。这些结构提供了对该基序在钙调节网络中的可能作用的见解。我们的研究为前人提出的剑桥桥模型提供了结构证据。
It is well-known that the opening of Ltype voltage gated calcium channels can be regulated by calmodulin (CaM). One of the main regulatory mechanisms is calcium-dependent inactivation (CDI), where binding of apo-CaM to the cytoplasmic C-terminal domain of the channel can effectively sense an increase in the local calcium ion concentration. Calcium-bound CaM can bind to the IQ-motif region of the C-terminal region and block the calcium channel, thereby providing a negative feedback mechanism that prevents the rise of cellular calcium concentrations over physiological limits. Recently, an additional Ca2+/CaM-binding motif (NSCaTE, N-terminal spatial Ca2+ transforming element) was identified in the amino terminal cytoplasmic region of Ca(v)1.2 and Ca(v)1.3. This motif exists only in Ca(v)1.2 and Ca(v)1.3 channels, and a pronounced N-lobe (Ca2+/CaM) CDI effect was found for Ca(v)1.3. To understand the molecular basis of this interaction, the complexes of Ca2+/CaM with the biosynthetically produced N-terminal region (residues 1-68) and NSCaTE peptide (residues 48-68) were investigated. We discovered that the NSCaTE motif in the N-terminal cytoplasmic region adopts an u-helical conformation, most likely due to its high alanine content. Additionally, the complex exhibits an unusual 1:2 protein:peptide stoichiometry when bound to Ca2+-CaM, and the N-lobe of CaM has a much stronger affinity for the peptide than the C-lobe. The complex structures of the isolated N- and C-lobe of Ca2+/CaM and the NSCaTE peptide were determined by nuclear magnetic resonance spectroscopy and data driven protein docking methods. Moreover, we also demonstrated that calcium binding protein 1, which competes with CaM for binding to the C-terminal cytoplasmic domain, binds only weakly to the NSCaTE region. The structures provide insights into the possible roles of this motif in the calcium regulatory network. Our study provides structural evidence for the CaM-bridge model proposed in previous studies.