Yadav Deepak Kumar, Ahoulou Effibe O, Anderson David E, Bej Aritra, Hell Johannes W, Ames James B
Department of Chemistry, University of California, Davis, California95616, United States.
Department of Pharmacology, University of California, Davis, California95616, United States.
Biochemistry. 2025 May 6;64(9):1933-1942. doi: 10.1021/acs.biochem.4c00880. Epub 2025 Apr 16.
The L-type voltage-gated Ca channel (Ca1.2) controls gene expression, cardiac contraction, and neuronal activity. The C-terminal cytosolic region of the Ca1.2 alpha subunit (α1C) contains two domains known as proximal and distal C-terminal regulatory domains (PCRD and DCRD), which have been suggested to control Ca-dependent channel inactivation (CDI). Previous studies identified a salt bridge interaction between PCRD and DCRD that might be central to channel function. In this study, we expressed and purified recombinant constructs of PCRD (residues 1680-1750) and DCRD (residues 2035-2089) in for NMR structural analysis. PCRD and DCRD each exhibit backbone NMR chemical shifts consistent with a random coil and lack of tertiary structure. A disordered random coil structure may explain the absence of electron density for these domains in recent cryo-EM structures of Ca1.2. Despite this lack of structure, fluorescence polarization binding assays reveal PCRD and DCRD each bind to the Ca1.2 IQ-motif (residues 1644-1668) with dissociation constants of 1.5 ± 1 and 14 ± 5 μM, respectively. Both PCRD and DCRD also become insoluble in the presence of high micromolar levels of the IQ peptide, consistent with each domain forming an insoluble complex with the IQ peptide. AlphaFold3 predicts that DCRD adopts a 3-helix bundle that binds to the helical IQ-motif, while PCRD was previously suggested to form a 4-helix bundle. We propose that the PCRD and DCRD bind to opposite sides of the helical IQ-motif, which may oppose calmodulin (CaM) binding to Ca1.2 and thus modulate channel CDI.
L型电压门控钙通道（Ca1.2）控制基因表达、心脏收缩和神经元活动。Ca1.2α亚基（α1C）的C末端胞质区域包含两个被称为近端和远端C末端调节结构域（PCRD和DCRD）的结构域，有人认为它们控制钙依赖性通道失活（CDI）。先前的研究确定了PCRD和DCRD之间的盐桥相互作用，这可能是通道功能的核心。在本研究中，我们表达并纯化了PCRD（第1680 - 1750位氨基酸残基）和DCRD（第2035 - 2089位氨基酸残基）的重组构建体，用于核磁共振结构分析。PCRD和DCRD各自表现出与无规卷曲一致的主链核磁共振化学位移，且缺乏三级结构。无序的无规卷曲结构可能解释了在最近的Ca1.2冷冻电镜结构中这些结构域没有电子密度的现象。尽管缺乏结构，但荧光偏振结合试验表明，PCRD和DCRD各自与Ca1.2 IQ模体（第1644 - 1668位氨基酸残基）结合，解离常数分别为1.5±1和14±5μM。在高微摩尔浓度的IQ肽存在下，PCRD和DCRD也都变得不溶，这与每个结构域与IQ肽形成不溶性复合物一致。AlphaFold3预测DCRD采用一个3螺旋束与螺旋状的IQ模体结合，而之前有人认为PCRD形成一个4螺旋束。我们提出，PCRD和DCRD与螺旋状IQ模体的相对两侧结合，这可能会阻止钙调蛋白（CaM）与Ca1.2结合，从而调节通道CDI。
