Piazza Michael, Guillemette J Guy, Dieckmann Thorsten
Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
Biochemistry. 2015 Mar 24;54(11):1989-2000. doi: 10.1021/bi501353s. Epub 2015 Mar 16.
The intracellular Ca²⁺ concentration is an important regulator of many cellular functions. The small acidic protein calmodulin (CaM) serves as a Ca²⁺ sensor and control element for many enzymes. Nitric oxide synthase (NOS) is one of the proteins that is activated by CaM and plays a major role in a number of key physiological and pathological processes. Previous studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the electron transfer between the reductase and oxygenase domains through a process that is thought to be highly dynamic. We have analyzed the structure and dynamics of complexes formed by peptides based on inducible NOS (iNOS) and endothelial NOS (eNOS) with CaM at Ca²⁺ concentrations that mimic the physiological basal (17 and 100 nM) and elevated levels (225 nM) found in mammalian cells using fluorescence techniques and nuclear magnetic resonance spectroscopy. The results show the CaM-NOS complexes have similar structures at physiological and fully saturated Ca²⁺ levels; however, their dynamics are remarkably different. At 225 nM Ca²⁺, the CaM-NOS complexes show overall an increase in backbone dynamics, when compared to the dynamics of the complexes at saturating Ca²⁺ concentrations. Specifically, the N-lobe of CaM in the CaM-iNOS complex displays a lower internal mobility (higher S²) and higher exchange protection compared to those of the CaM-eNOS complex. In contrast, the C-lobe of CaM in the CaM-eNOS complex is less dynamic. These results illustrate that structures of CaM-NOS complexes determined at saturated Ca²⁺ concentrations cannot provide a complete picture because the differences in intramolecular dynamics become visible only at physiological Ca²⁺ levels.
细胞内钙离子浓度是许多细胞功能的重要调节因子。小酸性蛋白钙调蛋白（CaM）作为许多酶的钙离子传感器和控制元件。一氧化氮合酶（NOS）是被CaM激活的蛋白质之一，在许多关键的生理和病理过程中起主要作用。先前的研究表明，CaM的作用就像一个开关，会导致NOS发生构象变化，从而通过一个被认为是高度动态的过程实现还原酶和加氧酶结构域之间的电子转移。我们利用荧光技术和核磁共振光谱，分析了基于诱导型NOS（iNOS）和内皮型NOS（eNOS）的肽段与CaM在模拟哺乳动物细胞中生理基础水平（17和100 nM）及升高水平（225 nM）的钙离子浓度下形成的复合物的结构和动力学。结果表明，CaM-NOS复合物在生理和完全饱和的钙离子水平下具有相似的结构；然而，它们的动力学却显著不同。与饱和钙离子浓度下复合物的动力学相比，在225 nM钙离子浓度时，CaM-NOS复合物的主链动力学总体增加。具体而言，与CaM-eNOS复合物相比，CaM-iNOS复合物中CaM的N叶显示出较低的内部流动性（较高的S²）和较高的交换保护。相反，CaM-eNOS复合物中CaM的C叶动态性较低。这些结果表明，在饱和钙离子浓度下测定的CaM-NOS复合物结构并不能提供完整的信息，因为分子内动力学差异仅在生理钙离子水平下才可见。
