Marino Valerio, Sulmann Stefan, Koch Karl-Wilhelm, Dell'Orco Daniele
Department of Life Sciences and Reproduction, Section of Biological Chemistry, University of Verona, Italy.
Department of Neurosciences, Biochemistry Group, University of Oldenburg, Germany.
Biochim Biophys Acta. 2015 Sep;1853(9):2055-65. doi: 10.1016/j.bbamcr.2014.10.026. Epub 2014 Nov 4.
The effects of physiological concentration of magnesium on the switch states of the neuronal calcium sensor proteins recoverin, GCAP1 and GCAP2 were investigated. Isothermal titration calorimetry was applied for binding studies. Circular dichroism spectroscopy was used to characterize protein thermal stability, secondary and tertiary structure in conditions of high and low [Ca²⁺], mimicking respectively the dark-adapted and light-exposed photoreceptor states during the phototransduction cascade. Further, molecular dynamics (MD) simulations were run to investigate the dynamical structural properties of GCAP1 in its activator, inhibitor and putative transitory states. Our results confirmed that Mg²⁺ is unable to trigger the typical Ca²⁺-induced conformational change of recoverin (myristoyl switch) while it decreases its thermal stability. Interestingly, Mg²⁺ seems to affect the conformation of GCAP2 both at high and low [Ca²⁺], however the variations are more substantial for myristoylated GCAP2 in the absence of Ca²⁺. GCAP1 is responsive to Mg²⁺ only in its low [Ca²⁺] state and Mg²⁺-GCAP1 tertiary structure slightly differs from both apo and Ca²⁺-bound states. Finally, MD simulations suggest that the GCAP1 state harboring one Mg²⁺ ion bound to EF2 acquires structural characteristics that are thought to be relevant for the activation of the guanylate cyclase. Moreover, all the putative Mg²⁺-bound states of myristoylated GCAP1 are structurally less flexible than Ca²⁺-bound states. GCAP1 acquires a more compact tertiary structure that is less accessible to the solvent, thereby inducing a different conformation to the myristoyl moiety, which might be crucial for the activation of the guanylate cyclase. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.
研究了生理浓度的镁对神经元钙传感器蛋白恢复蛋白、鸟苷酸环化酶激活蛋白1（GCAP1）和鸟苷酸环化酶激活蛋白2（GCAP2）开关状态的影响。采用等温滴定量热法进行结合研究。圆二色光谱用于表征在高[Ca²⁺]和低[Ca²⁺]条件下蛋白质的热稳定性、二级和三级结构，分别模拟光转导级联过程中暗适应和光暴露的光感受器状态。此外，进行了分子动力学（MD）模拟以研究GCAP1在其激活剂、抑制剂和假定的过渡状态下的动态结构特性。我们的结果证实，Mg²⁺无法触发恢复蛋白典型的Ca²⁺诱导的构象变化（肉豆蔻酰开关），同时它会降低其热稳定性。有趣的是，Mg²⁺似乎在高[Ca²⁺]和低[Ca²⁺]时都会影响GCAP2的构象，然而在无Ca²⁺时，对于肉豆蔻酰化的GCAP2，变化更为显著。GCAP1仅在其低[Ca²⁺]状态下对Mg²⁺有反应，并且Mg²⁺-GCAP1的三级结构与脱辅基和Ca²⁺结合状态均略有不同。最后，MD模拟表明，GCAP1状态下一个Mg²⁺离子与EF2结合，获得了被认为与鸟苷酸环化酶激活相关的结构特征。此外，肉豆蔻酰化GCAP1的所有假定Mg²⁺结合状态在结构上比Ca²⁺结合状态更不灵活。GCAP1获得了更紧凑的三级结构，溶剂更难接近，从而诱导肉豆蔻酰部分形成不同的构象，这可能对鸟苷酸环化酶的激活至关重要。本文是名为：第13届欧洲钙研讨会的特刊的一部分。
