Structural effects of Mg2+ on the regulatory states of three neuronal calcium sensors operating in vertebrate phototransduction

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 [Ca(2+)], 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 Mg(2+) is unable to trigger the typical Ca(2+)-induced conformational change of recoverin (myristoyl switch) while it decreases its thermal stability. Interestingly, Mg(2+) seems to affect the conformation of GCAP2 both at high and low [Ca(2+)], however the variations are more substantial for myristoylated GCAP2 in the absence of Ca(2+). GCAP1 is responsive to Mg(2+) only in its low [Ca(2+)] state and Mg(2+)-GCAP1 tertiary structure slightly differs from both apo and Ca(2+)-bound states. Finally, MD simulations suggest that the GCAP1 state harboring one Mg(2+) ion bound to EF2 acquires structural characteristics that are thought to be relevant for the activation of the guanylate cyclase. Moreover, all the putative Mg(2+)-bound states of myristoylated GCAP1 are structurally less flexible than Ca(2+)-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.

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. (C) 2014 Elsevier B.V. All rights reserved.