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.
Keywords: Conformational switch; Guanylate cyclase-activating protein; Magnesium; Molecular dynamics; Neuronal calcium sensor; Phototransduction.
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