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

Abstract

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.

Structural Characterization of Ferrous Ion Binding to Retinal Guanylate Cyclase Activator Protein 5 from Zebrafish Photoreceptors

“…4D), consistent with possible Fe 2+ binding at EF2. In the presence of physiological Mg 2+ levels (1 mM), Mg 2+ interferes with Ca 2+ -binding to GCAP5 16 and is known to bind to EF2 in GCAP1 17,29 . To test whether Fe 2+ can bind to EF2 in place of Mg 2+ , an HSQC spectrum of Mg 2+ -bound GCAP5 was recorded in the presence and absence of saturating Fe 2+ (Fig.…”

“…7A) may protect photoreceptor cells from apoptosis and possibly prevent retinal degeneration. Mutations in GCs and/or GCAPs that cause constitutive GC activation are genetically linked to retinal degeneration 29, 37-41 . Also, the retinal degeneration 3 (RD3) protein (that binds to GC in place of GCAPs and prevents GC activation) is important for turning off basal cyclase activity during the trafficking of GC to the outer segment 42 .…”

Mutations in PLS1 , encoding fimbrin, cause autosomal dominant nonsyndromic hearing loss

“…In silico amino acid substitutions (E264 to K; L238 to R, or F128 to S) were performed using PyMol (The PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC) and the resulting structural models of each fimbrin mutant were energy‐minimized in two steps, similarly to what was done previously (Marino & Dell’Orco, ; Marino, Sulmann, Koch, & Dell’Orco, ), the only difference being that the minimization was performed with unconstrained motion for backbone and side‐chain atoms both for the first steepest descent algorithm step and for the following steps performed according to the conjugate gradients algorithm.…”

CaF2nanoparticles as surface carriers of GCAP1, a calcium sensor protein involved in retinal dystrophies

“…Binding stoichiometry of GCAP1 interacting with NP was tested by isothermal titration calorimetry (ITC) as described earlier for other binding processes involving NCS proteins. 18,29,30 Briefly, ITC was performed using a VP-ITC instrument from MicroCal (Northampton, MA, USA) at T = 25°C. Purified GCAP1 or the mutant D100E was present in the recording cell in a decalcified buffer composed of 5 mM Tris-HCl pH 7.5 and 150 mM KCl.…”

“…15 Among Ca 2+ sensor proteins operating in the phototransduction cascade, guanylate cyclase-activating proteins (GCAPs) are able to bind both Ca 2+ and Mg 2+ and thereby switch between activators and inhibitors of the target enzyme guanylate cyclase GC-E (from now on indicated as GC). [16][17][18][19][20] GC uses GTP as a substrate to synthesize cyclic GMP (cGMP), another important second messenger crucial for the recovery of the photoresponse upon illumination. Indeed, cGMP synthesis is finely regulated in a Ca 2+dependent manner by GCAPs, which inhibit the GC at high [Ca 2+ ] (∼250-800 nM), where the three functional Ca 2+ -binding EF hand motifs (EF2, EF3 and EF4; see Fig.…”