Biophysical properties of different GCAP1 Mutants associated with cone dystrophy

In the photoreceptor cell many processes are regulated by proteins, which are directly affected by the intracellular Ca2+ concentration. Among these calcium sensor proteins the guanylate cyclase-activating proteins (GCAPs) play a crucial role in the restoration of the dark adapted state of the photoreceptor cell. Subtle changes of the biophysical properties of GCAPs due to mutations can lead to a disturbed restoration of the dark adapted state and therefore to retinal dysfunction. Different mutations in the gene encoding for GCAP1 were found in patients suffering from cone dystrophies. How do these mutations change the biophysical and biochemical properties of the GCAPs? To address this question we heterologously expressed and purified the proteins and accessed these properties using different techniques. Ca2+ binding constants and -enthalpies of the GCAP1 mutants causing cone dystrophy, namely E89K, D100E, L151F and G159V were determined by isothermal titration calorimetry (ITC), showing a shift of Ca2+ affinity and binding enthalpy for all the mutants compared to the wildtype. The Ca2+-related conformational change was monitored by a recently presented technique based on the surface plasmon resonance phenomenon (SPR). With this technique a real time determination of conformational transition in GCAPs was achieved. In addition, the calcium sensor protein in muscle cells, troponin C, was used for control recordings. For example, the Ca2+-induced change of the hydrodynamic shell of troponin C and of GCAPs were determined by dynamic light scattering (DLS). Our results using the SPR technique opened up a broad range of applications, not limited to calcium sensor proteins in the phototransduction cascade

Structural effects of Mg2+ on the regulatory states of Neuronal Calcium Sensors operating in vertebrate phototransduction

Several lines of evidence suggest that free Mg2+ plays an important role in phototransduction, as the Neuronal Calcium Sensors (NCS) Recoverin and Guanylate Cyclase Activating Proteins 1 and 2 (GCAP1 and GCAP2) are also capable of binding Mg2+ via their EF-hand motifs. Previous studies showed that a Mg2+ -bound state is required for GCAP1 in order to activate GC and that Recoverin binds Mg2+ without triggering its physiological conformational change. No structural studies were performed so far about GCAP2, for which the effects that Mg2+ could exert were only hypothesized. Here we compared the effects of physiological [Mg2+] (1 mM) on the switch states of these three NCS in their myristoylated (myr) and non myristoylated (nonmyr) form over the extreme conditions of high and low [Ca2+], mimicking respectively dark and light states of the photoreceptor cell. We performed Circular Dichroism spectroscopy measurements to assess the differences in thermal stability, secondary and tertiary structure of all NCS in the aforementioned conditions. Intrinsic fluorescence spectroscopy titrations and Isothermal Titration Calorimetry were performed for monitoring the binding of Mg2+ to GCAP2. Molecular dynamics simulations (200 ns, all-atom force field) were performed to assess structural properties of GCAP1 in putatively activator, inhibitor and transitory states. Our results confirm that Mg2+ is unable to trigger the physiological conformational change of Recoverin (myristoyl switch) and that it decreases its thermal stability. Mg2+ induces a conformational change in GCAP2 both at high and low [Ca2+], however these variations are more substantial for apo-myrGCAP2. Apo-GCAP1 is responsive to Mg2+, acquiring a different tertiary structure from both apo and Ca2+-bound states, though this difference is lost when Ca2+ is saturating. GCAP1 seems to be stabilized by the presence of Mg2+ in solution, more notably its Ca2+-bound form. Molecular dynamics simulations point out that myrGCAP1 has a highly flexible loop (125-135) when at least one divalent cation is bound to EF-3. In line with experimental data, this is sufficient to stabilize the entire structure. Moreover all simulated transitory states show very similar dynamic properties, which differ from both apo and Ca2+- or Mg2+- loaded forms

Conformational Changes in Calcium-Sensor Proteins under Molecular Crowding Conditions.

Fundamental components of signaling pathways are switch modes in key proteins that control start, duration, and ending of diverse signal transduction events. A large group of switch proteins are Ca2+ sensors, which undergo conformational changes in response to oscillating intracellular Ca2+ concentrations. Here we use dynamic light scattering and a recently developed approach based on surface plasmon resonance to compare the protein dynamics of a diverse set of prototypical Ca2+ -binding proteins including calmodulin, troponin C, recoverin, and guanylate cyclase-activating protein. Surface plasmon resonance biosensor technology allows monitoring conformational changes under molecular crowding conditions, yielding for each Ca2+ -sensor protein a fingerprint profile that reflects different hydrodynamic properties under changing Ca2+ conditions and is extremely sensitive to even fine alterations induced by point mutations. We see, for example, a correlation between surface plasmon resonance, dynamic light scattering, and size-exclusion chromatography data. Thus, changes in protein conformation correlate not only with the hydrodynamic size, but also with a rearrangement of the protein hydration shell and a change of the dielectric constant of water or of the protein-water interface. Our study provides insight into how rather small signaling proteins that have very similar three-dimensional folding patterns differ in their Ca2+ -occupied functional state under crowding conditions

Impact of cone dystrophy-related mutations in GCAP1 on a kinetic model of phototransduction.

Cone dystrophy-related mutations in guanylate cyclase-activating protein 1 (GCAP1) are known to cause severe disturbance of their Ca2+-sensing properties affecting also their regulatory modes. However, crucial biochemical properties of mutant GCAP1 forms have not been fully elucidated and regulatory parameters of GCAP1 mutants have not been considered within the context of a comprehensive description of the phototransduction cascade kinetics. We investigated therefore the structure-function relationships of four dystrophy-relevant point mutations in GCAP1 harboring the following amino acid substitutions: E89K, D100E, L151F, and G159V. All mutations decrease the catalytic efficiency in regulating the target guanylate cyclase and decrease the affinity of Ca2+-binding in at least one, but in most cases two EF-hand Ca2+-binding sites. Although the wild type and mutants of GCAP1 displayed large differences in Ca2+-binding and regulation, circular dichroism (CD) spectroscopy revealed that all proteins preserved an intact secondary and tertiary structure with a significant rearrangement of the aromatic residues upon binding of Ca2+. To gain insight into the dynamic changes of cyclic GMP levels in a photoreceptor cell, we incorporated parameters describing the regulation of target guanylate cyclase by GCAP1 mutants into a comprehensive kinetic model of phototransduction. Modeling led us to conclude that the contribution of GCAP1 to the dynamic synthesis of cyclic GMP in rod cells would depend on the expression level of the wild-type form. Although the synthesis rate controlled by GCAP1 remains at a constant level, in the case of high expression levels of cone-dystrophy GCAP1 mutants it would not contribute at all to shaping the cGMP rate, which becomes dynamically regulated solely by the other present Ca2+-sensor GCAP2

Unveiling biochemical and physiological consequences of cone dystrophy-related mutations in GCAP1

Purpose Cone dystrophies are often associated with altered levels of calcium (Ca2+) and cyclic GMP (cGMP), the second messengers operating in the phototransduction cascade in rod and cone photoreceptors. By using a multiscale approach, we investigated the biochemical and physiological effects of four pathogenic point mutations identified in the guanylate cyclase-activating protein 1 (GCAP1), leading to the amino acid substitutions E89K, D100E, L151F and G159V. Methods Structure-function relationships were studied by biophysical methods, including circular dichroism to monitor secondary and tertiary structural changes in GCAP1 variants upon binding of Ca2+ and isothermal titration calorimetry to monitor the thermodynamics of Ca2+-binding. Experimental parameters describing the regulation of the target enzyme guanylate cyclase 1 (GC) by each GCAP1 variant were incorporated into a comprehensive kinetic model of phototransduction, in order to assess the effect of each individual point mutation on the whole cell response. Results Wild type and cone dystrophy-related point mutations in GCAP1 showed large differences in Ca2+-binding and GC regulation but, except for E89K, the structural effects of all the tested mutations are minor and involve mostly a slight rearrangement of aromatic residues in the Ca2+-bound form. System-level modeling suggests that the main effect of all point mutations on the photoresponse kinetics is a perturbation of the photocurrent shape consisting in increased amplitude and prolonged duration. However, the effect is strongly dependent on the expression levels of pathogenic GCAP1 forms as compared to the wild-type form. Conclusion Our data suggest that a multiscale approach combining biochemistry, biophysics and systems biology strategies allows a deep molecular understanding of dysfunctional states in photoreceptors in cone-dystrophy conditions. In particular, we conclude that the contribution of GCAP1 to the dynamic synthesis of cGMP in rod cells depends on the expression level of the wild type form, and in the case of high expression levels of cone-dystrophy GCAP1 mutants it would not contribute at all to shaping the cGMP rate, which becomes dynamically regulated solely by the other present Ca2+-sensor GCAP2

Fingerprint profile of cone dystrophy related GCAP1 mutants

Purpose Photoreceptor cells efficiently respond to changing light conditions on a millisecond timescale by a well-balanced interplay of two second messengers, cGMP and calcium. Calcium sensor proteins like the guanylate cyclase-activating proteins (e.g. GCAP1 and GCAP2 in mammalians) control the synthesis of cGMP in a calcium-dependent manner and in astep-by-step calcium relay mode of action. Mutations in the gene GUCA1A encoding GCAP1 correlate with human cone dystrophies and are known to cause an imbalance of the calcium and cGMP homeostasis. Here we investigate the biophysical and biochemical properties of the GCAP1 mutants E89K, D100E, L151F and G159V, which are constitutive activators of photoreceptor guanylate cyclase (GC). Methods GCAP1 wildtype (WT) and mutants were heterologously expressed and purified. Hydrodynamic properties and calcium-binding parameters of GCAP1 variants were investigated by dynamic light scattering, isothermal titration calorimetry and size exclusion chromatography. Calcium-induced conformational changes were monitored by surface plasmon resonance. Catalytic parameters were determined by enzymatic assays using the target guanylate cyclase. Results Calcium-binding studies revealed three functional EF-hand calcium-binding sites in all mutants, but two EF-hands showed a several-fold lower affinity in the mutants than in WT GCAP1. Interestingly, the EF-hand with the highest affinity remained nearly unchanged. Changes in protein conformation correlated with data from dynamic light scattering and size exclusion chromatography showing a rearrangementof the protein hydration shell and a change of the dielectric constant of the protein-water interface. All mutations decrease the catalytic efficiency in regulating the target GC. Conclusion Point mutations of the calcium sensor GCAP1 have strong, but differential impacts on the biophysical and biochemical properties enabling the formulation of a fingerprint profile of each mutant. Thus, we further tested the consequences of dystrophy-related mutations in a kinetic model of phototransduction, in which we can access the cGMP synthesis rate resulting from either GCAP1 or GCAP2 during a photoresponse. The computational analysis revealed that the synthesis rate controlled by GCAP1 remains at a constant level, but it would not at all contribute to the shaping of the photoresponse. The latter would prominently be regulated by GCAP2