The dimerization domain in outer segment guanylate cyclase is a Ca\ub2\u207a-sensitive control switch module.

Membrane-bound guanylate cyclases harbor a region called the dimerization or linker domain, which aids the enzymes in adopting an optimal monomer-monomer arrangement for catalysis. One subgroup of these guanylate cyclases is expressed in rod and cone cells of vertebrate retina, and mutations in the dimerization domain of rod outer segment guanylate cyclase 1 (ROS-GC1, encoded by the GUCY2D gene) correlate with retinal cone-rod dystrophies. We investigate how a Q847L/K848Q double mutation, which was found in patients suffering from cone-rod dystrophy, and the Q847L and K848Q single-point mutations affect the regulatory mechanism of ROS-GC1. Both the wild type and mutants of heterologously expressed ROS-GC1 were present in membranes. However, the mutations affected the catalytic properties of ROS-GC1 in different manners. All mutants had higher basal guanylate cyclase activities but lower levels of activation by Ca(2+)-sensing guanylate cyclase-activating proteins (GCAPs). Further, incubation with wild-type GCAP1 and GCAP2 revealed for all ROS-GC1 mutants a shift in Ca(2+) sensitivity, but activation of the K848Q mutant by GCAPs was severely impaired. Apparent affinities for GCAP1 and GCAP2 were different for the double mutant and the wild type. Circular dichroism spectra of the dimerization domain showed that the wild type and mutants adopt a prevalently \u3b1-helical structure, but mutants exhibited lower thermal stability. Our results indicate that the dimerization domain serves as a Ca(2+)-sensitive control module. Although it is per se not a Ca(2+)-sensing unit, it seems to integrate and process information regarding Ca(2+) sensing by sensor proteins and regulator effector affinity

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