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.