title: Impact of cone dystrophy-related mutations in GCAP1 on a kinetic model of phototransduction.
abstract: Cone dystrophy-related mutations in guanylate cyclase-activating protein 1 (GCAP1) are known to cause severe disturbance of their Ca(2+)-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 Ca(2+)-binding in at least one, but in most cases two EF-hand Ca(2+)-binding sites. Although the wild type and mutants of GCAP1 displayed large differences in Ca(2+)-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 Ca(2+). 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 Ca(2+)-sensor GCAP2.
authors.affiliation: Section of Biological Chemistry, Department of Life Sciences and Reproduction, University of Verona, 37134 Verona, Italy, daniele.dellorco@univr.it.
authors.forename: Daniele
authors.initials: D
authors.lastname: Dell'Orco
authors.forename: Stefan
authors.initials: S
authors.lastname: Sulmann
authors.forename: Patrick
authors.initials: P
authors.lastname: Zägel
authors.forename: Valerio
authors.initials: V
authors.lastname: Marino
authors.forename: Karl-Wilhelm
authors.initials: KW
authors.lastname: Koch
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D055162:Biocatalysis; D002118:Calcium / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D051604:Guanylate Cyclase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D055537:Light Signal Transduction; D051379:Mice; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012160:Retina / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D051604:Guanylate Cyclase-Activating Proteins; D011994:Recombinant Proteins; D006152:Cyclic GMP; D002118:Calcium