title: Dysfunction of cGMP signalling in photoreceptors by a macular dystrophy-related mutation in the calcium sensor GCAP1.
abstract: Macular dystrophy leads to progressive loss of central vision and shows symptoms similar to age-related macular degeneration. Genetic screening of patients diagnosed with macular dystrophy disclosed a novel mutation in the GUCA1A gene, namely a c.526C > T substitution leading to the amino acid substitution p.L176F in the guanylate cyclase-activating protein 1 (GCAP1). The same variant was found in three families showing an autosomal dominant mode of inheritance. For a full functional characterization of the L176F mutant we expressed and purified the mutant protein and measured key parameters of its activating properties, its Ca2+/Mg2+-binding, and its Ca2+-induced conformational changes in comparison to the wildtype protein. The mutant was less sensitive to changes in free Ca2+, resulting in a constitutively active form under physiological Ca2+-concentration, showed significantly higher activation rates than the wildtype (90-fold versus 20-fold) and interacted with an higher apparent affinity with its target guanylate cyclase. However, direct Ca2+-binding of the mutant was nearly similar to the wildtype; binding of Mg2+ occurred with higher affinity. We performed molecular dynamics simulations for comparing the Ca2+-saturated inhibiting state of GCAP1 with the Mg2+-bound activating states. The L176F mutant exhibited significantly lower flexibility, when three Ca2+ or two Mg2+ were bound forming probably the structural basis for the modified GCAP1 function.
authors.affiliation: Department of Neuroscience, Biochemistry Group, University of Oldenburg, Oldenburg, Germany.
authors.forename: Farina
authors.initials: F
authors.lastname: Vocke
authors.affiliation: Molecular Genetics Laboratory, Institute for Ophthalmic Research, University of Tübingen, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Weisschuh
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, Verona, Italy and.
authors.forename: Valerio
authors.initials: V
authors.lastname: Marino
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, Verona, Italy and.
authors.forename: Silvia
authors.initials: S
authors.lastname: Malfatti
authors.affiliation: Scheie Eye Institute, Department of Ophthalmology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Samuel G
authors.initials: SG
authors.lastname: Jacobson
authors.affiliation: Molecular Genetics Laboratory, Institute for Ophthalmic Research, University of Tübingen, Germany.
authors.forename: Charlotte M
authors.initials: CM
authors.lastname: Reiff
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, Verona, Italy and.
authors.forename: Daniele
authors.initials: D
authors.lastname: Dell'Orco
authors.affiliation: Department of Neuroscience, Biochemistry Group, University of Oldenburg, Oldenburg, Germany.
authors.forename: Karl-Wilhelm
authors.initials: KW
authors.lastname: Koch
mesh_terms: D000293:Adolescent; D000328:Adult; D002118:Calcium / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism*; D005260:Female; D051604:Guanylate Cyclase-Activating Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008268:Macular Degeneration / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008297:Male; D008875:Middle Aged; D056004:Molecular Dynamics Simulation; D009154:Mutation / Q000235:genetics*; D010375:Pedigree; D011487:Protein Conformation; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism* / Q000473:pathology; D055815:Young Adult
chemical_list: C496736:GUCA1A protein, human; D051604:Guanylate Cyclase-Activating Proteins; D006152:Cyclic GMP; D002118:Calcium