Dysfunction of cGMP signalling in photoreceptors by a macular dystrophy-related mutation in the calcium sensor GCAP1

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 Ca(2+)/Mg(2+)-binding, and its Ca(2+)-induced conformational changes in comparison to the wildtype protein. The mutant was less sensitive to changes in free Ca(2+), resulting in a constitutively active form under physiological Ca(2+)-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 Ca(2+)-binding of the mutant was nearly similar to the wildtype; binding of Mg(2+ )occurred with higher affinity. We performed molecular dynamics simulations for comparing the Ca(2+)-saturated inhibiting state of GCAP1 with the Mg(2+)-bound activating states. The L176F mutant exhibited significantly lower flexibility, when three Ca(2+ )or two Mg(2+ )were bound forming probably the structural basis for the modified GCAP1 function.