Missense mutations affecting Ca2+-coordination in GCAP1 lead to cone-rod dystrophies by altering protein structural and functional properties
Guanylate cyclase activating protein 1 (GCAP1) is a neuronal calcium sensor (NCS) involved in the early biochemical steps underlying the phototransduction cascade. By switching from a Ca2+-bound form in the dark to a Mg2+-bound state following light activation of the cascade, GCAP1 triggers the acti...
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| Published in: | Biochimica et biophysica acta. Molecular cell research 2020-10, Vol.1867 (10), p.118794-118794, Article 118794 |
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| Main Authors: | , , , , |
| Format: | Article |
| Language: | eng |
| Online Access: | Get full text |
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| Summary: | Guanylate cyclase activating protein 1 (GCAP1) is a neuronal calcium sensor (NCS) involved in the early biochemical steps underlying the phototransduction cascade. By switching from a Ca2+-bound form in the dark to a Mg2+-bound state following light activation of the cascade, GCAP1 triggers the activation of the retinal guanylate cyclase (GC), thus replenishing the levels of 3′,5′-cyclic monophosphate (cGMP) necessary to re-open CNG channels. Here, we investigated the structural and functional effects of three missense mutations in GCAP1 associated with cone-rod dystrophy, which severely perturb the homeostasis of cGMP and Ca2+. Substitutions affect residues directly involved in Ca2+ coordination in either EF3 (D100G) or EF4 (E155A and E155G) Ca2+ binding motifs. We found that all GCAP1 variants form relatively stable dimers showing decreased apparent affinity for Ca2+ and blocking the enzyme in a constitutively active state at physiological levels of Ca2+. Interestingly, by corroborating spectroscopic experiments with molecular dynamics simulations we show that beside local structural effects, mutation of the bidentate glutamate in an EF-hand calcium binding motif can profoundly perturb the flexibility of the adjacent EF-hand as well, ultimately destabilizing the whole domain. Therefore, while Ca2+-binding to GCAP1 per se occurs sequentially, allosteric effects may connect EF hand motifs, which appear to be essential for the integrity of the structural switch mechanism in GCAP1, and perhaps in other NCS proteins.
•GCAP1 activates retinal guanylate cyclase (GC) in a Mg2+-bound state and inhibits it when Ca2+-bound•Retinal dystrophies-related variants of GCAP1 prevent the molecular switch and generally decrease its Ca2+-affinity•Mutations of the bidentate glutamate in the Ca2+-binding loop cause constitutive activation of GC at physiological [Ca2+]•EF3 and EF4 cation binding motifs are allosterically connected with one another as suggested by extensive MD simulations•Alteration of GCAP1 structure and function causes the dysregulation of Ca2+ and cGMP homeostasis in photoreceptors |
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| ISSN: | 0167-4889 1879-2596 |