title: Two retinal dystrophy-associated missense mutations in GUCA1A with distinct molecular properties result in a similar aberrant regulation of the retinal guanylate cyclase.
abstract: Two recently identified missense mutations (p. L84F and p. I107T) in GUCA1A, the gene coding for guanylate cyclase (GC)-activating protein 1 (GCAP1), lead to a phenotype ascribable to cone, cone-rod and macular dystrophies. Here, we present a thorough biochemical and biophysical characterization of the mutant proteins and their distinct molecular features. I107T-GCAP1 has nearly wild-type-like protein secondary and tertiary structures, and binds Ca(2+) with a >10-fold lower affinity than the wild-type. On the contrary, L84F-GCAP1 displays altered tertiary structure in both GC-activating and inhibiting states, and a wild type-like apparent affinity for Ca(2+). The latter mutant also shows a significantly high affinity for Mg(2+), which might be important for stabilizing the GC-activating state and inducing a cooperative mechanism for the binding of Ca(2+), so far not been observed in other GCAP1 variants. Moreover, the thermal stability of L84F-GCAP1 is particularly high in the Ca(2+)-bound, GC-inhibiting state. Molecular dynamics simulations suggest that such enhanced stability arises from a deeper burial of the myristoyl moiety within the EF1-EF2 domain. The simulations also support an allosteric mechanism connecting the myristoyl moiety to the highest-affinity Ca(2+) binding site EF3. In spite of their remarkably distinct molecular features, both mutants cause constitutive activation of the target GC at physiological Ca(2+). We conclude that the similar aberrant regulation of the target enzyme results from a similar perturbation of the GCAP1-GC interaction, which may eventually cause dysregulation of both Ca(2+) and cyclic GMP homeostasis and result in retinal degeneration.
authors.affiliation: Department of Neurological, Biomedical and Movement Sciences, Section of Biological Chemistry.
authors.forename: Valerio
authors.initials: V
authors.lastname: Marino
authors.affiliation: Department of Neurosciences, Biochemistry Group, University of Oldenburg, 26111 Oldenburg, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Scholten
authors.affiliation: Department of Neurosciences, Biochemistry Group, University of Oldenburg, 26111 Oldenburg, Germany.
authors.forename: Karl-Wilhelm
authors.initials: KW
authors.lastname: Koch
authors.affiliation: Department of Neurological, Biomedical and Movement Sciences, Section of Biological Chemistry, Centre for BioMedical Computing (CBMC), University of Verona, 37134 Verona, Italy and daniele.dellorco@univr.it.
authors.forename: Daniele
authors.initials: D
authors.lastname: Dell'Orco
mesh_terms: D000818:Animals; D002118:Calcium; D002413:Cations, Divalent / Q000378:metabolism; D005786:Gene Expression Regulation; D006162:Guanylate Cyclase / Q000235:genetics / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins / Q000235:genetics* / Q000378:metabolism; D006358:Hot Temperature; D008274:Magnesium; D056004:Molecular Dynamics Simulation; D020125:Mutation, Missense; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability; D058499:Retinal Dystrophies / Q000235:genetics* / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; C496736:GUCA1A protein, human; D051604:Guanylate Cyclase-Activating Proteins; D006162:Guanylate Cyclase; D008274:Magnesium; D002118:Calcium