Publication Date:
2015-11-06
Description:
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.
Print ISSN:
0964-6906
Electronic ISSN:
1460-2083
Topics:
Biology
,
Medicine
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