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Missense mutations affecting Ca2+-coordination in GCAP1 lead to cone-rod dystrophies by altering protein structural and functional properties

DOI: 10.1016/j.bbamcr.2020.118794 DOI Help

Authors: Giuditta Dal Cortivo (University of Verona) , Valerio Marino (University of Verona) , Francesco Boni (CNR-IBF, Istituto di Biofisica; Università di Milano) , Mario Milani (CNR-IBF, Istituto di Biofisica; Università di Milano) , Daniele Dell'Orco (University of Verona)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochimica Et Biophysica Acta (bba) - Molecular Cell Research

State: Published (Approved)
Published: July 2020
Diamond Proposal Number(s): 20221

Abstract: 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.

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: B21-High Throughput SAXS

Added On: 14/07/2020 10:40

Discipline Tags:

Health & Wellbeing Biochemistry Neurology Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Scattering Small Angle X-ray Scattering (SAXS)