The Crystal Structure of GCAP3 Suggests Molecular Mechanism of GCAP-linked Cone Dystrophies

Abstract

SummaryAbsorption of light by visual pigments initiates the phototransduction pathway that results in degradation of the intracellular pool of cyclic-GMP (cGMP). This hydrolysis promotes the closing of cGMP-gated cation channels and consequent hyperpolarization of rod and cone photoreceptor cell membranes. Guanylate Cyclase Activating Proteins (GCAPs) are a family of proteins that regulate retinal guanylate cyclase (GC) activity in a Ca 2+ -dependent manner. At high [Ca 2+ ], typical of the dark-adapted state (~500 nM), GCAPs inhibit retinal GCs. At the low [Ca 2+ ] (~50 nM) that occur after the closing of cGMP-gated channels, GCAPs activate retinal GCs to replenish dark-state cGMP levels. Here, we report the crystal structure of unmyristoylated human GCAP3 with Ca 2+ bound. GCAP3 is an EF-hand Ca 2+ -binding protein with Ca 2+ bound to EF2, 3 and 4, while Ca 2+ binding to EF-hand 1 is disabled. GCAP3 contains two domains with the EF-hand motifs arranged in a tandem array similar to GCAP2 and members of the recoverin subfamily of Ca 2+ -binding proteins. Residues not involved in Ca 2+ binding, but conserved in all GCAPs, cluster around EF1 in the N-terminal domain and may represent the interface with GCs. Five point mutations in the closely related GCAP1 have been linked to the etiology of cone dystrophies. These residues are conserved in GCAP3 and the structure suggests important roles for these amino acids. We present a homology model of GCAP1 based on GCAP3 that offers insight into the molecular mechanism underlying the autosomal dominant cone dystrophies produced by GCAP1 mutations.

Activation and Inhibition of Photoreceptor Guanylyl Cyclase by Guanylyl Cyclase Activating Protein 1 (GCAP-1)

“…Moreover, EF-hands 1, 2, and 3 are even less likely to undergo a major change between Mg 2ϩ and Ca 2ϩ forms, because EF-2 and EF-3 are both capable of maintaining the high-affinity interaction with the cyclase in either Mg 2ϩ or Ca 2ϩ form. To date, only a partial Ca 2ϩ -bound structure of two other GCAPs was established (47,48), but to properly understand the mechanism and the structural basis for the RetGC regulation, one would need to compare, potentially rather subtle, differences between Mg 2ϩ -and Ca 2ϩ -bound GCAP-1.…”

Chemistry and Biology of Vision

“…Key myristoylated Ca 2ϩ -binding proteins involved in phototransduction, namely guanylate cyclase-activating proteins (52), have been visualized at high resolution by NMR and crystallographic methods to reveal their internal architecture, but only in their Ca 2ϩ -bound forms (53)(54)(55). More advanced studies have been performed on another myristoylated photoreceptor protein called recoverin.…”

Identification of Target Binding Site in Photoreceptor Guanylyl Cyclase-activating Protein 1 (GCAP1)

“…Structures of Ca 2ϩ -liganded GCAP1 (Fig. 1A), GCAP2, and GCAP3 have been solved mostly by NMR spectroscopy and x-ray crystallography (22)(23)(24), and the principal role of GCAPs in physiology and retinal diseases is well understood (2,25,26). GCAPs have different Ca 2ϩ sensitivities and target specificities.…”