Molecular properties of human guanylate cyclase-activating protein 2 (GCAP2) and its retinal dystrophy-associated variant G157R

Abstract

In murine and bovine photoreceptors, guanylate cyclase-activating protein 2 (GCAP2) activates retinal guanylate cyclases (GCs) at low Ca²⁺ levels, thus contributing to the Ca²⁺/cGMP negative feedback on the cyclase together with its paralog guanylate cyclase-activating protein 1, which has the same function but different Ca²⁺ sensitivity. In humans, a GCAP2 missense mutation (G157R) has been associated with inherited retinal degeneration (IRD) via an unknown molecular mechanism. Here, we characterized the biochemical properties of human GCAP2 and the G157R variant, focusing on its dimerization and the Ca²⁺/Mg²⁺-binding processes in the presence or absence of N-terminal myristoylation. We found that human GCAP2 and its bovine/murine orthologs significantly differ in terms of oligomeric properties, cation binding, and GC regulation. Myristoylated GCAP2 endothermically binds up to 3 Mg²⁺ with high affinity and forms a compact dimer that may reversibly dissociate in the presence of Ca²⁺. Conversely, nonmyristoylated GCAP2 does not bind Mg²⁺ over the physiological range and remains as a monomer in the absence of Ca²⁺. Both myristoylated and nonmyristoylated GCAP2 bind Ca²⁺ with high affinity. At odds with guanylate cyclase-activating protein 1 and independently of myristoylation, human GCAP2 does not significantly activate retinal GC1 in a Ca²⁺-dependent fashion. The IRD-associated G157R variant is characterized by a partly misfolded, molten globule-like conformation with reduced affinity for cations and prone to form aggregates, likely mediated by hydrophobic interactions. Our findings suggest that GCAP2 might be mostly implicated in processes other than phototransduction in human photoreceptors and suggest a possible molecular mechanism for G157R-associated IRD.

Keywords: GCAP; GUCA1B; cGMP; calcium-binding proteins; guanylate cyclase (guanylyl cyclase); neurodegenerative disease; phototransduction; retina; retinal degeneration; vision.

Figure 1

Three-dimensional structure of nmGCAP2 and assessment of the molecular mass of GCAP2 variants by MALDI-TOF MS.A, Cartoon representation of the homology model of human nmGCAP2 (built on bovine nmGCAP2 template, PDB entry 1JBA (8)), colored in a red-to-purple rainbow according to the sequence. Ca²⁺ are displayed as red spheres, and IRD-associated R157 variant is shown as white sticks with N atoms highlighted in blue. EF1 to EF4 are labeled, together with residue R157. The protein view is rotated by 90° clockwise along the x-axis in the right panel. MALDI spectra of (B) nmGCAP2, (C) mGCAP2, and (D) G157R with the molecular mass corresponding to each peak. GCAP2, guanylate cyclase–activating protein 2; IRD, inherited retinal dystrophy; mGCAP2, myristoylated GCAP2; nmGCAP2, nonmyristoylated GCAP2.

Figure 2

Effects of Mg²⁺and Ca²⁺on the apparent molecular mass and the hydrodynamic diameter of GCAP2 variants. Analytical SEC profiles of ∼40-μM (A) nmGCAP2 and (B) mGCAP2 in the presence of 500-μM EGTA + 1-mM Mg²⁺ (blue) or 1-mM Mg²⁺ + 500-μM Ca²⁺ (red). Hydrodynamic diameter estimation by DLS of ∼40-μM (C) nmGCAP2 and (D) mGCAP2 in the presence of 500-μM EGTA + 1-mM Mg²⁺ (blue) or 1-mM Mg²⁺ + 500-μM Ca²⁺ (red). DLS, dynamic light scattering; GCAP2, guanylate cyclase–activating protein; mGCAP2, myristoylated GCAP2; nmGCAP2, nonmyristoylated GCAP2; SEC, size-exclusion chromatography.

Figure 3

Effects of Mg²⁺andCa²⁺on mGCAP2 dimerization. Analytical gel-filtration chromatograms of 5-μM (black), 10-μM (red), 20-μM (blue), 40-μM (green), and 80-μM (orange) mGCAP2 in the presence of (A) 500-μM EGTA + 1-mM Mg²⁺ and (B) 1-mM Mg²⁺ + 500-μM Ca²⁺. Insets show the apparent molecular mass as a function of the mGCAP2 concentration together with the optimal fitting to a hyperbolic curve (see Experimental procedures). GCAP2, guanylate cyclase–activating protein 2; mGCAP2, myristoylated GCAP2.

Figure 4

Conformational changes of GCAP2 variants upon cation binding monitored by CD spectroscopy. Near-UV CD spectra of 30-μM (A) nmGCAP2, (B) mGCAP2, and (C) G157R in the presence of 500-μM EGTA (black) and after addition of 1-mM Mg²⁺ (blue) and 1-mM Ca²⁺ (red). Far-UV CD spectra of 8-μM (D) nmGCAP2, (E) mGCAP2, and (F) G157R in the presence of 300-μM EGTA (black) and after addition of 1-mM Mg²⁺ (blue) and 600-μM Ca²⁺ (red). GCAP2, guanylate cyclase–activating protein 2; mGCAP2, myristoylated GCAP2; nmGCAP2, nonmyristoylated GCAP2.

Figure 5

Cation-dependent thermal stability of GCAP2 variants monitored by CD spectroscopy. Thermal denaturation profiles of 8-μM (A) nmGCAP2, (B) mGCAP2, and (C) G157R in the presence of 300-μM EGTA (black), 300-μM EGTA + 1-mM Mg²⁺ (blue) and 1-mM Mg²⁺ + 600-μM Ca²⁺ (red). When possible, profiles were fitted to a 4-parameter Hill sigmoid, yielding the T_m values reported in Table 2. GCAP2, guanylate cyclase–activating protein 2; mGCAP2, myristoylated GCAP2; nmGCAP2, nonmyristoylated GCAP2.

Figure 6

Conformational changes of GCAP2 variants upon ion binding assessed by proteolytic cleavage. Limited proteolysis of GCAP2 variants after 10-min incubation with trypsin (60:1 molar ratio) in the presence of 1-mM EDTA, 500-μM EGTA + 1-mM Mg²⁺, or 1-mM Mg²⁺ + 1-mM Ca²⁺. GCAP2, guanylate cyclase–activating protein 2; mGCAP2, myristoylated GCAP2; nmGCAP2, nonmyristoylated GCAP2.

Figure 7

Variations in hydrophobicity of GCAP2 variants upon cation binding investigated by ANS fluorescence. Fluorescence spectra of 30-μM ANS and 2-μM (A) nmGCAP2, (B) mGCAP2, and (C) G157R in the presence of 500-μM EGTA (black) and after sequential additions of 1-mM Mg²⁺ (blue) and 1-mM Ca²⁺ (red). The spectrum of ANS is shown as a green dashed line in panel A. ANS, anilinonaphthalene-1-sulfonic acid; GCAP2, guanylate cyclase–activating protein 2; mGCAP2, myristoylated GCAP2; nmGCAP2, nonmyristoylated GCAP2.

Figure 8

Effects of Mg²⁺and Ca²⁺on the electrophoretic mobility of GCAP2 variants. SDS-PAGE of 20-μM GCAP2 variants in the presence of 5-mM EDTA, 5-mM EGTA + 1-mM free Mg²⁺, or 1-mM Mg²⁺ + 1-mM Ca²⁺. GCAP2, guanylate cyclase–activating protein 2; mGCAP2, myristoylated GCAP2; nmGCAP2, nonmyristoylated GCAP2.

Figure 9

Metal cations binding to GCAP2 variants measured by ITC. Representative ITC titrations of 20-μM nmGCAP2 (A–C) and mGCAP2 (D–F). Mg²⁺ titrations of (A) nmGCAP2 and (D) mGCAP2. Ca²⁺ titrations of (B) nmGCAP2 and (E) mGCAP2 in the absence and in the presence (C) and (F) of 1-mM Mg²⁺. Upper panels show heat pulses, and lower panels represent molar enthalpy changes relative to each injection. When possible, data were fitted to a two- or three-sequential binding sites (see Experimental procedures), yielding apparent dissociation constants (K_D), enthalpy changes (ΔH), and entropy changes (−TΔS) reported in Tables 3 and 4. GCAP2, guanylate cyclase–activating protein 2; ITC, isothermal titration calorimetry; mGCAP2, myristoylated GCAP2; nmGCAP2, nonmyristoylated GCAP2.