Structure of Guanylyl Cyclase Activator Protein 1 (GCAP1) Mutant V77E in a Ca2+-free/Mg2+-bound Activator State

Abstract

GCAP1, a member of the neuronal calcium sensor subclass of the calmodulin superfamily, confers Ca(2+)-sensitive activation of retinal guanylyl cyclase 1 (RetGC1). We present NMR resonance assignments, residual dipolar coupling data, functional analysis, and a structural model of GCAP1 mutant (GCAP1(V77E)) in the Ca(2+)-free/Mg(2+)-bound state. NMR chemical shifts and residual dipolar coupling data reveal Ca(2+)-dependent differences for residues 170-174. An NMR-derived model of GCAP1(V77E) contains Mg(2+) bound at EF2 and looks similar to Ca(2+) saturated GCAP1 (root mean square deviations = 2.0 Å). Ca(2+)-dependent structural differences occur in the fourth EF-hand (EF4) and adjacent helical region (residues 164-174 called the Ca(2+) switch helix). Ca(2+)-induced shortening of the Ca(2+) switch helix changes solvent accessibility of Thr-171 and Leu-174 that affects the domain interface. Although the Ca(2+) switch helix is not part of the RetGC1 binding site, insertion of an extra Gly residue between Ser-173 and Leu-174 as well as deletion of Arg-172, Ser-173, or Leu-174 all caused a decrease in Ca(2+) binding affinity and abolished RetGC1 activation. We conclude that Ca(2+)-dependent conformational changes in the Ca(2+) switch helix are important for activating RetGC1 and provide further support for a Ca(2+)-myristoyl tug mechanism.

Keywords: calcium; calcium-binding protein; calorimetry; guanylate cyclase (guanylyl cyclase); nuclear magnetic resonance (NMR); phototransduction; vision.

FIGURE 1.

Amino acid sequence alignment of bovine GCAP1, recoverin, and NCS-1. Secondary structural elements (α-helices and β-strands) were derived from NMR analyses (22, 37). The four EF-hands (EF1, EF2, EF3 and EF4) are highlighted green, red, cyan, and yellow.

FIGURE 2.

GCAP1 mutant GCAP1^V77E binds to Mg²⁺ and Ca²⁺ as measured by ITC and NMR (inset). ITC binding isotherms recorded at 30 °C are shown for Mg²⁺ binding (A) and Ca²⁺ binding (B) to GCAP1^V77E. Binding isotherms were fit to a sequential model (solid line), and fitting parameters are given in “Results.” Downfield spectral regions of ¹H,¹⁵N HSQC spectra of Mg²⁺-bound GCAP1^V77E (inset, A) and Ca²⁺-bound GCAP1^V77E (inset, B) are also shown. The downfield peak assigned to Gly-69 indicates Mg²⁺ is bound at EF2 (inset, A), whereas peaks assigned to Gly-69, Gly-105, and Gly-149 indicate Ca²⁺ is bound at EF2, EF3, and EF4.

FIGURE 3.

NMR spectroscopy of GCAP1^WT, GCAP1^V77E, and mutants (SGL and ΔLeu-174). Two-dimensional (¹H,¹⁵N HSQC) NMR spectra of ¹⁵N-labeled wild type GCAP1 in the Ca²⁺-free/Mg²⁺-bound state (A), GCAP1^V77E in the Ca²⁺-free/Mg²⁺-bound state (B), GCAP1^V77E in the Ca²⁺-bound state (C), and GCAP1 mutants (SGL, black; ΔLeu-174, red) in the Ca²⁺-bound state (D). Spectra were obtained at 37 °C. A downfield resonance at ∼10.5 ppm for Ca²⁺-free/Mg²⁺-bound GCAP1^V77E is assigned to a conserved glycine residue (Gly-69) in EF2 and indicates Mg²⁺ is bound at EF2. Downfield resonances (at 10.45, 10.47, and 10.55 ppm) for Ca²⁺-bound GCAP1^V77E are assigned to conserved glycine residues (Gly-105, Gly-69, and G149) and indicate that three Ca²⁺ are bound per protein at EF2, EF3, and EF4. Sequence-specific resonance assignments for Ca²⁺-free/Mg²⁺-bound V77E are indicated by the peak labels. Complete NMR assignments were deposited in the BMRB (accession no. 26688).

FIGURE 4.

RDC structural analysis of GCAP1^V77E. ¹H,¹⁵N IPAP (inphase/antiphase)-HSQC spectra of Ca²⁺-free/Mg²⁺-bound GCAP1^V77E in the absence (A) and presence (B) of 12 mg/ml Pf1 phage. Spectral splittings for the isotropic condition (J_NH) versus the anisotropic condition (J_NH + D_NH) are marked by vertical lines and were used to calculate RDCs as described under “Experimental Procedures.” C, RDCs calculated from the structure of Ca²⁺-free/Mg²⁺-bound GCAP1^V77E in Fig. 5 are plotted versus the RDCs measured in Fig. 4B and show good agreement (Q-factor = 0.28 and an R-factor = 0.95 (40)).

FIGURE 5.

NMR-derived structure of Ca²⁺-free/Mg²⁺-bound GCAP1^V77E (PDB ID 2NA0). The main chain structure of Ca²⁺-free/Mg²⁺-bound GCAP1^V77E (A) and the same view rotated by 180 degrees (B) show four EF-hands (colored as in Fig. 1) packed in a globular arrangement very similar to what is seen for Ca²⁺-bound GCAP1 (20). The secondary structural elements are labeled as defined in Fig. 1. The Ca²⁺ switch helix (α10) is highlighted in orange, bound Mg²⁺ is in blue, and the N-terminal myristoyl group is in magenta.

FIGURE 6.

Ca²⁺-induced conformational changes in GCAP1. Shown in Ca²⁺-dependent amide chemical shift difference (Ca²⁺-free minus Ca²⁺-bound) plotted versus residue number (A) and chemical shift difference mapped onto the main chain structure (B). Residues Thr-171 and Leu-174 exhibited the largest Ca²⁺-induced chemical shift differences. Residues (Thr-62, Phe-140, Leu-151, Val-160, Leu-170, Thr-171, Leu-174) with a chemical shift difference higher than 0.8 are colored red. Residues (Ala-52, Tyr-55, Asp-68, Ile-115, Ala-118, Ser-141, Ser-152, Glu-158, Gln-161, Asp-168) with a chemical shift difference between 0.5 and 0.8 are colored magenta. Residues with a chemical shift difference <0.5 are colored light blue. C, close-up view of the Ca²⁺ switch helix (α10, orange) that is elongated by one turn in Ca²⁺-free/Mg²⁺-bound GCAP1^V77E (left) compared with Ca²⁺-bound GCAP1^WT (right). The angle between EF2 exiting helix (red) and EF3 entering helix (cyan) at the domain interface increased slightly (dotted line) due to Ca²⁺-dependent interactions with the Ca²⁺ switch helix.

FIGURE 7.

Single amino acid residue insertion in the Ca²⁺ switch helix affects metal sensor properties of GCAP1. A, Ca²⁺ binding isotherm for wild type (black, ●) and SGL (red, ●) GCAP1. Ca²⁺ binding was assayed using titration of 20 μm GCAP1 in the presence of Fluo4FF as described under “Experimental Procedures.” B–D, tryptophan fluorescence titrations for monitoring metal-dependent conformational change in wild type (black, ●) and SGL (red, ●) GCAP1 caused by Ca²⁺ (B and C) or Mg²⁺ (D) binding. B and C, Ca²⁺ titration in the absence (B) or in the presence (C) of 10 mm Mg²⁺. AU, absorbance units. D, Mg²⁺ titration. E, RetGC1 activation in vitro by wild type (black, ●) and SGL (red, ●) GCAP1 in the presence of 1 mm Mg²⁺ was assayed as described under “Experimental Procedures.”

FIGURE 8.

Effect of a single amino acid residue deletion in Ca²⁺ switch helix on metal sensor properties of GCAP1. A–C, tryptophan fluorescence titrationsfor monitoring Ca²⁺-dependent conformational change in ΔArg-172 (■) (A), ΔSer-173 (▴) (B), or ΔLeu-174 (♦) (C) GCAP1 in the absence (red symbols) or in the presence (blue symbols) of 10 mm Mg²⁺. AU, absorbance units. D and E, comparison of the Ca²⁺-dependent (D) or Mg²⁺-dependent (E) Trp fluorescence change in the wild type (black, ●), ΔArg-172 (□) (A), ΔSer-173 (▴) (B), or ΔLeu-174 (♢); no Mg²⁺ added in D. F, dose dependence of RetGC1 activation in vitro by wild type (black, ●) ΔArg-172 (red, □), ΔSer-173 (red, ▴), or ΔLeu-174 (red ♢) GCAP1 in the presence of 6 mm free Mg²⁺ and 2 mm EGTA; the data points were fitted using Synergy Kaleidagraph 4 software assuming a standard Michaelis hyperbolic function.