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

Abstract

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.Guanylyl cyclase activating proteins (GCAPs) 2 belong to the neuronal calcium sensor (NCS) branch of the calmodulin superfamily (1-3) and regulate Ca 2ϩ -sensitive activity of retinal guanylyl cyclase (RetGC) in rod and cone cells (4 -6). Phototransduction in retinal rods and cones is modulated by intracellular Ca 2ϩ sensed by GCAPs (7,8), and defects in Ca 2ϩ signaling by GCAPs are linked to retinal diseases (9). GCAP proteins in the Ca 2ϩ -free/Mg 2ϩ -bound state activate RetGC (10), whereas Ca 2ϩ -bound GCAPs inhibit RetGC at high Ca 2ϩ levels maintained in the dark (11-13).The GCAPs (GCAP1 (6), GCAP2 (14), GCAP3 (15), and GCAP4 -8 (16) are all ϳ200-amino acid residue proteins containing a covalently attached N-terminal myristoyl group and four EF-hand motifs (EF1 through EF4; Fig. 1 RetGC (10,18,19). The x-ray crystal structure of Ca 2ϩ -bound GCAP1 (20) and NMR structure of GCAP2 (21) showed that the four EF-hands form two semiglobular domains (EF1 and EF2 in the N-domain and EF3 and EF4 in the C-domain); Ca 2ϩ is bound at EF2, EF3, and EF4, and the N-terminal myristoyl group in GCAP1 is buried inside the Ca 2ϩ -bound protein flanked by hydrophobic residues at the N and C termini (see the red residues in Fig. 1 Experimental ProceduresExpression and Purification of GCAP1 and Mutants-Mutations were introduced in a bovine GCAP1 coding plasmid using a "splice by overlap extension" approach as previously described (24). Myristoylated GCAP1 and its mutants were produced in Escherichia coli strain harboring yeast N-myristoyl

Structural Characterization of Ferrous Ion Binding to Retinal Guanylate Cyclase Activator Protein 5 from Zebrafish Photoreceptors

“…Mg 2+ bound to EF2 (magenta) is supported by the downfield NMR resonance assigned to Gly68 in EF2 of Mg 2+ -bound GCAP5 (Fig. S2), which is similar to that observed for Mg 2+ -bound GCAP1 17 . Covalently attached myristic acid is depicted by a space-filling model (brown).…”

“…S2). In the absence of Fe 2+ , the Mg 2+ -bound GCAP5 exhibited a downfield peak at 10.65 ppm assigned to Gly68 that represents Mg 2+ binding to EF2 like that observed for GCAP1 17 . This downfield peak becomes significantly broadened upon adding a saturating concentration of Fe 2+ , consistent with Fe 2+ binding to EF2 in place of Mg 2+ when the Fe 2+ concentration exceeds that of Mg 2+ .…”

“…4D), consistent with possible Fe 2+ binding at EF2. In the presence of physiological Mg 2+ levels (1 mM), Mg 2+ interferes with Ca 2+ -binding to GCAP5 16 and is known to bind to EF2 in GCAP1 17,29 . To test whether Fe 2+ can bind to EF2 in place of Mg 2+ , an HSQC spectrum of Mg 2+ -bound GCAP5 was recorded in the presence and absence of saturating Fe 2+ (Fig.…”

“…6D). The intermolecular contact involving Val76 was also implicated in the dimerization of GCAP1, because mutating this Val residue to Glu abolishes dimerization of GCAP1 17, 19, 34 .…”

“…The experimental conditions are defined under Materials and Methods and were the same as described previously for GCAP1 17 .…”