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

2015, Journal of Biological Chemistry

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 EFhand (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. 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). Mg 2ϩ binds to GCAP1 in place of Ca 2ϩ when cytosolic Ca 2ϩ levels are below 50 nM in light-activated photoreceptor cells (17). This Ca 2ϩfree/Mg 2ϩ-bound GCAP1 is called the activator form because it activates 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). The structure of the physiological activator form of GCAPs (Mg 2ϩ-bound/Ca 2ϩ-free state) is currently unknown. The structure of the Ca 2ϩ-free/Mg 2ϩ-bound activator state of GCAP1 has remained elusive, in part because it tends to aggregate under conditions for NMR or x-ray crystallography (22). Here, we present a NMR structural analysis of Ca 2ϩ-free/ Mg 2ϩ-bound GCAP1 mutant that has Val-77 replaced by Glu (called GCAP1 V77E). The GCAP1 V77E mutant retains functional Mg 2ϩ and Ca 2ϩ binding with intact tertiary structure. However, unlike the dimeric wild type GCAP1, GCAP1 V77E is monomeric in solution and remains soluble under NMR conditions. Our NMR analysis indicates that Ca 2ϩ-free/Mg 2ϩbound GCAP1 V77E is overall structurally similar to that of Ca 2ϩ-saturated GCAP1 (root mean square deviations of 2.0 Å), except that Mg 2ϩ is bound at EF2, and the other EF-hands are unoccupied. The largest Ca 2ϩ-dependent structural differences in GCAP1 are seen for residues in EF4 and the adjacent helical region (residues 164-174, called Ca 2ϩ switch helix). We propose that the Ca 2ϩ switch helix may serve as a conduit that relays Ca 2ϩ-induced structural changes in EF4 to the RetGC binding site in the N-terminal domain, which provides further support of a Ca 2ϩ-myristoyl tug mechanism (23). Experimental Procedures Expression 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 * This work was supported by National Institutes of Health Grants EY012347 (to J. B. A.), EY11522 (to A. M. D.), and RR11973 (UC Davis NMR). This work was also supported by a Pennsylvania Department of Health CURE Formula grant (to A. M. D.) The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The atomic coordinates and structure factors (code 2NA0) have been deposited in the Protein Data Bank (http://wwpdb.org/). A complete list of NMR assignments for Ca 2ϩ-free/Mg 2ϩ-bound GCAP1 V77E has been deposited in the Biological Magnetic Resonance Bank (BMRB) (accession no. 26688).