Ca 2+ ‐dependent conformational changes in bovine GCAP‐2

Dizhoor

2004

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Guanylyl cyclase-activating proteins (GCAPs) are calcium sensor proteins of the EF-hand superfamily that inhibit retinal photoreceptor membrane guanylyl cyclase (retGC) in the dark when they bind Ca 2؉ but activate retGC when Ca 2؉ dissociates from GCAPs in response to light stimulus. We addressed the difference in exposure of GCAP-2 structure to protein kinase and a protease as indicators of conformational change caused by binding and release of Ca 2؉ . We have found that unlike its homolog, GCAP-1, the C terminus of GCAP-2 undergoes phosphorylation by cyclic nucleotidedependent protein kinases (CNDPK) present in the retinal extract and rapid dephosphorylation by the protein phosphatase PP2C present in the retina. Inactivation of the CNDPK phosphorylation site in GCAP-2 by substitutions S201G or S201D, as well as phosphorylation or thiophosphorylation of Ser 201 , had little effect on the ability of GCAP-2 to regulate retGC in reconstituted membranes in vitro. At the same time, Ca 2؉ strongly inhibited phosphorylation of the wild-type GCAP-2 by retinal CNDPK but did not affect phosphorylation of a constitutively active Ca 2؉ -insensitive GCAP-2 mutant. Partial digestion of purified GCAP-2 with Glu-C protease revealed at least two sites that become exposed or constrained in a Ca 2؉ -sensitive manner. The Ca 2؉ -dependent conformational changes in GCAP-2 affect the areas around Glu 62 residue in the entering helix of EFhand 2, the areas proximal to the exiting helix of EF-hand 3, and Glu 136 -Glu 138 between EF-hand 3 and EF-hand 4. These changes also cause the release of the C-terminal Ser 201 from the constraint caused by the Ca 2؉ -bound conformation.Photoisomerized rhodopsin triggers hydrolysis of cGMP through the activation of the G-protein transducin, which stimulates cGMP phosphodiesterase and thus causes cGMPgated channels to close (see Refs. 1-3 for review). Rods and cones rapidly recover to their resting potential after excitation induced by a brief non-saturating exposure to light. They also adapt to a constant background illumination by decreasing the amplitude and accelerating the kinetics of their electrical responses to a standard test flash (see Refs. 1-3 for review). Reopening of the cGMP-gated channels during recovery and light adaptation requires acceleration of cGMP synthesis by guanylyl cyclase, a process controlled by the level of intracellular free Ca 2ϩ . In the dark, Ca 2ϩ extruded from the photoreceptor outer segment by a Na ϩ /K ϩ ,Ca 2ϩ exchanger re-enters the outer segment through the open cGMP-gated channels so that the free intracellular outer segment Ca 2ϩ concentrations reach 250 nM in mammals and 350 -450 nM in lower vertebrates (4 -6). In the light, cGMP is hydrolyzed, and these channels close while the exchanger continues to extrude Ca 2ϩ ions from the photoreceptor outer segment; therefore, Ca 2ϩ concentrations decrease nearly 10-fold (4 -7). Ca 2ϩ -binding proteins GCAP-1 1 and GCAP-2 activate retGC1 and retGC2 (and their homologs found in photoreceptors of different vertebr...

Section: Ca 2ϩ -Dependent Conformational Changes In Gcap-2 50347mentioning

confidence: 53%

Ca2+-dependent Conformational Changes in Guanylyl Cyclase-activating Protein 2 (GCAP-2) Revealed by Site-specific Phosphorylation and Partial Proteolysis

Dizhoor

2004

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“…Surprisingly, the GCAP proteins do not possess a Ca 2ϩ -myristoyl switch (17,49,50), and the N-terminal myristoyl group remains buried inside the protein in both Ca 2ϩ -free and Ca 2ϩ -bound GCAPs (17,51). Although NMR and crystal structures are known for Ca 2ϩ -bound GCAPs (20 -22, 52), before this study little was known structurally about the Mg 2ϩ -bound/Ca 2ϩ -free activator state of GCAPs and how Ca 2ϩ -induced conformational changes in GCAPs control cyclase activation.…”

Section: Discussionmentioning

confidence: 99%

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

Lim

Olshevskaya

et al. 2016

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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

“…In contrast to the Ca 2ϩ -myristoyl switch in recoverin (58,59), GCAPs retain their fatty moiety inside the protein globule (24,60). Moreover, GCAP1 does not undergo a major rearrangement of its main chain upon metal binding and displays rather modest Ca 2ϩ -induced changes in chemical shifts as detected by NMR spectroscopy for its inhibitor versus activator states (54,56).…”

Section: Discussionmentioning

confidence: 99%

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

Lim

Ames

et al. 2014

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Background: GCAP1 regulates cGMP synthesis in photoreceptors in response to light. Results: Mutagenesis of the entire GCAP1 surface reveals its guanylyl cyclase interface. Conclusion:The interface forms a compact patch that enables both primary binding to and allosteric activation of the target enzyme. Significance: Guanylyl cyclase activation by GCAP1 is indispensable for vision and survival of photoreceptors.