Calcium-Myristoyl Tug Is a New Mechanism for Intramolecular Tuning of Calcium Sensitivity and Target Enzyme Interaction for Guanylyl Cyclase-activating Protein 1: DYNAMIC CONNECTION BETWEEN N-FATTY ACYL GROUP AND EF-HAND CONTROLS CALCIUM SENSITIVITY (original) (raw)
Related papers
The Journal of biological chemistry, 2001
Guanylyl cyclase activator proteins (GCAPs) are calcium-binding proteins closely related to recoverin, neurocalcin, and many other neuronal Ca(2+)-sensor proteins of the EF-hand superfamily. GCAP-1 and GCAP-2 interact with the intracellular portion of photoreceptor membrane guanylyl cyclase and stimulate its activity by promoting tight dimerization of the cyclase subunits. At low free Ca(2+) concentrations, the activator form of GCAP-2 associates into a dimer, which dissociates when GCAP-2 binds Ca(2+) and becomes inhibitor of the cyclase. GCAP-2 is known to have three active EF-hands and one additional EF-hand-like structure, EF-1, that deviates form the EF-hand consensus sequence. We have found that various point mutations within the EF-1 domain can specifically affect the ability of GCAP-2 to interact with the target cyclase but do not hamper the ability of GCAP-2 to undergo reversible Ca(2+)-sensitive dimerization. Point mutations within the EF-1 region can interfere with both t...
Retinal Guanylyl Cyclase-Activating Protein 1 and 2
Encyclopedia of Signaling Molecules, 2016
Historical Background Retinal guanylyl cyclases (RetGCs) in retinal rod and cone photoreceptors are regulated by a family of EF-hand Ca 2+ sensor proteins called guanylyl cyclase-activating proteins (GCAP1-8) that belong to the neuronal calcium sensor (NCS) family. Mammalian GCAPs (GCAP1 and GCAP2) activate RetGCs at low Ca 2+ levels in light-activated photoreceptor cells and inhibit RetGC activity at higher Ca 2+ levels in darkadapted photoreceptors. The Ca 2+-sensitive RetGC activity controlled by GCAPs is an important mechanism of visual recovery and light adaptation of phototransduction. Mutations in either RetGCs or GCAPs that disable this Ca 2+-sensitive cyclase activity are genetically linked to retinal disease. Here I review atomic-level structures of GCAP1 in both Ca 2+-free/Mg 2+-bound (activator) and Ca 2+-saturated (inhibitory) states, as well as the structure of Ca 2+-saturated GCAP2. The structure of GCAP2 reveals an exposed N-terminus that may be important for Ca 2+-dependent membrane anchoring of the myristoyl group. By contrast, the structures of Ca 2+-free and Ca 2+-bound forms of GCAP1 each contain a covalently attached myristoyl group that is sequestered in a hydrophobic protein cavity formed by helices at both the N-and C-terminus. Hence, myristoylated GCAP1 is not targeted to bilayer membranes. The Ca 2+-free activator form of GCAP1 contains Mg 2+ bound at the second EF-hand (EF2) that is essential for activating RetGC. The Ca 2+ saturated form of GCAP1 contains Ca 2+ bound at EF2, EF3, and EF4. Ca 2+-dependent conformational changes are most apparent in EF2 and in the Ca 2+ switch helix (residues 169-174) and will be discussed in terms of a proposed mechanism for Ca 2+-dependent activation of retinal guanylyl cyclases.
1999
Guanylyl cyclase-activating proteins (GCAPs are 23-kDa Ca 2؉-binding proteins belonging to the calmodulin superfamily. Ca 2؉-free GCAPs are responsible for activation of photoreceptor guanylyl cyclase during light adaptation. In this study, we characterized GCAP1 mutants in which three endogenous nonessential Trp residues were replaced by Phe residues, eliminating intrinsic fluorescence. Subsequently, hydrophobic amino acids adjacent to each of the three functional Ca 2؉-binding loops were replaced by reporter Trp residues. Using fluorescence spectroscopy and biochemical assays, we found that binding of Ca 2؉ to GCAP1 causes a major conformational change especially in the region around the EF3-hand motif. This transition of GCAP1 from an activator to an inhibitor of GC requires an activation energy E a ؍ 9.3 kcal/mol. When Tyr 99 adjacent to the EF3-hand motif was replaced by Cys, a mutation linked to autosomal dominant cone dystrophy in humans, Cys 99 is unable to stabilize the inactive GCAP1-Ca 2؉ complex. Stopped-flow kinetic measurements indicated that GCAP1 rapidly loses its bound Ca 2؉ (k ؊1 ؍ 72 s ؊1 at 37°C) and was estimated to associate with Ca 2؉ at a rate (k 1 > 2 ؋ 10 8 M ؊1 s ؊1) close to the diffusion limit. Thus, GCAP1 displays thermodynamic and kinetic properties that are compatible with its involvement early in the phototransduction response. The Ca 2ϩ-binding motif termed EF-hand, introduced by Nockolds et al. (1), refers to the helix-loop-helix structure responsible for selective high affinity (K d Ͻ10 Ϫ5 M) Ca 2ϩ binding. EF-hand motifs, reliably predictable based on primary sequence and present in one to eight copies in some polypeptides, have been identified in over 500 Ca 2ϩ-binding proteins. Neuronal Ca 2ϩ-binding proteins (NCBPs) 1 are a subset of the
Identification of Target Binding Site in Photoreceptor Guanylyl Cyclase-activating Protein 1 (GCAP1)
Journal of Biological Chemistry, 2014
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. Retinal guanylyl cyclase (RetGC)-activating proteins (GCAPs) regulate visual photoresponse and trigger congenital retinal diseases in humans, but GCAP interaction with its target enzyme remains obscure. We mapped GCAP1 residues comprising the RetGC1 binding site by mutagenizing the entire surface of GCAP1 and testing the ability of each mutant to bind RetGC1 in a cell-based assay and to activate it in vitro. Mutations that most strongly affected the activation of RetGC1 localized to a distinct patch formed by the surface of non-metalbinding EF-hand 1, the loop and the exiting helix of EF-hand 2, and the entering helix of EF-hand 3. Mutations in the binding patch completely blocked activation of the cyclase without affecting Ca 2؉ binding stoichiometry of GCAP1 or its tertiary fold. Exposed residues in the C-terminal portion of GCAP1, including EF-hand 4 and the helix connecting it with the N-terminal lobe of GCAP1, are not critical for activation of the cyclase. GCAP1 mutants that failed to activate RetGC1 in vitro were GFP-tagged and co-expressed in HEK293 cells with mOrange-tagged RetGC1 to test their direct binding in cyto. Most of the GCAP1 mutations introduced into the "binding patch" prevented co-localization with RetGC1, except for Met-26, Lys-85, and Trp-94. With these residues mutated, GCAP1 completely failed to stimulate cyclase activity but still bound RetGC1 and competed with the wild type GCAP1. Thus, RetGC1 activation by GCAP1 involves establishing a tight complex through the binding patch with an additional activation step involving Met-26, Lys-85, and Trp-94. Retinal membrane guanylyl cyclase (RetGC) 2 and RetGCactivating proteins (GCAPs) play a critical role in the physiol
2021
Neuronal calcium sensors play a crucial role in different pathways of Ca2+-mediated neurotransmission. Among them guanylate cyclase-activating protein 1 (GCAP1) is expressed only in photoreceptors and activates or inhibits retinal guanylate cyclase 1 (retGC1) depending on cellular Ca2+ concentrations during phototransduction. To date, 22 pathogenic mutations responsible for retinal dystrophy have been associated to GCAP1, but a complete picture of the molecular determinants of the disease is still missing. The only crystal structure available so far is the wt Ca2+-bound monomeric homologue from chicken and no cure exists for retinal dystrophy. In this work I report for the first time that the recombinant human GCAP1 is characterized by a highly dynamic monomer-dimer equilibrium, whose dissociation constant is influenced by salt concentration and by the nature of the divalent ion bound. Surprisingly, I discovered that also the chicken protein shows a similar mechanism, suggesting tha...
Journal of Biological Chemistry, 1999
Guanylyl cyclase activating protein-2 (GCAP-2) is a Ca 2؉-sensitive regulator of phototransduction in retinal photoreceptor cells. GCAP-2 activates retinal guanylyl cyclases at low Ca 2؉ concentration (<100 nM) and inhibits them at high Ca 2؉ (>500 nM). The light-induced lowering of the Ca 2؉ level from ϳ500 nM in the dark to ϳ50 nM following illumination is known to play a key role in visual recovery and adaptation. We report here the three-dimensional structure of unmyristoylated GCAP-2 with three bound Ca 2؉ ions as determined by nuclear magnetic resonance spectroscopy of recombinant, isotopically labeled protein. GCAP-2 contains four EF-hand motifs arranged in a compact tandem array like that seen previously in recoverin. The root mean square deviation of the main chain atoms in the EF-hand regions is 2.2 Å in comparing the Ca 2؉-bound structures of GCAP-2 and recoverin. EF-1, as in recoverin, does not bind calcium because it contains a disabling Cys-Pro sequence. GCAP-2 differs from recoverin in that the calcium ion binds to EF-4 in addition to EF-2 and EF-3. A prominent exposed patch of hydrophobic residues formed by EF-1 and EF-2 (Leu 24 , Trp 27 , Phe 31 , Phe 45 , Phe 48 , Phe 49 , Tyr 81 , Val 82 , Leu 85 , and Leu 89) may serve as a target-binding site for the transmission of calcium signals to guanylyl cyclase.
Frontiers in molecular neuroscience, 2014
Neuronal calcium sensor (NCS) proteins, a sub-branch of the calmodulin superfamily, are expressed in the brain and retina where they transduce calcium signals and are genetically linked to degenerative diseases. The amino acid sequences of NCS proteins are highly conserved but their physiological functions are quite different. Retinal recoverin controls Ca(2) (+)-dependent inactivation of light-excited rhodopsin during phototransduction, guanylyl cyclase activating proteins 1 and 2 (GCAP1 and GCAP2) promote Ca(2) (+)-dependent activation of retinal guanylyl cyclases, and neuronal frequenin (NCS-1) modulates synaptic activity and neuronal secretion. Here we review the molecular structures of myristoylated forms of NCS-1, recoverin, and GCAP1 that all look very different, suggesting that the attached myristoyl group helps to refold these highly homologous proteins into different three-dimensional folds. Ca(2) (+)-binding to both recoverin and NCS-1 cause large protein conformational c...
Biochemistry, 1999
Regulation of cAMP and cGMP production is a fundamental step in a broad range of signal transduction systems, including phototransduction. To identify regions within photoreceptor guanylyl cyclase 1 (GC1) that interact with GC-activating proteins (GCAPs), we synthesized the intracellular fragment of GC1, residues 491-1110, as a set of 15 amino acid long, partially overlapping peptides on the surface of individual pins arranged in a microtiter plate format. This pin assay identified 8 peptides derived from different regions of the GC1 intracellular domain that bind GCAPs. Peptide variants containing these sequences were synthesized as free peptides and tested for their ability to inhibit GC1 stimulation by GCAPs. A free peptide, 968 GTFRMRHMPEVPVRIRIG, from the catalytic domain of GC1 was the strongest inhibitor of GCAP1/GCAP2-mediated activation. In native GC1, this polypeptide fragment is likely to form a loop between R-helix 3 and-strand 4. When this region in GC1 was replaced by the corresponding sequence of GCAP-insensitive GC type A, GCAPs did not stimulate the GC1 mutant. The corresponding loops in related adenylyl cyclase (AC) are involved in the activating and inhibiting interactions with G sR and G iR , respectively. Thus, despite interacting with different activating proteins, both AC and GC activity may be modulated through their respective regions within catalytic domains.
Activation of Retinal Guanylyl Cyclase-1 by Ca2+-binding Proteins Involves Its Dimerization
Journal of Biological Chemistry, 1999
Retinal guanylyl cyclase-1 (retGC-1), a key enzyme in phototransduction, is activated by guanylyl cyclase-activating proteins (GCAPs) if [Ca 2؉ ] is less than 300 nM. The activation is believed to be essential for the recovery of photoreceptors to the dark state; however, the molecular mechanism of the activation is unknown. Here, we report that dimerization of retGC-1 is involved in its activation by GCAPs. The GC activity and the formation of a 210-kDa cross-linked product of retGC-1 were monitored in bovine rod outer segment homogenates, GCAPs-free bovine rod outer segment membranes and recombinant bovine retGC-1 expressed in COS-7 cells. In addition to recombinant bovine GCAPs, constitutively active mutants of GCAPs that activate retGC-1 in a [Ca 2؉ ]-independent manner and bovine brain S100b that activates retGC-1 in the presence of ϳ10 M [Ca 2؉ ] were used to investigate whether these activations take place through a similar mechanism, and whether [Ca 2؉ ] is directly involved in the dimerization. We found that a monomeric form of retGC-1 (ϳ110 kDa) was mainly observed whenever GC activity was at basal or low levels. However, the 210-kDa product was increased whenever the GC activity was stimulated by any Ca 2؉ -binding proteins used. We also found that [Ca 2؉ ] did not directly regulate the formation of the 210-kDa product. The 210-kDa product was detected in a purified GC preparation and did not contain GCAPs even when the formation of the 210-kDa product was stimulated by GCAPs. These data strongly suggest that the 210-kDa cross-linked product is a homodimer of retGC-1. We conclude that inactive retGC-1 is predominantly a monomeric form, and that dimerization of retGC-1 may be an essential step for its activation by active forms of GCAPs.
Biochemistry, 1998
Guanylate cyclase-activating protein 1 (GCAP1), a photoreceptor-specific Ca 2+-binding protein, activates retinal guanylate cyclase 1 (GC1) during the recovery phase of phototransduction. In contrast to other Ca 2+-binding proteins from the calmodulin superfamily, the Ca 2+-free form of GCAP1 stimulates the effector enzyme. In this study, we analyzed the Ca 2+-dependent changes in GCAP1 structure by limited proteolysis and mutagenesis in order to understand the mechanism of Ca 2+-sensitive modulation of GC1 activity. The change from a Ca 2+-bound to a Ca 2+-free form of GCAP1 increased susceptibility of Ca 2+-free GCAP1 to proteolysis by trypsin. Sequencing data revealed that in the Ca 2+-bound form, only the N-terminus (myristoylated Gly 2-Lys 9) and C-terminus (171-205 fragment) of GCAP1 are removed by trypsin, while in the Ca 2+-free form, GCAP1 is readily degraded to small fragments. Successive inactivation of each of the functional EF loops by site-directed mutagenesis showed that only EF3 and EF4 contribute to a Ca 2+-dependent inactivation of GCAP1. GCAP1(E 75 D,E 111 D,E 155 D) mutant did not bind Ca 2+ and stimulated GC1 in a [Ca 2+ ]-independent manner. GCAP1 and GCAP2, but not S-100 , a high [Ca 2+ ] free activator of GC1, competed with the triple mutant at high [Ca 2+ ] free , inhibiting GC1 with similar IC 50 's. These competition results are consistent with comparable affinities between GC1 and GCAPs. Our data suggest that GCAP1 undergoes major conformational changes during Ca 2+ binding and that EF3 and EF4 motifs are responsible for changes in the GCAP1 structure that converts this protein from the activator to the inhibitor of GC1. Calcium ions, Ca 2+ , play a crucial role in cellular signaling. Because they are nondegradable, several systems have evolved to regulate the cellular concentration of free Ca 2+ ([Ca 2+ ] free), including intracellular compartmentalization/ sequestration, pumping to the extracellular space, and buffering by Ca 2+-binding proteins. Some of these Ca 2+-binding proteins are also poised to take advantage of transient changes in [Ca 2+ ] free to affect properties of regulatory enzymes and ion channels. In cells that lower their internal [Ca 2+ ] free upon excitation, such as rod and cone photoreceptor cells, distinct types of proteins have evolved that act as activators of effector enzymes when they are in the Ca 2+free state. Guanylate cyclase-activating proteins, GCAP1 1 and GCAP2, were found to fulfill such functions in the regulation of photoreceptor guanylate cyclase (GC1) (1-4). GCAP1 and GCAP2 are acidic, ∼23-kDa, homologous proteins that contain three functional high-affinity, EF-hand Ca 2+-chelating motifs (reviewed in ref 5). At low [Ca 2+ ] free , GCAPs increase the activity of GC1 (6) at least 10-fold (1) by an unknown mechanism. GCAP1 forms a stable complex with GC1, independent of [Ca 2+ ] free. The GC1/GCAP1 complex may switch between two conformations, active and inactive, with the binding or dissociation of Ca 2+ (2, 7). GCAP2 may translocate from the cytosol to the membranebound cyclase when it is free of Ca 2+ and stimulates GC1 activity (8). The N-terminal fatty acid-acylated regions of both GCAPs show weak sequence conservation, and the function of this region remains speculative. It is possible that the N-terminus is flexible and exposed, providing hydrophobic tethering to the membranes for the most efficient stimulation of GC1, as proposed for GCAP1 by Otto-Bruc et al. (9), but this modification is functionally unrelated in the GC1 stimulation by GCAP2, as proposed by Olshevskaya et al. (8).