Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

Retinal Guanylyl Cyclase-Activating Protein 1 and 2

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_101702

Synonyms

Historical Background

Retinal guanylyl cyclases (RetGCs) in retinal rod and cone photoreceptors are regulated by a family of EF-hand Ca2+ 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 Ca2+ levels in light-activated photoreceptor cells and inhibit RetGC activity at higher Ca2+ levels in dark-adapted photoreceptors. The Ca2+-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 Ca2+-sensitive cyclase activity are genetically linked to retinal disease. Here I review atomic-level structures of GCAP1 in both Ca2+-free/Mg2+-bound (activator) and Ca2+-saturated (inhibitory) states, as well as the structure of Ca2+-saturated GCAP2. The structure of GCAP2 reveals an exposed N-terminus that may be important for Ca2+-dependent membrane anchoring of the myristoyl group. By contrast, the structures of Ca2+-free and Ca2+-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 Ca2+-free activator form of GCAP1 contains Mg2+ bound at the second EF-hand (EF2) that is essential for activating RetGC. The Ca2+ saturated form of GCAP1 contains Ca2+ bound at EF2, EF3, and EF4. Ca2+-dependent conformational changes are most apparent in EF2 and in the Ca2+ switch helix (residues 169–174) and will be discussed in terms of a proposed mechanism for Ca2+-dependent activation of retinal guanylyl cyclases.

Introduction

Visual excitation of retinal rod and cone photoreceptors is mediated by a phototransduction cascade that hydrolyzes cGMP, which closes cGMP-gated cation channels on the plasma membrane, resulting in membrane hyperpolarization (see review Pugh et al. 1997). The light-induced hyperpolarization rapidly recovers back to the resting dark state when the light stimulus is removed in a process known as visual recovery. The recovery phase of phototransduction involves replenishing the cGMP levels (Burns and Baylor 2002) by the Ca2+ sensitive activation (Koch and Stryer 1988) of retina-specific guanylyl cyclases (RetGCs) (Dizhoor et al. 1994; Lowe et al. 1995). The Ca2+-dependent activity of RetGC is controlled by intracellular domains (Duda et al. 2005) that interact with soluble EF-hand Ca2+ sensor proteins, called guanylyl cyclase-activating proteins (GCAP1 and GCAP2) (Dizhoor et al. 1994; Dizhoor et al. 1995; Palczewski et al. 1994) (Fig. 1). The GCAP proteins activate RetGC in light-activated photoreceptors (low Ca2+ levels) and inhibit RetGC in dark-adapted photoreceptors (high Ca2+ levels). The Ca2+-dependent regulation of RetGC by the GCAPs is an important mechanism of visual recovery.
Retinal Guanylyl Cyclase-Activating Protein 1 and 2, Fig. 1

Amino acid sequence alignment of GCAP1 and GCAP2. EF-hand motifs (EF1 green, EF2 red, EF3 cyan, and EF4 yellow); N-terminal and C-terminal helices (violet); and GCAP1 residues that contact the myristoyl group (magenta) are highlighted. Swiss Protein database accession numbers are P46065 (bovine GCAP1) and P51177 (bovine GCAP2)

Mutations in the EF-hand motifs of GCAP1 that disable Ca2+ binding (but do not affect Mg2+ binding) cause GCAP1 to constitutively activate RetGC in rods and cones. A number of these mutations (Y99C and E155G) are genetically linked to various retinal diseases (Jiang and Baehr 2010). These mutations (Y99C (Payne et al. 1998) and E155G (Wilkie et al. 2001)) lower the Ca2+ binding affinity outside the photoreceptor Ca2+ concentration range, which causes the Ca2+-free/Mg2+-bound GCAP1 activator state to persist even at high Ca2+ levels in dark-adapted rods. These mutants (that are unable to bind Ca2+) cause persistent activation of RetGC. The GCAP mutants that constitutively activate RetGC then cause elevated cGMP levels in photoreceptor cells that promote apoptosis and disease.

Atomic-Level Structures of GCAP1 and GCAP2

NMR Structure of Ca2+-Bound GCAP2

The NMR structure of the Ca2+-saturated form of unmyristoylated GCAP2 (Fig. 2a) was the first atomic resolution structure of a GCAP protein (Ames et al. 1999). The first 22 amino acids from the N-terminus and the last 19 residues from the C-terminus in unmyristoylated GCAP2 could not be resolved by NMR (see dotted lines in Fig. 2a). The structure of the core region of GCAP2 (residues 23–185) contained 4 EF-hands whose structure was broadly similar to the corresponding EF-hands in Ca2+-bound recoverin (Ames et al. 1997). The overall RMSD was 2.4 angstroms for the EF-hand region when comparing the main chain atoms of recoverin versus GCAP2. An important structural difference between GCAP2 and recoverin is that Ca2+ is bound to EF2, EF3, and EF4 in GCAP2, in contrast to recoverin where Ca2+ is bound only at EF2 and EF3. The binding of Ca2+ to EF4 in GCAP2 may explain the 100-fold higher Ca2+ binding affinity for GCAP2 compared to that of recoverin. The lack of N-terminal myristoylation in the GCAP2 NMR structure led to the destabilization of two key structural elements: an N-terminal helix (residues 5 – 15) and C-terminal helix (residues 175 -183; highlighted violet in Fig. 1) observed in the crystal structure of myristoylated GCAP1 (Fig. 2b). Both the N-terminal and C-terminal helices in GCAP1 form critical contacts with the N-terminal myristoyl group (Fig. 2b, d) that enable the myristoyl group to remain sequestered inside the protein in both Ca2+-free and Ca2+-bound states, which prevents GCAP1 from having a Ca2+-myristoyl switch. By contrast, the N-terminal myristoyl group in GCAP2 is exposed to the exterior in the presence of lipid bilayer membranes (Theisgen et al. 2011), which could enable the myristoyl group to anchor GCAP2 to membranes (Margetic et al. 2014).
Retinal Guanylyl Cyclase-Activating Protein 1 and 2, Fig. 2

Atomic level structures of Ca2+-saturated unmyrisoylated GCAP2 (PDB ID: 1JSA) (a), Ca2+-saturated GCAP1 (PDB ID: 2R2I) (b), and Ca2+-free/Mg2+-bound GCAP1 (PDB ID: 2NA0) (c). Close-up view of the myristate-binding pocket in GCAP1 (d). EF-hands and terminal helices that contact the myristoyl group are colored as defined in Fig. 1. The N-terminal myristoyl group is colored magenta. Bound Ca2+ and Mg2+ are colored orange and purple, respectively

Crystal Structure of Ca2+-Bound GCAP1 with Sequestered Myristoyl Group

The x-ray crystal structure of Ca2+-saturated form of GCAP1 (Fig. 2b) showed the N-terminal myristoyl group to be sequestered inside the protein (Stephen et al. 2007). The four EF-hands in GCAP1 (Figs. 1 and 2b) are grouped into two globular domains: the N-domain is comprised of EF1 and EF2 (residues 18–83) and the C-domain is comprised of EF3 and EF4 (residues 88–161). Ca2+ is bound to GCAP1 at EF2, EF3, and EF4, and the structure of each Ca2+-bound EF-hand in GCAP1 (Fig. 2b) adopts the familiar open conformation of EF-hands as seen in calmodulin and other Ca2+-bound EF-hand proteins. Indeed, the interhelical angles for each Ca2+-bound EF-hand in GCAP1 are nearly identical to those of GCAP2 (Fig. 2a) and recoverin. Although the internal structure of each EF-hand in GCAP1 is similar to that of GCAP2, the overall three-dimensional packing arrangement of the four EF-hands is somewhat different in GCAP1 compared to GCAP2. A unique structural feature of GCAP1 is that the N-terminal α-helix (residues 5–15) upstream of EF1 and C-terminal helix (residues 175–183) downstream of EF4 are held closely together by their mutual interaction with the N-terminal myristoyl group (Fig. 2d). Thus, the covalently attached myristoyl group in GCAP1 is sequestered within a unique environment inside the Ca2+-bound protein. The myristoyl group attached to GCAP1 makes contacts with N-terminal residues (V9, L12, and F42) and the C-terminal helix (L174, V178, and I181) (Fig. 2d). In essence, the myristoyl group serves to bridge both the N-terminal and C-terminal ends of the protein, which explains how Ca2+-induced conformational changes in the C-terminal domain (particularly in EF4) might be transmitted to a possible target binding site in EF1. A Ca2+-myristoyl tug mechanism has been proposed to explain how Ca2+-induced conformational changes in EF4 serve to “tug” on the adjacent C-terminal helix that connects structurally to the myristoyl group and EF1. This tug mechanism helps explain how Ca2+-induced structural changes in EF4 might be relayed to the cyclase-binding region in EF1. The Ca2+-induced structural changes involving the C-terminal helix might also be related to Ca2+-dependent phosphorylation of S201 in GCAP2.

NMR Structure of Ca2+-Free/Mg2+-Bound GCAP1 Mutant V77E

The atomic level structure of Ca2+-free/Mg2+-bound activator form of GCAP1 or GCAP2 is currently not known. The difficulty has been that Ca2+-free/Mg2+-bound GCAP proteins form dimers and higher order protein oligomers that causes considerable sample heterogeneity at high protein concentrations needed for NMR or to make crystals for x-ray crystallography. Ca2+-dependent dimerization of GCAP2 has been suggested to be important for activating the cyclase (Olshevskaya et al. 1999). Protein dimerization was also reported for GCAP1, and GCAP1 mutants that prevent protein dimerization also abolish its ability to activate RetGC, suggesting that dimerization of Ca2+-free/Mg2+-bound activator state might be important for activating RetGC.

A GCAP1 mutant, V77E (called GCAP1V77E), that abolishes protein dimerization was used recently to solve the NMR structure of Ca2+-free/Mg2+-bound GCAP1V77E (Lim et al. 2016). The NMR structure of Ca2+-free/Mg2+-bound GCAPV77E is shown in Fig. 2c. The overall structure of Ca2+-free/Mg2+-bound GCAPV77E is similar to the crystal structure of Ca2+-bound GCAP1 (root mean squared deviation of main chain atoms is 2.0 Ǻ when comparing the two structures). Surprisingly, the structures of EF3 and EF4 devoid of Ca2+ are both nearly identical to the structures of Ca2+-bound EF3 and EF4. Thus, EF3 and EF4 adopt a preformed open conformation even in the absence of Ca2+. This preformed open structure of EF3 and EF4 might explain the 100-fold higher Ca2+-binding of affinity for GCAP1 compared to the Ca2+ sensor proteins like recoverin that undergo large Ca2+-induced conformational changes (Ames et al. 1997). In contrast to EF3 and EF4, the structure of Ca2+-free/Mg2+-bound GCAP1V77E contains Mg2+ bound at EF2, and the structure of Mg2+-bound EF2 is somewhat different from that of Ca2+-bound EF2. The interhelical angle of Mg2+-bound EF2 (114°) is slightly more closed than the interhelical angle of Ca2+-bound EF2 (110°). Thus, the Ca2+-induced opening of EF2 might be functionally important for regulating RetGC. The largest Ca2+-induced structural change in GCAP1 is observed in the Ca2+ switch helix (residues 169–174 highlighted red in Fig. 3), which is one-half turn longer in the Ca2+-free state. The structure of the Ca2+ switch helix contains two residues (T171 and L174) that exhibit Ca2+-dependent solvent accessibility. T171 is exposed in the Ca2+-free structure, whereas it becomes buried and makes contact with L92 in the Ca2+-bound structure. Conversely, L174 is buried and makes contact with L92 in the Ca2+-free structure, but it switches to a solvent-exposed environment in the Ca2+-bound structure. The Ca2+-dependent contacts formed by both T171 and L174 may be important for switching GCAP1 from the Ca2+-free activator to the Ca2+-bound inhibitor states. We suggest that Ca2+-induced shortening of the Ca2+ switch helix may play a role in modulating Ca2+-dependent contacts with RetGC.
Retinal Guanylyl Cyclase-Activating Protein 1 and 2, Fig. 3

Ca2+-induced conformational changes in GCAP1. Structures of Ca2+-free/Mg2+-bound GCAP1V77E activator state (a) and Ca2+-saturated GCAP1 inhibitor state (b). The largest Ca2+-induced structural change is seen for the Ca2+-switch helix (residues 169 – 174) highlighted in red

Summary

The structural information for GCAP1 above provides insight into the activation mechanism of RetGC (Fig. 4). RetGC is known to function as a dimer that binds to dimeric GCAP1 in a 2:2 complex. The GCAP1 dimerization site resides in the C-domain (light green oval in Fig. 4). GCAP1 residues in the N-domain (dark green oval in Fig. 4) have been shown to make direct contact with the kinase homology domain (KHD) in RetGC (blue oval in Fig. 4). In light-adapted photoreceptors (low Ca2+ levels), GCAP1 is in the Ca2+-free/Mg2+-bound activator state and has an elongated Ca2+ switch helix (red box in Fig. 4) that causes an extended orientation between the two domains in GCAP1. The N-domain orientation in Ca2+-free GCAP1 causes the kinase homology domain in RetGC to point inward to facilitate the association of the dimerization domains within the RetGC dimer. The dimeric association of the dimerization domain then facilitates dimerization of the downstream catalytic domain (yellow circles in Fig. 4) to form a functional cyclase active site. Ca2+-binding to GCAP1 is proposed to cause a Ca2+-induced shortening of the Ca2+ switch helix (red helix) that in turn causes a reorientation of the N-domain (dark green oval) with respect to the C-domain (light green oval). This Ca2+-induced domain swiveling within GCAP1 promotes a Ca2+-myristoyl tug mechanism (see red arrows in Fig. 4) that causes GCAP1 to tug on KHD within RetGC. The Ca2+-induced movement of the KHD (blue arrow) disrupts association of the downstream dimerization domain that in turn breaks up the catalytic domain that may inactive the enzyme. To more rigorously test this model, future studies will be needed to determine the structure of GCAP1 bound to RetGC. Future studies are also needed to determine the structure of the GCAP1 dimer and whether Ca2+-induced changes in quaternary structure of the GCAP1 dimer might have a regulatory role.
Retinal Guanylyl Cyclase-Activating Protein 1 and 2, Fig. 4

Schematic diagram of retinal guanylyl cyclase activation by GCAP1. The domains in RetGC are labeled and highlighted in color: extracellular domain (ECD, purple), transmembrane domain (TMD, black), juxtamembrane domain (JMD, orange), kinase homology domain (KHD, blue), dimerization domain (DD, gray), and catalytic domain (yellow). The binding of Ca2+ (orange circles) to GCAP1 causes a shortening of the Ca2+ switch helix (red) that in turn causes swiveling of the two domains in GCAP1 (represented by ovals colored light green (C-domain) and dark green (N-domain)). The Ca2+-induced domain swiveling in GCAP1 promotes a Ca2+-myristoyl tug mechanism (see red arrows and myristoyl group in magenta) that causes GCAP1 to tug on the KHD domain. The Ca2+-induced movement of the KHD domain (blue arrow) disrupts association of the downstream dimerization domain that in turn breaks up the catalytic domain to inactive the enzyme

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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of California at DavisDavisUSA