Retinal Guanylyl Cyclase-Activating Protein 1 and 2
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.
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
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.
- Olshevskaya EV, Ermilov AN, Dizhoor AM (1999) Dimerization of guanylyl cyclase-activating protein and a mechanism of photoreceptor guanylyl cyclase activation. J Biol Chem. 274:25583–87.Google Scholar