Molecular properties of human guanylate cyclase–activating protein 2 (GCAP2) and its retinal dystrophy–associated variant G157R
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100619
ISSN1083-351X
AutoresAnna Avesani, Valerio Marino, Serena Zanzoni, Karl‐Wilhelm Koch, Daniele Dell’Orco,
Tópico(s)Ubiquitin and proteasome pathways
ResumoIn murine and bovine photoreceptors, guanylate cyclase–activating protein 2 (GCAP2) activates retinal guanylate cyclases (GCs) at low Ca2+ levels, thus contributing to the Ca2+/cGMP negative feedback on the cyclase together with its paralog guanylate cyclase–activating protein 1, which has the same function but different Ca2+ sensitivity. In humans, a GCAP2 missense mutation (G157R) has been associated with inherited retinal degeneration (IRD) via an unknown molecular mechanism. Here, we characterized the biochemical properties of human GCAP2 and the G157R variant, focusing on its dimerization and the Ca2+/Mg2+-binding processes in the presence or absence of N-terminal myristoylation. We found that human GCAP2 and its bovine/murine orthologs significantly differ in terms of oligomeric properties, cation binding, and GC regulation. Myristoylated GCAP2 endothermically binds up to 3 Mg2+ with high affinity and forms a compact dimer that may reversibly dissociate in the presence of Ca2+. Conversely, nonmyristoylated GCAP2 does not bind Mg2+ over the physiological range and remains as a monomer in the absence of Ca2+. Both myristoylated and nonmyristoylated GCAP2 bind Ca2+ with high affinity. At odds with guanylate cyclase–activating protein 1 and independently of myristoylation, human GCAP2 does not significantly activate retinal GC1 in a Ca2+-dependent fashion. The IRD-associated G157R variant is characterized by a partly misfolded, molten globule-like conformation with reduced affinity for cations and prone to form aggregates, likely mediated by hydrophobic interactions. Our findings suggest that GCAP2 might be mostly implicated in processes other than phototransduction in human photoreceptors and suggest a possible molecular mechanism for G157R-associated IRD. In murine and bovine photoreceptors, guanylate cyclase–activating protein 2 (GCAP2) activates retinal guanylate cyclases (GCs) at low Ca2+ levels, thus contributing to the Ca2+/cGMP negative feedback on the cyclase together with its paralog guanylate cyclase–activating protein 1, which has the same function but different Ca2+ sensitivity. In humans, a GCAP2 missense mutation (G157R) has been associated with inherited retinal degeneration (IRD) via an unknown molecular mechanism. Here, we characterized the biochemical properties of human GCAP2 and the G157R variant, focusing on its dimerization and the Ca2+/Mg2+-binding processes in the presence or absence of N-terminal myristoylation. We found that human GCAP2 and its bovine/murine orthologs significantly differ in terms of oligomeric properties, cation binding, and GC regulation. Myristoylated GCAP2 endothermically binds up to 3 Mg2+ with high affinity and forms a compact dimer that may reversibly dissociate in the presence of Ca2+. Conversely, nonmyristoylated GCAP2 does not bind Mg2+ over the physiological range and remains as a monomer in the absence of Ca2+. Both myristoylated and nonmyristoylated GCAP2 bind Ca2+ with high affinity. At odds with guanylate cyclase–activating protein 1 and independently of myristoylation, human GCAP2 does not significantly activate retinal GC1 in a Ca2+-dependent fashion. The IRD-associated G157R variant is characterized by a partly misfolded, molten globule-like conformation with reduced affinity for cations and prone to form aggregates, likely mediated by hydrophobic interactions. Our findings suggest that GCAP2 might be mostly implicated in processes other than phototransduction in human photoreceptors and suggest a possible molecular mechanism for G157R-associated IRD. The phototransduction cascade in vertebrates is finely regulated by the subtle changes in intracellular Ca2+, which follow the closure of cyclic nucleotide–gated (CNG) channels upon the light-induced activation of the 3′,5′- cGMP phosphodiesterase 6 (PDE) (1Koch K.W. Dell'Orco D. Protein and signaling networks in vertebrate photoreceptor cells.Front. Mol. Neurosci. 2015; 8: 67Crossref PubMed Scopus (58) Google Scholar). Guanylate cyclase–activating proteins (GCAPs) are among the neuronal calcium sensors (NCSs) that permit such fine regulation and tune the light sensitivity of rods and cones, thus contributing to shape their photoresponses (1Koch K.W. Dell'Orco D. Protein and signaling networks in vertebrate photoreceptor cells.Front. Mol. Neurosci. 2015; 8: 67Crossref PubMed Scopus (58) Google Scholar, 2Palczewski K. Polans A.S. Baehr W. Ames J.B. 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Kinetic analysis of the interaction between the Ca2+-bound and the Ca2+-free guanylyl cyclase activating proteins (GCAPs) and recombinant photoreceptor guanylyl cyclase 1 (RetGC-1).Biochemistry. 2004; 43: 13796-13804Crossref PubMed Scopus (41) Google Scholar, 6Peshenko I.V. Olshevskaya E.V. Azadi S. Molday L.L. Molday R.S. Dizhoor A.M. Retinal degeneration 3 (RD3) protein inhibits catalytic activity of retinal membrane guanylyl cyclase (RetGC) and its stimulation by activating proteins.Biochemistry. 2011; 50: 9511-9519Crossref PubMed Scopus (32) Google Scholar) and are able to detect changes in intracellular [Ca2+]. In the low intracellular Ca2+ phase, when CNG channels are closed because of PDE-mediated cGMP depletion, GCAPs stimulate the production of cGMP. When cGMP levels are sufficiently high, CNG channels reopen, restoring the submicromolar Ca2+ levels typical of dark-adapted cells. Finally, GCAPs switch to a Ca2+-bound, GC-inhibiting state, bringing the catalytic activity down to or below the basal levels (1Koch K.W. Dell'Orco D. Protein and signaling networks in vertebrate photoreceptor cells.Front. Mol. Neurosci. 2015; 8: 67Crossref PubMed Scopus (58) Google Scholar, 7Koch K.W. Dell'Orco D. A calcium-relay mechanism in vertebrate phototransduction.ACS Chem. Neurosci. 2013; 4: 909-917Crossref PubMed Scopus (56) Google Scholar). GCAPs share a high degree of sequence identity, and the available three-dimensional structures of three isoforms (8Ames J.B. Dizhoor A.M. Ikura M. Palczewski K. Stryer L. Three-dimensional structure of guanylyl cyclase activating protein-2, a calcium-sensitive modulator of photoreceptor guanylyl cyclases.J. Biol. Chem. 1999; 274: 19329-19337Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 9Lim S. Peshenko I.V. Olshevskaya E.V. Dizhoor A.M. Ames J.B. 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Up to three GCAPs isoforms have been discovered in mammal photoreceptors, and six to eight in teleost fish (11Palczewski K. Sokal I. Baehr W. Guanylate cyclase-activating proteins: Structure, function, and diversity.Biochem. Biophys. Res. Commun. 2004; 322: 1123-1130Crossref PubMed Scopus (75) Google Scholar), thus raising the question as to the physiological meaning of such apparent redundancy. GCAPs display different affinity for Ca2+ (12Dell'Orco D. Sulmann S. Linse S. Koch K.W. Dynamics of conformational Ca2+-switches in signaling networks detected by a planar plasmonic device.Anal. Chem. 2012; 84: 2982-2989Crossref PubMed Scopus (31) Google Scholar) and are able to replace Ca2+ with Mg2+ (13Dizhoor A.M. Olshevskaya E.V. Peshenko I.V. Mg2+/Ca2+ cation binding cycle of guanylyl cyclase activating proteins (GCAPs): Role in regulation of photoreceptor guanylyl cyclase.Mol. Cell Biochem. 2010; 334: 117-124Crossref PubMed Scopus (76) Google Scholar, 14Peshenko I.V. Dizhoor A.M. 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Besides their established activity in the regulation of the phototransduction cascade in murine and bovine photoreceptor outer segments, GCAP1 and GCAP2 have also been localized in the synaptic layer (18Dizhoor A.M. Olshevskaya E.V. Henzel W.J. Wong S.C. Stults J.T. Ankoudinova I. Hurley J.B. Cloning, sequencing, and expression of a 24-kDa Ca(2+)-binding protein activating photoreceptor guanylyl cyclase.J. Biol. Chem. 1995; 270: 25200-25206Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 19Frins S. Bonigk W. Muller F. Kellner R. Koch K.W. Functional characterization of a guanylyl cyclase-activating protein from vertebrate rods. Cloning, heterologous expression, and localization.J. Biol. Chem. 1996; 271: 8022-8027Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), especially suggesting for GCAP2 a Ca2+-dependent mediation of the synaptic ribbon plasticity (27Schmitz F. 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Retinitis pigmentosa with EYS mutations is the most prevalent inherited retinal dystrophy in Japanese populations.J. Ophthalmol. 2015; 2015: 819760Crossref PubMed Scopus (41) Google Scholar). The mutation affects a highly conserved residue in the EF4 loop (Fig. 1A), compatible with a decrease in the affinity for Ca2+ of that binding site. The mutant has been studied in vivo by expressing the bovine equivalent to human G157R as a transgene in a mouse model of rods GCAP1/2 KO (42Lopez-Begines S. Plana-Bonamaiso A. Mendez A. Molecular determinants of guanylate cyclase activating protein subcellular distribution in photoreceptor cells of the retina.Sci. Rep. 2018; 8: 2903Crossref PubMed Scopus (8) Google Scholar). The study revealed that the mutation caused a significant retention of GCAP2 in the inner segment, which is thought to result in photoreceptor cell death and severe retinal degeneration (42Lopez-Begines S. Plana-Bonamaiso A. Mendez A. Molecular determinants of guanylate cyclase activating protein subcellular distribution in photoreceptor cells of the retina.Sci. Rep. 2018; 8: 2903Crossref PubMed Scopus (8) Google Scholar, 43Lopez-del Hoyo N. Fazioli L. Lopez-Begines S. Fernandez-Sanchez L. Cuenca N. Llorens J. de la Villa P. Mendez A. Overexpression of guanylate cyclase activating protein 2 in rod photoreceptors in vivo leads to morphological changes at the synaptic ribbon.PLoS One. 2012; 7e42994Crossref PubMed Scopus (14) Google Scholar). To date, no comprehensive biochemical investigation of the molecular properties of human GCAP2 has been performed and no evaluation of the alterations of such properties brought by the G157R variant was available. In this work, we present a thorough characterization of the structural and functional properties of human GCAP2 and its inherited retinal dystrophy (IRD)-associated G157R variant. NCS proteins are subjected to a post-translational modification where a fatty acid (typically myristic acid) is covalently bound to the N-terminal Gly, thus conferring the protein-specific features such as membrane binding or modulation of the Ca2+ sensitivity (44Burgoyne R.D. Neuronal calcium sensor proteins: Generating diversity in neuronal Ca2+ signalling.Nat. Rev. Neurosci. 2007; 8: 182-193Crossref PubMed Scopus (392) Google Scholar, 45Burgoyne R.D. Weiss J.L. The neuronal calcium sensor family of Ca2+-binding proteins.Biochem. J. 2001; 353: 1-12Crossref PubMed Scopus (379) Google Scholar). To evaluate the effect of myristoylation on structure–function properties of human GCAP2, we expressed and purified both myristoylated and nonmyristoylated variants and assessed the presence of the post-translational modification by MALDI-TOF MS. The theoretical molecular mass (MM) of nonmyristoylated GCAP2 (nmGCAP2), based on the canonical protein sequence (UniProt entry: Q9UMX6), is reported in Table S1, which also reports the predicted MM of the myristoylated variant (myristoylated GCAP2 [mGCAP2]) both without and with the Gly-to-Arg substitution (G157R). The difference in the main peaks detected by MALDI-TOF spectra for mGCAP2 and nmGCAP2 (212.2 Da) is compatible with the effective myristoylation of the protein. Noticeably, no band ascribable to nmGCAP2 was visible in the mGCAP2 MALDI-TOF spectrum (Fig. 1B), thus pointing to a very efficient myristoylation process. The same analysis performed on the G157R variant showed in a peak at 23.7 kDa (Fig. 1D), compatible with a Gly-to-Arg mutation and successful myristoylation, as judged by the substantial correspondence between measured peaks and theoretical values (Table S1). Interestingly, MALDI-TOF spectra showed the presence of dimeric forms in samples of all variants (Fig. 1, B–D and Table S1). NCS proteins are characterized by heterogeneous oligomeric states (23Ames J.B. Dimerization of neuronal calcium sensor proteins.Front. Mol. Neurosci. 2018; 11: 397Crossref PubMed Scopus (7) Google Scholar), and it has been reported that the bovine GCAP2 undergoes Ca2+-dependent dimerization (46Olshevskaya E.V. Ermilov A.N. Dizhoor A.M. Dimerization of guanylyl cyclase-activating protein and a mechanism of photoreceptor guanylyl cyclase activation.J. Biol. Chem. 1999; 274: 25583-25587Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). We investigated whether a similar behavior could be observed for its human ortholog. By performing analytical size-exclusion chromatography (SEC) in the presence of Mg2+ or Ca2+ on both myristoylated and nonmyristoylated forms, we assessed the effect of cation binding and the post-translational modification on the oligomeric state. The elution profiles of nmGCAP2 showed a main peak at 15.2 ml in the presence of Mg2+, which shifted to 14 ml in the concomitant presence of Ca2+ (Fig. 2A), thus resulting in an estimated MM of 25 and 58 kDa, respectively (see Experimental procedures). On the other hand, SEC profiles of mGCAP2 (Fig. 2B) displayed a cation-independent elution peak at 14.7 ml, corresponding to a MM of 35 kDa, which could be related to a different oligomeric state of the protein in Mg2+ or Ca2+ saturating conditions. Dynamic light scattering (DLS) conveniently allows measuring the change in the hydrodynamic diameter of calcium sensor proteins resulting from the structural rearrangement upon cation binding, as well as to evaluate any potential time-dependent aggregation propensity by monitoring the mean count rate (MCR) along the time. Results from DLS experiments showed an increase of the hydrodynamic diameter upon Ca2+ binding for both GCAP2 forms (Fig. 2, C and D), although to a different extent. Indeed, nmGCAP2 exhibited a 0.79-nm increase in the hydrodynamic diameter (6.71 versus 7.50 nm, Table 1, p-value < 0.001), whereas mGCAP2 displayed a 1.06-nm increase upon Ca2+ binding (6.02 versus 7.08 nm, Table 1, p-value < 0.001). Nevertheless, such increase in the hydrodynamic diameter can be ascribable to a conformational change rather than to an aggregation process, as shown by the stability of the MCR over the 90-min experimental time (Fig. S1). Interestingly, in the absence of cations, mGCAP2 displayed an abrupt increase in the MCR and wide oscillations around an approximately 40% higher value compared with the Mg2+ or Ca2+ saturating conditions, without evidence of aggregation trends (Fig. S1) at odds with nmGCAP2 that, in the apo-form, showed very stable and low MCR values, indicative of a particularly stable colloidal suspension. The same experiments performed with the IRD-associated G157R variant did not allow the estimation of the hydrodynamic diameter because of the high polydispersity index (0.44 ± 0.07 < polydispersity index <0.54 ± 0.08) of the colloidal suspension, indicative of a high tendency to rapidly form aggregates and/or of low protein stability.Table 1Hydrodynamic diameter estimation of GCAP2 variantsProteinStated ± σ (nm) [n]PdI ± σnmGCAP2Mg2+6.71 ± 0.08 [39]0.37 ± 0.08Mg2+ + Ca2+7.50 ± 0.15 [41]0.31 ± 0.04mGCAP2Mg2+6.02 ± 0.06 [55]0.28 ± 0.04Mg2+ + Ca2+7.08 ± 0.10 [38]0.30 ± 0.03d, hydrodynamic diameter; n, number of measurements; PdI, polydispersity index; σ, standard error. Open table in a new tab d, hydrodynamic diameter; n, number of measurements; PdI, polydispersity index; σ, standard error. To investigate the dimerization process of the physiologically relevant myristoylated form of GCAP2 in the presence of cations, the analytical SEC profiles of mGCAP2 were collected at an increasing protein concentration. In the presence of the physiological concentration of Mg2+, the elution peak displayed a minor shift of the apparent mass from 31.9 kDa at 5 μM mGCAP2 to 34.3 kDa at concentrations higher than 10 μM (Fig. 3A). On the other hand, in the presence of both Mg2+ and Ca2+, the main elution peak shifted compatibly with an increase of mass from 24 kDa (at 5 μM) to 33.8 kDa at 80-μM mGCAP2 (Fig. 3B), moreover, each peak displayed a shoulder at higher MM with the intensity proportional to the protein concentration. By fitting the apparent MM as a function of the mGCAP2 concentration to a hyperbolic curve (Fig. 3, inset), it was possible to obtain a cation-specific apparent KD of dimerization. Indeed, in the presence of Mg2+ mGCAP2 was found to dimerize with an apparent KD < 1 μM (R2= 0.89), thus suggesting that with physiological [Mg2+] in conditions of very low [Ca2+] mGCAP2 is a dimer. On the other hand, in the presence of both Mg2+ and Ca2+, the estimated apparent KD of dimerization shifted to 55.4 μM (R2= 0.99), thus pointing toward a Ca2+-dependent dimerization of mGCAP2. The same experiments performed on G157R showed that, despite having the same nominal concentration, all chromatographic profiles (Fig. S2) were considerably lower in intensity than the WT. Such decrease in absorbance pointed toward a massive cation-independent aggregation process, which would render the chromatograms collected at > 20 μM protein almost undetectable, in line with the behavior observed in DLS measurements. CD spectroscopy can be used to monitor the conformational changes of NCS proteins upon metal binding, by exploiting the differential absorption of circularly polarized light of the chiral centers of the protein. Specifically, the CD signal in the near UV (250–320 nm) arises from the microenvironment of aromatic residues, thus representing a fingerprint of the protein tertiary structure, whereas in the far UV (200–250 nm), the optically active peptide bonds provide information about the secondary structure elements. The near-UV CD spectrum of apo-nmGCAP2 (Fig. 4A, black trace) showed a relatively intense positive signal in all three bands corresponding to aromatic residues (Phe, Tyr, and Trp), indicating that the protein is significantly structured even in the a
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