J. Biochem. 114, 535-540 (1993) Recoverin Alters Its Surface Properties Depending on Both Calcium-Binding and N-Terminal Myristoylation1 Mikio Kataoka, Ken'ichi Mihara, and Fumio Tokunaga Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560 Received for publication, June 16, 1993 The solution structure and calcium-dependent structural changes of recoverin, a 23kDa calcium binding protein of vertebrate photoreceptors, have been studied by small-angle X-ray scattering and CD, as well as the effect of N-terminal myristoylation. The CD spectrum is not affected by N-terminal myristoylation, but strongly affected by Ca", indicating that N-terminal myristoylation alone does not cause a conformational change. The major conformational change in recoverin induced by Ca2+ is characterized as a decrease in the a-helical content of the protein and an increase in global size upon removal of Ca". In the presence of Ca", unmyristoylated recoverin is monomeric and globular in solution, while N-terminal myristoylation brings about aggregation. In the absence of Ca", unmyristoylated recoverin tends to aggregate, while myristoylated recoverin becomes monomeric and globular. These observations indicate that recoverin changes its surface properties depending on both calcium binding and N-terminal myristoylation. Melittin interacts non-specifically only with the myristoylated recoverin in the absence of Ca2+. This may be indicative of the properties of the interaction between recoverin and its normal physiological target enzyme. Many cellular functions are regulated by intracellular free duction cascade, depending on the free Ca" concentration, calcium ions. Calcium receptor proteins acting as molecular rather than mediate direct regulation of guanylate cyclase switches mediate these regulatory processes. The calcium or phosphodiesterase activity (8, 9). In fact, the function receptor proteins detect changes in the intracellular free and mechanism of recoverin as a Ca2+-dependent guanylate calcium ion concentration which are produced by primary cyclase activator were denied by the discoverers them- stimuli, and alter their interaction with their target en selves (10). Although uncertainty remains as to the iden zymes. A common structural motif present in many cal tity of the physiological target enzyme of recoverin (8, 9, cium receptor proteins is known as the EF-hand (1). To 11), recoverin is an EF-hand calcium regulatory protein. understand the mechanism of action of these EF-hand A further important and interesting feature of recoverin proteins, it is essential to characterize calcium induced is that its N-terminus is heterogeneously acylated with one changes in their structures. of four types of fatty acyl residues including myristoyl Recoverin is a 23kDa EF-hand calcium binding protein (C14:0), C14:1, C14:2, and C12:0 (12). N-terminal acyl isolated from bovine retinal rod (2, 3). The protein was residues would be expected to play a role in protein- firstly discovered as a calcium-dependent modulator of membrane interactions (11, 13), rather than in protein- retinal guanylate cyclase (2, 3). The enzyme cascade protein interactions (14). Ray et al. (15) demonstrated that following the absorption of light by rhodopsin causes the the fluorescence emission spectra of recoverin are respon closure of a cyclic GMP dependent membrane channel, sive to both myristoylation and the binding of Ca2+, sugges which hyperpolarizes the plasma membrane, and lowers ting that both the N-terminal fatty acyl residues and the cytosolic Ca2+ concentration (4-6). Reopening of the calcium binding alter the conformation of recoverin. channel, which is induced by binding of cyclic GMP, is We investigated the conformational change in recoverin required for recovery to the dark state. Since cyclic GMP is upon binding of calcium, as well as the effect of myristoyla tion on its solution structure by small-angle X-ray scatter- hydrolyzed by phosphodiesterase during photoexcitation, the synthesis of cyclic GMP by guanylate cyclase and ing (SAXS) and CD. Although SAXS is a lower resolution inhibition of phosphodiesterase are necessary for the technique compared with X-ray crystallography, it is useful for detecting conformational changes under various solvent recovery. It was suggested that recoverin activated guan conditions, which is frequently impossible to achieve with ylate cyclase at lower Ca2+ concentrations (2). On the other hand, a homologous protein, S-modulin, was isolated from protein crystals. In fact, calcium-dependent conformational changes in calmodulin, a typical EF-hand calcium modula frog retinal rod as a calcium-dependent modulator of tor, have been demonstrated with this technique, including phosphodiesterase (7). Recent studies suggested that recoverin-like proteins modulate termination of the trans the effect of binding of target peptides (16-21). The conformational change in calmodulin on the binding of both 1A part of this study was supportedby Grants-in-Aidfrom the calcium and target peptides has been confirmed subse Ministryof Education, Science and Cultureof Japan to M.K.and F.T. quently by both NMR solution structure analysis (22) and Abbreviations: DTT, dithiothreitol; MW, molecular weight; Rg, X-ray crystallography (23). radius of gyration;SAXS, small-angle X-ray scattering. V,,! '_14, No. 4, 1993 535 536 M. Kataoka et al. SAXS measurements indicate that recoverin is globular tained at five or more protein concentrations (17). The final in solution, in contrast to the dumbbell shape of calmodulin extrapolated data were analyzed by Guinier analysis (26) and troponin C. Calcium binding brings about a confor and the indirect Fourier transform method of Moore (27). mational change in recoverin. N-terminal myristoylation Data analyses were performed with a personal computer, alone does not bring about a major conformational change in PC9801, or an ACOS 2020 of the Computer Center of recoverin, but appears to alter the surface properties of Osaka University. The details of the measurements and the recoverin. The effect of the binding of recoverin to its target analysis techniques have been presented previously (17- protein on its solution structure is physiologically impor 19). tant. Since we have no information about the true target of Measurement of CD Spectra-CD spectra were mea recoverin, we have tried to examine the effect of binding to sured with a JASCO spectropolarimeter, model J-500A melittin, a bee venom peptide, as a model. An effect of (JASCO, Tokyo), controlled with a personal computer, melittin was observed only for the myristoylated recoverin PC9801, using a quartz cell of 1mm path length. The in the absence of Ca 2+. sample concentration was 0.1 or 0.2mg/ml. Each spectrum was collected 4 times. The temperature was maintained at MATERIALS AND METHODS 20°C by circulating thermostated water through the cuvette holder. Preparation of Recoverin•\Both myristoylated and unmyristoylated recoverins were expressed in Escherichia RESULTS coli, and purified as described by Ray et al. (15). The purified recoverins were generous gifts from Drs. S. CD spectra of unmyristoylated and myristoylated recover Zozulya and L. Stryer at Stanford University. ins show a conformational change in the protein on the Preparation of Samples for X-Ray Scattering-Stock binding of calcium, although the myristoyl modification solutions of recoverins were concentrated with a Centricon- alone does not bring about a major conformational change 10 (Amicon, Danvers, MA) to ca. 30 mg/ml. The final (Fig. 1). The change in the spectrum on binding Ca2+ is concentrations were determined by measuring the dry essentially the same for both unmyristoylated and myris weight. Melittin was purchased from Sigma and purified by toylated recoverins. The spectral change is characterized as HPLC before use (24). Samples of recoverin alone or of recoverin mixed with equimolar amounts of melittin were dialyzed against one of the solutions described below with a Spectra/por 4 dialysis membrane. The dialysis of 0.2 to 0.5ml samples was performed, at 4`C, against three changes of 100ml of the described solutions, the first two changes being after 24 h each, and the final dialysis being for 72 h to ensure complete equilibration. At the end of the final dialysis, the samples were removed from the dialysis tubing, and aliquots were quantitatively diluted with the final dialysis fluid to produce a concentration series ranging from 3 to 30mg/ml. The dialysis solutions all contained 100mM KCl, 20mM Tris, 1mM DTT, 1mM MgCl2, and 5% glycerol (pH8.0). For samples in the presence of calcium, the initial dialysis fluid included an additional 1mM CaCl2; the subsequent two changes of dialysis fluid each contained 0.1mM CaC12. For samples in the absence of calcium, the initial dialysis fluid contained 5mM EGTA, and subsequent changes contained 1mM EGTA. The final dialysis fluids were used for the measurements of the backgrounds. Small-Angle X-Ray Scattering Measurements -SAXS measurements were performed with a SAXES camera installed at BL10C of the Photon Factory at Tsukuba using synchrotron radiation (19, 25). The samples were measured in a specially designed cell with quartz windows that were 15 ,u m thick, 10mm wide, and 3mm high. The path length was 1mm. Samples were maintained at 20°C by circulating thermostated water through the cell holder. The exposure times were 15min for samples with lower concentrations than 8 g/ml, and 10min for the others. Scattering profiles were