From the
Institut für Biologische Informationsverarbeitung IBI-2 (Biologische Strukturforschung) and the ¶Institut für Biologische Informationsverarbeitung IBI-1, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany and the
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119992 Moscow, Russia
Received for publication, January 15, 2003 , and in revised form, April 8, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
At its N terminus recoverin is heterogeneously acylated, the prevailing modification being a myristoyl chain (15). The observation of a Ca2+-dependent partitioning of recoverin to membranes led to the proposal that it underwent a Ca2+-myristoyl switch (16). The mechanics of this switch were unraveled by determining the solution structures of Ca2+-free and Ca2+-bound myristoylated recoverin via NMR spectroscopy. In the Ca2+-free state of recoverin (T state) the myristoyl moiety is buried within a hydrophobic pocket, whereas in the Ca2+-bound form (R state), the acyl group is extruded and thus available for interaction with other proteins or insertion into a lipid bilayer (1719). Moreover, the myristoyl chain has been proposed to act as an intrinsic allosteric effector modifying the conformational equilibrium between T and R states (20).
Ca2+ binds to recoverin in a sequential fashion, i.e. occupation of high affinity EF-hand 3 is followed by filling of low affinity EF-hand 2. An intermediate state with Ca2+ bound solely to EF-3 can be prepared using myristoylated recoverin harboring the mutation E85Q, which virtually abolishes Ca2+ binding to EF-hand 2. Recently the three-dimensional structure of the myristoylated E85Q mutant was determined by NMR spectroscopy (21). It was shown to exhibit a hybrid fold with the N- and C-terminal domains resembling the corresponding portions of Ca2+-free and Ca2+-bound myristoylated wild-type recoverin, respectively. Consequently, the myristoyl group is still sequestered within a hydrophobic cavity and only partially unclamped. Biochemical studies on the myristoylated E85Q mutant confirmed that it can only bind one Ca2+ at saturating concentration (21, 22). Intriguingly, a fraction of myristoylated E85Q recoverin binds to membranes already at low free Ca2+ (29 µM), indicating that the presence of lipid bilayers may shift the conformational equilibrium toward a form of the E85Q mutant in which the myristoyl chain is exposed (22).
The presence of the N-terminal acyl group lowers the apparent Ca2+ affinity and adds cooperativity to the Ca2+ binding mode of recoverin (6). Because in the Ca2+-saturated R state the myristoyl group is exposed to the solvent, the x-ray structure of non-myristoylated recoverin in the presence of Ca2+ has been suggested to represent a good approximation to the R form of the myristoylated protein (23, 24). We therefore speculate that the non-myristoylated E85Q mutant may represent a model of the myristoylated analog in the presence of membranes. Despite the lack of evidence for involvement of nonacylated recoverin in signal transduction, non-myristoylated forms of the protein are valuable models to delineate the pathways of Ca2+-induced conformational changes.
In the present study we address the impact of the myristoyl group on the Ca2+-dependent conformational transition in recoverin. To this end, we have determined the x-ray structure of the non-myristoylated E85Q mutant and compared it to the non-myristoylated wild-type as well as to the myristoylated mutant published recently (21). Furthermore, to gain more insight into the biochemical properties of the non-acylated E85Q variant, we investigated key functional parameters such as Ca2+ binding, association with hydrophobic matrices, and inhibition of rhodopsin kinase. Finally we asked how the biochemical properties of the E85Q mutant correlate with the structural information that we obtained on this mutant.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
45Ca2+ Binding AssayBinding of 45Ca2+ to wild-type recoverin and the E85Q mutant was investigated as described previously (21, 22). In summary, 50 or 100 µM protein was dissolved in 20 mM HEPES-KOH, pH 7.5, 100 mM NaCl, and 1 mM DTT and transferred to Centricon 10 devices (Amicon). Radioactive 45CaCl2 was added, the samples were centrifuged for 1 min (7000 rpm, tabletop centrifuge Eppendorf model 5415), and radioactivity of the filtrate was counted (free Ca2+). Next, non-radioactive CaCl2 was added and the centrifugation procedure was repeated. Protein-bound Ca2+ versus free Ca2+ was determined from the excess Ca2+ in the protein sample over that present in the ultrafiltrate. Data were analyzed as described previously (22).
Phenyl-agarose Binding AssayThe phenyl-agarose binding assay was performed according to a published procedure (16). Briefly, 2 µM wild-type recoverin or mutant E85Q were mixed with 100 µl phenyl-agarose (Sigma) and incubated at 37 °C (Eppendorf thermomixer 5436, 1,000 rpm) for 15 min in 20 mM HEPES, pH 7.5, 150 mM NaCl, 20 mM MgCl2,1mM DTT, 3 mM EGTA, and 050 mM CaCl2 (total volume, 1000 µl). The mixture was centrifuged for 15 min (14,000 rpm, table-top centrifuge Eppendorf model 5415), and protein concentration in the supernatant was determined by a Bradford protein assay (Bio-Rad).
Rhodopsin Kinase AssayThe assay mixture in a final volume of 50 µl contained 10 µM rhodopsin (urea-washed ROS), 20 mM Tris-HCl, pH 7.5, 2 mM MgCl2,1mM [-32P]ATP (30100 dpm/pmol), 1 mM DTT, 1 mM PMSF, and 0.3 unit of rhodopsin kinase. Where appropriate, wild-type recoverin or the E85Q mutant and a Ca2+/bisaminobromophenoxyethane tetraacetate buffer were added (20). The reaction was initiated by addition of ATP, and samples were incubated in continuous light for 30 min at 37 °C. Incubation was terminated by adding 1 ml of 10% (w/v) trichloroacetic acid. The resulting precipitate was collected by centrifugation and washed 34 times with 1 ml of 10% trichloroacetic acid; the pellet was used for Cerenkov counting.
Recoverin CrystallizationNon-myristoylated recombinant bovine recoverin was crystallized using the hanging-drop setup in a buffer containing 100 mM Tris-HCl, pH 8.0, 1 mM CaCl2,and1mM MgCl2 with the reservoir solution additionally containing 7080% saturated ammonium sulfate. Crystals measuring 150250 µm in each dimension grew within 46 weeks under these conditions.
Data CollectionCrystallographic datasets were collected at 100 K. Because crystals could not be grown in the presence of cryoprotectants, they were soaked in reservoir solution containing 5, 10, and 15% (v/v) glycerol for 10 min each and 30% for 10 s prior to cryocooling.
Native data were recorded at beamline ID14-1 of the European Synchrotron Radiation Facility (Grenoble, France) tuned to a wavelength of 0.934 Å on an ADSC-Q4R detector (ADSC-Quantum). Data processing, including reflections up to 1.5- and 1.9-Å resolution for wild-type and mutant, respectively, was carried out using MOSFLM (26) and SCALA, the latter of which is part of the CCP4 software suite (27).
Structure SolutionBoth recoverin structures were determined by molecular replacement using a single dataset each. The crystal structure of non-myristoylated recombinant bovine recoverin reported previously (23) was used as a starting model for refinement of the wild-type structure. Following preliminary refinement using the CNS software package (28), all regions poorly defined in electron density were removed from the model and re-established in an iterative process, including several cycles of positional refinement and manual rebuilding using the program O (29). To minimize model bias in the refinement process, electron density was modified using the DM package (CCP4), including solvent flattening, histogram mapping, and Sayre's equation. This finally allowed for portions of the structure not well-defined in 2FoFc maps (i.e. residues 26 and 201202) to be built as well. A total of 85 crystallographic water molecules could be determined in the structure.
The resulting improved wild-type recoverin model served as a starting point for determination of the E85Q mutant structure, which was performed using essentially the same strategy. In this case, residues 26, 7577, and 198202 could not be properly defined even by the use of density modification algorithms. The number of water molecules in this structure was 76. For statistics on data collection and refinement refer to Table I.
|
According to Ramachandran plots generated with PROCHECK (CCP4), both models exhibit good geometry with 98.4 and 100%, respectively, of the residues in the allowed regions. Exceptions in the wild-type structure include serines 4 and 6 located in the N-terminal tail poorly defined in the 2FoFc map and aspartate 74, which is part of the Ca2+ binding EF-2 loop. Figures were generated using MOL-SCRIPT (30) and RASTER3D (31) using secondary structure assignments as given by the DSSP program (32). Surface representations were prepared with VMD (33). For depicting solution structures determined by NMR spectroscopy the individual structure exhibiting the lowest r.m.s. deviation from the mean, as determined with MOLMOL (34), was selected from the ensemble. Calculation of EF-hand interhelical angles was performed using the program INTERHLX (obtained from nmr.uhnres.utoronto.ca/ikura/datasoft.html).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Interaction with Phenyl-agaroseBinding of recoverin to phenyl-agarose is thought to depend on the Ca2+-induced exposure of hydrophobic residues and not on the presence of the myristoyl group (16). We applied this assay to test whether the non-myristoylated E85Q variant could bind to phenyl-agarose in a fashion similar to the wild-type (Fig. 2). In fact, the mutant protein did not display any binding activity at all. By contrast, wild-type recoverin included as a control bound to the hydrophobic matrix with half-saturation occurring at 1.5 µM Ca2+.
|
Inhibition of Rhodopsin KinaseOne more characteristic feature of recoverin is its capability of inhibiting rhodopsin kinase in a Ca2+-dependent manner (911, 35, 36). Myristoylation of recoverin is dispensable for this activity, because the non-acylated protein inhibits rhodopsin kinase in a manner very similar to the myristoylated form (37). Thus, it is suggested that calcium triggers exposure of an interaction site that mediates inhibition of GRK1 by recoverin and is different from its N-terminal myristoyl group. We tested whether the mutant could inhibit rhodopsin kinase in a Ca2+-dependent manner. As shown in Fig. 3, the E85Q mutant was not capable of inhibiting GRK1 to a significant extent. In contrast, the nonmyristoylated wild-type used as a positive control inhibited rhodopsin kinase as expected.
|
Crystal Structure of Wild-type Recoverin and the E85Q MutantFor elucidating the role of the myristoyl modification as an intrinsic modulator of the Ca2+-induced conformational transition in recoverin, structural studies on non-myristoylated analogs are particularly useful. As a first approach, to establish a positive control system, we have re-investigated the crystallographic structure of recombinant non-myristoylated bovine recoverin to a higher resolution (1.5 Å) than has been described previously (1.9 Å (23)). As a result, we were able to trace backbone and side-chain density in a larger portion of the molecule, particularly in the N- and C-terminal parts and the EF-2 loop. In the extreme termini (residues 26 and 201202), use of conventional 2FoFc and FoFc maps did not allow for satisfactory density allocation. However, based on density modification strategies, tentative coordinates are included for these stretches as well. The overall fold of the molecule is mostly identical to the published structure with a root mean square (r.m.s.) deviation of 0.45 Å for main-chain atoms and 1.03 Å for all non-hydrogen atoms. Likewise, the EF-2 Ca2+ binding site was found to be unoccupied despite the presence of 1 mM Ca2+ in the crystallization mother liquor. This is probably due to the high concentration of ammonium sulfate (approx. 3 M) in the solution, because it is well established that high ionic strength leads to electrostatic screening of ionic binding sites. Thus the Ca2+ activity is decreased into a range supporting binding to the high affinity EF-3 but not to the low affinity EF-2 site. Moreover, a model calculation of free Ca2+ in the presence of 3 M sulfate lead to an estimate well below 1 µM, which is clearly insufficient for Ca2+ binding to EF-2. The absence of Ca2+ in EF-2 in the crystal structures should therefore be viewed as a reflection of the ion concentrations in the samples and not an indication of an artifact due to crystal packing interactions. Although the non-liganded EF-2 loop is quite well defined in our crystal structure (mean atomic B factor 22.4 Å2 for residues 7282 compared with 21.7 Å2 for the entire structure), we cannot exclude that it exhibits increased flexibility in solution.
Using crystallization conditions similar to those for the wild-type protein, crystals of the E85Q mutant diffracting to 1.9 Å were obtained. In this case, the entire polypeptide chain could be traced with the exception of residues 26, 7577, and 198202, which are poorly defined in electron density. The overall fold of the mutant protein turned out to be very similar to our wild-type structure with an r.m.s. deviation over the portions well-defined in both structures (residues 774 and 78197) of 0.30 Å for main-chain atoms and 0.65 Å for all non-hydrogen atoms. This similarity is not surprising, because even the crystal structure of wild-type recoverin does not show any Ca2+ binding to EF-hand 2 (see above), which is disabled in the E85Q mutant. As has already been noted for the wild-type crystal structure, this fold is quite similar to the solution structure of the myristoylated protein with two Ca2+ bound (19). During the course of the present study, an NMR structure of the myristoylated E85Q mutant was published (21). Intriguingly, there are striking differences between the two structures of E85Q (Fig. 4).2 In the myristoylated variant (shown in red), the conformation of the N-terminal domain is similar to the solution structure of the Ca2+-free myristoylated wild-type protein, i.e. the T state (18), whereas in our non-myristoylated mutant (blue) it resembles the R state fold of the myristoylated wild-type with two Ca2+ (19). Specifically, the relatively limited conformational changes in the C-terminal domain as well as rotation of the two domains with respect to each other near Gly96, both of which are believed to be induced by Ca2+ binding to EF-3, are common to both structures. In contrast, rotation around Gly42 (indicated by an arrowhead in Fig. 4), leading to a considerable displacement of the N-terminal -helix and the entering helix of EF-1, is only observed in the non-acylated mutant. These observations suggest that the unmodified protein possesses the intrinsic capability of undergoing almost the entire Ca2+-induced conformational transition upon Ca2+ binding to EF-3, whereas in the myristoylated form the N-terminal domain is locked in the T state until Ca2+ is bound to EF-2.
|
Because in Ca2+-saturated recoverin the myristoyl group is extruded and does not significantly interact with the protein moiety (19), we decided to use the NMR structure of the myristoylated protein with two Ca2+ as a model for the corresponding non-myristoylated analog. Although this is a reasonable assumption, equivalence of tertiary folds cannot be unequivocally proven in the absence of a structure of Ca2+-saturated non-myristoylated recoverin. This stated, the differences between our crystallographic structure of non-myristoylated recoverin with one Ca2+ and the solution structure of the myristoylated protein with two Ca2+ should be mainly related to Ca2+ binding to EF-2. The most significant structural alterations are highlighted in Fig. 5. Establishment of the Ca2+ co-ordination sphere requires "flipping" of the Ca2+-binding loop (residues 7282, region 1), which is associated with a decrease in the EF-2 interhelical angle (Table II, below). The resulting movement of the domain linker (residues 95101, region 2) by 5 Å causes a steric clash with a stretch of residues connecting the N-terminal
-helix to the entering helix of EF-1 (residues 1824, region 3), which forces a concerted movement of this loop. Consequently, the orientation of the N-terminal helix (region 4) also changes considerably. Comparison of these two recoverin structures thus enables us to trace structural implications of Ca2+ binding to EF-2 throughout the entire domain. Moreover, as the structure of the nonacylated E85Q mutant is virtually identical to the non-acylated wild-type, both of which contain one Ca2+ bound to EF-3, these effects of EF-2 occupation probably account for the functional inactivity of the mutant, as discussed below.
|
|
The interhelical angle defined by the entering and exiting helices is a sensitive indicator of conformational changes in EF-hand modules (39). Table II gives a compilation of this parameter for EF-hands 1 through 4 for all recoverin structures currently available. The angle defined by the exiting helix of EF-2 and the entering helix of EF-3 has been included for monitoring the arrangement of the N-terminal domain relative to the C-terminal portion. The values listed support our conclusion that, upon occupation of EF-3 in the non-myristoylated protein, EF-1 adopts a configuration reminiscent of the sterical arrangement in myristoylated recoverin with two Ca2+. At the same time, the interhelical angle in EF-2 is similar to the value in the myristoylated E85Q mutant and is thus representative of a non-liganded EF-2 in the T/R intermediate state. The difference in EF-2 interhelical angle between these structures on the one hand and the Ca2+-free protein on the other may indicate a slight conformational change in EF-2, which is associated with Ca2+ binding to EF-3 and the resulting re-arrangement of the domain interface. With respect to the C-terminal domain, interpretation of the results is more complicated. For example, the angle of EF-3 in the crystal structures containing one Ca2+, although clearly different from the unoccupied EF-3 in Ca2+-free myristoylated recoverin, is still not identical to the myristoylated structures with one or two Ca2+ ions. Moreover, the extent of the rotation around Gly96 in non-myristoylated E85Q recoverin, which ultimately results from Ca2+ binding to EF-3, is more similar to the myristoylated protein with two Ca2+ than to the myristoylated E85Q mutant with one Ca2+. These observations indicate that the increased conformational rigidity of the N-terminal domain in the presence of the buried myristoyl group on the one hand limits the magnitude of the domain rotation but on the other hand in some way facilitates the Ca2+-induced "opening" movement of EF-3. Thus, the bidirectional transfer of conformational information across the domain interface is significantly modulated by the presence of the N-terminal myristoyl moiety.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we have determined the crystallographic structure of non-myristoylated recoverin bearing the E85Q substitution which virtually abolishes Ca2+ binding to EF-hand 2. An improved structure of the wild-type protein at 1.5-Å resolution allowed us to trace larger portions of the molecule than was previously possible. The overall fold of the wild-type and mutant recoverin is almost identical, which can be explained by the absence of Ca2+ binding to EF-2 even in the wild-type under the crystallization conditions employed. Thus, in both cases the protein is trapped in an intermediate state reflecting conformational changes induced by Ca2+ binding to EF-3. Several conclusions can be drawn from the intriguing similarity between these two structures and the solution structure of the fully Ca2+-occupied myristoylated protein.
First, the Ca2+-induced conformational transition initially observed in the NMR structures of myristoylated recoverin appear for the most part not to require Ca2+ binding to EF-2 in the non-myristoylated protein. Instead, upon Ca2+ binding to EF-3 both domains adopt a characteristic R state fold, including rotational movements about Gly42 and Gly96 (Fig. 4). This conclusion relies on the assumption that in the Ca2+-free state recoverin structure is largely independent of the myristoylation state. Unfortunately, structural information on non-acylated Ca2+-free recoverin is currently unavailable. Nonetheless, there are several lines of indirect evidence supporting this view. Dizhoor et al. (20) have analyzed the susceptibility of acylated and non-acylated recoverin to tryptic cleavage in the presence and absence of Ca2+. They found Ca2+ to increase resistance toward degradation, this effect being even more pronounced with the non-acylated protein. In a CD-spectroscopic study performed by Kataoka et al. (40), the Ca2+-induced increase in ellipticity at = 208 nm and
= 222 nm was reported to be independent of the myristoylation state. Finally, binding of the fluorescent dye 1-anilinonaphthalene-8-sulfonate by recoverin increases dramatically upon Ca2+ binding in both the myristoylated and the non-myristoylated state (41). These data indicate that the Ca2+-induced conformational changes in unmodified recoverin are at least comparable to those observed for the native acylated protein. Of course, we cannot exclude the possibility that the presence of the myristoyl moiety may induce minor structural changes in the Ca2+-free protein, the NMR structure of which has revealed a tight interaction of the acyl chain with its hydrophobic pocket. Indeed, when comparing deuterium exchange rates of myristoylated and non-myristoylated recoverin in the presence and absence of Ca2+, Neubert et al. (42) found the myristoylation state to affect isotope exchange in certain recoverin fragments, although for both the myristoylated and the non-myristoylated protein, significant effects of Ca2+ binding were observed. Attempts to crystallize Ca2+-free non-myristoylated recoverin have thus far been unsuccessful.
A second conclusion from our data concerns the role of the myristoyl group. As stated above, the conformational changes in the C-terminal domain induced by occupancy of EF-3 and the subsequent reorganization at the domain interface tend to destabilize the T state in the N-terminal part even in the absence of Ca2+ binding to EF-2. This effect is counterbalanced in the presence of the myristoyl moiety, which is able to stabilize the arrangement of the N-terminal domain in the Ca2+-free state. As a result, in the myristoylated protein binding of Ca2+ to EF-2 is essential to shift the conformational equilibrium toward the R state. In this model, Ca2+ binding to EF-2 would not directly drive the rotation about Gly42, resulting in the N terminus "pulling" the myristoyl group out of its hydrophobic pocket, as previously suggested, but is likely to destabilize the hydrophobic interactions of the myristoyl with its environment, rendering the buried state thermodynamically less favorable. This would ultimately allow the intrinsic tension of the domain to be relieved, partly by means of the aforementioned rotation.
In the absence of the myristoyl modification, the structural impact of Ca2+ binding to EF-2 is greatly reduced (Fig. 5). Nonetheless, it is absolutely required to attain the R state, because the E85Q mutant neither displays binding to phenyl-agarose (Fig. 2) nor is able to inhibit rhodopsin kinase (Fig. 3).
A thorough comparison of available recoverin structures offers possible explanations for this behavior. Fig. 6 illustrates the pattern of polarity on the solvent-accessible surface computed for various recoverin structures. Upon Ca2+ binding, the myristoylated protein exposes a large hydrophobic patch (blue circle), which is made up of aromatic (Phe23, Trp31, Phe35, Phe56, Phe57, Phe83) and aliphatic (Leu28, Ile44, Leu81, Leu90) side chains and is surrounded by charged and polar residues. Although not discernible in the myristoylated E85Q mutant, in the non-myristoylated structures this patch is formed in its general outline, but the pattern still changes further in the fully Ca2+-occupied protein. Specifically, the terminal hydroxyl oxygen of Tyr86, which is located in the center of the hydrophobic patch and is readily solvent-accessible in non-myristoylated recoverin with one Ca2+, becomes almost completely buried upon Ca2+ incorporation into EF-2. Because this hydrophobic region is a good candidate for mediating the Ca2+-induced binding of recoverin to phenyl-agarose, these observations may explain why the E85Q mutant (largely irrespective of myristoylation) does not efficiently bind to this hydrophobic matrix in the presence of Ca2+.
|
A similar approach was used to understand the different capabilities of the same recoverin variants to inhibit rhodopsin kinase in a Ca2+-dependent manner. In a recent mutational study on the frog recoverin homolog S-modulin, Tachibanaki et al. (43) identified seven conserved amino acid residues that were proposed to constitute an interaction surface for rhodopsin kinase. Based on the degree of preservation of Ca2+-induced conformational changes as assessed by CD spectroscopy, the authors defined "probable" (residues Phe23, Glu27, Phe56, and Thr93) and "possible" (residues Thr21, Phe57, and Lys101) interaction sites. In Fig. 7 these groups of residues (colored red and orange, respectively) are mapped to the solvent-accessible surface in the recoverin structures under consideration. It is evident that these crucial side chains localize near each other only upon Ca2+ binding to EF-2 and EF-3 in the myristoylated protein. The conformational transition, which is allowed in the acylated E85Q mutant upon EF-3 occupation, does not establish the interaction surface, and even the non-acylated wild-type and mutant proteins containing one Ca2+, while displaying an overall fold resembling the native R state, do not present the same sterical arrangement of these critical residues. Fig. 8 emphasizes individual side-chain displacements occurring in the N-terminal domain of non-myristoylated recoverin upon binding of Ca2+ to EF-2 and thus accounting for the differences in surface properties between Rec * 1 Ca and myr-Rec * 2 Ca shown in Figs. 6 and 7.
|
|
The three-dimensional structures of several other members of the neuronal calcium sensor family have been determined thus far. These include GCAP-2 (44), neurocalcin (45), human (46), and yeast frequenin (47). Although the overall topology of their folds resembles the structure of recoverin, they also differ in several aspects. For example, an attached myristoyl group can adopt diverse functions, and although some proteins such as recoverin, hippocalcin, and neurocalcin exhibit a Ca2+-myristoyl switch, others like GCAP-1, GCAP-2, and frequenin apparently do not (4850). Diversity of Ca2+ signaling via neuronal calcium sensor proteins is also mirrored by the variety of target proteins, including various kinases, membrane-bound guanylate cyclases, adenylate cyclases, ion channels, and cytoskeletal proteins. Our results indicate that subtle changes in surface properties can dramatically alter the biochemical characteristics of recoverin, such as interaction with phenyl-agarose and rhodopsin kinase inhibition. In line with these conclusions, minor differences in surface exposition of amino acid side chains might well account for the diversity of neuronal calcium sensor signaling.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Deutsche Forschungsgemeinschaft (to K.-W. K.), a grant from the Forschungszentrum Jülich for visiting scientists (to I. I. S. and P. P. Ph.), the Ludwig Institute for Cancer Research (to P. P. Ph.), "International Projects" of Ministry of Industry and Science, Russian Federation (to P. P. Ph.), and the Russian Foundation for Basic Research (Grants 00-04-48332 and 00-04-48332). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|| To whom correspondence should be addressed. Tel.: 49-2461-61-3255; Fax: 49-2461-61-4216; E-mail: k.w.koch{at}fz-juelich.de.
1 The abbreviations used are: GRK, G-protein-coupled receptor kinase; DTT, dithiothreitol; GCAP, guanylate cyclase activating protein; myr-, myristoylated; PMSF, phenylmethylsulfonyl fluoride; Rec, wild-type recoverin; Rec[E85Q], recoverin with E85Q substitution; Rec * 1 Ca, recoverin with Ca2+ bound to EF-3; Rec * 2 Ca, recoverin with Ca2+ bound to EF-2 and EF-3; ROS, rod outer segments; r.m.s., root mean square.
2 Comparative structural representations included in this report display crystallographic structures along with NMR structures. We are aware of the possibility of artifacts, e.g. due to crystal packing interactions (38), that in certain cases may limit comparability of structures determined by different methods. However, due to the large extent of the majority of structural differences we refer to, we are confident that the conclusions drawn are nonetheless significant.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|