From the Department of Biochemistry and Molecular
Biology, University of New Hampshire, Durham, New Hampshire 03824 and
§ Zentrum für Umweltforschung and Umwelttechnologie
(UFT), Abt. III Bioorganische Chemie, Universität Bremen,
D-28359 Bremen, Federal Republic of Germany
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ABSTRACT |
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The cGMP-specific phosphodiesterase (PDE) of retinal photoreceptors is a central regulatory enzyme in the visual transduction pathway of vertebrate vision. Although the mechanism of activation of PDE by transducin is well understood, the role of the noncatalytic cGMP binding sites located on the catalytic subunits of PDE remains obscure. We report here for the first time the molecular basis of the noncovalent interactions between cGMP and the high affinity, noncatalytic cGMP binding sites of frog photoreceptor PDE.
None of the tested cGMP analogs were able to bind with greater affinity than cGMP itself, and the noncatalytic sites were unable to bind cAMP. The major determinant for discrimination of cGMP over cAMP is in the N-1/C-6 region of the purine ring of cGMP where hydrogen bonding probably stabilizes the selective binding of cGMP. Substitutions at the C-2 position demonstrate that this region of the molecule plays a secondary but significant role in stabilizing cGMP binding to PDE through hydrogen bond interactions. The unaltered hydrogen at the C-8 position is also important for high affinity binding. A significant interaction between the binding pocket and the ribose ring of cGMP occurs at the 2'-hydroxyl position. Steric constraints were greatest in the C-8 and possibly the C-6/N-1 regions, whereas the C-2/N-3 and C-2' regions tolerated bulky substituents better. Several lines of evidence indicate that the noncatalytic site binds cGMP in the anti-conformation. The numerous noncovalent interactions between cGMP and the noncatalytic binding pocket of the photoreceptor PDE described in this study account for both the high affinity for cGMP and the high level of discrimination of cGMP from other cyclic nucleotides at the noncatalytic site.
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INTRODUCTION |
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Visual excitation of vertebrate retinal photoreceptors begins with the absorption of light by the visual pigment (opsin) which is followed by activation of a G-protein, transducin. This results in activation of a photoreceptor-specific cGMP phosphodiesterase (PDE).1 Increased cGMP hydrolysis lowers the cytoplasmic cGMP concentration which is believed to cause closure of the cGMP-gated ion channel in the plasma membrane and the generation of an electrical response (for reviews, see Refs. 1-5).
The rod photoreceptor PDE holoenzyme is a heterotetramer consisting of
two non-identical catalytic subunits ( and
) and two small
subunits (
) that serve to inhibit catalytic activity. Cone
photoreceptors have a highly homologous PDE that consists of two
identical catalytic subunits (
') and two cone-specific
'
subunits. In addition to containing the active site for cGMP hydrolysis, the catalytic subunits of rod and cone PDEs also possess noncatalytic cGMP binding sites (6). Two other classes of PDEs, namely
the cGMP-stimulated PDE (PDE2) and the cGMP-specific PDE (PDE5), are
also known to contain noncatalytic binding sites for cGMP. In the case
of PDE2, binding of cGMP at the noncatalytic site allosterically
activates cyclic nucleotide hydrolysis at the active site (7, 8). For
PDE5, the noncatalytic cGMP binding site has been proposed to
indirectly regulate catalytic activity of the enzyme via protein
phosphorylation (for review, see Ref. 9).
The function of the noncatalytic cGMP binding sites on the
photoreceptor PDE (classified as PDE6) is currently not well
understood. Recent evidence has supported the idea that binding of cGMP
to noncatalytic sites on amphibian rod PDE enhances the affinity of the
inhibitory subunits for binding to the catalytic subunits (10).
Conversely, displacement or removal of the
subunits from PDE by
G-protein activation causes a marked decrease in the binding affinity
of cGMP with these binding sites (11, 12). These results suggest that
the noncatalytic cGMP binding sites may be the locus for an additional
type of regulation of PDE during visual transduction.
To better understand the functional significance of these noncatalytic binding sites, we have examined the structural requirements for the binding of cGMP to the binding pocket of the noncatalytic sites on rod PDE. We have employed a series of derivatives of cGMP modified in particular positions of the cGMP molecule to identify the types of interactions that stabilize the high affinity binding of cGMP to noncatalytic sites on PDE. Cyclic nucleotide analogs have been successfully employed to define the topology of the nucleotide binding site in several proteins, including the cAMP-dependent protein kinase (13, 14), the cGMP-dependent protein kinase (15), the cyclic nucleotide-gated ion channels (16-19), as well both catalytic (20, 21) and noncatalytic (9, 22-24) binding sites on the various classes of PDEs.
Our results with the noncatalytic cGMP binding site of frog rod PDE demonstrate that the high affinity binding of cGMP is a consequence of multiple sites of interaction between the cGMP molecule and the binding pocket of the enzyme. In particular, the C-6, N-1, C-2, and C-8 positions of the purine ring, the cyclic phosphate moiety, and the 2'-hydroxyl group of the ribose ring all contribute to stabilizing specific binding to cGMP. Furthermore, the noncatalytic site of the rod PDE shows the greatest ability to discriminate cGMP from cAMP and the highest binding affinity of any cyclic nucleotide binding protein studied to date.
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EXPERIMENTAL PROCEDURES |
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Materials-- Frogs were obtained from Charles Sullivan, Inc. Multiscreen filtration plates (MAHA N45) were purchased from Millipore. Scintillation fluid (Ultima Gold) was obtained from Packard Instrument Co. [3H]cGMP was obtained from NEN Life Science Products. All chemicals other than cyclic nucleotide analogs were obtained from Sigma.
Cyclic Nucleotides and Derivatives-- cGMP, cIMP (8), cXMP (19), cAMP (7), thymidine 3',5'-cyclic monophosphate, uridine 3',5'-cyclic monophosphate, cytidine 3',5'-cyclic monophosphate, 8-Br-cGMP (29), 2'-O-monobutyryl-cGMP (47), 2'-deoxy-cGMP (46), and 5'-GMP (43) were purchased from Sigma. (Rp)-cGMPS (37) and (Sp)-cGMPS (38) were purchased from Biolog Life Science Institute, Bremen. We also acknowledge the kind gifts of the following cGMP analogs: compound 27 (F. Seela, Osnabrück); compounds 30, 31, and 36 (G. Weimann, Boehringer Mannheim); compounds 32 and 33 (S. H. Francis, Vanderbilt University); and compound 44 (M. Morr, Braunschweig). All nucleotides in this study showed a purity of >98-99.5% and less than 0.1% cGMP contamination, as determined by analytical HPLC using a 250 × 4-mm, 10-µm LiChrosorb RP 18 column (Merck).
Synthesis-- Previously reported methods were used to synthesize compounds 1 (25), 2, 4, 5, 28, 29 (26), 39, 40, 41 (27), 35 (28), 42 and 48,2 and 45 (29). Compounds 9, 11-18, 20-26, 34 were prepared with modifications to the method of Michal et al. (30).
Preparation of 06-Benzyl-guanosine and O6-Benzyl-cGMP (3)-- O6-Benzyl-cGMP (3) was synthesized according to the methods of Chae et al. (31) and Genieser et al. (26) with significant modifications. In brief, O6-benzyl-guanosine was prepared by reacting 2-NH2-6-Cl-purine riboside (1.7 g, 5.63 mmol) with sodium benzoxide (6.21 mmol) in 20 ml of dry benzyl alcohol. The alkylating reagent was generated by pretreatment of the alcohol with NaH (149 mg, 6.2 mmol) at 0-25 °C for 2 h. This mixture was stirred at room temperature for 30 h and then evaporated to dryness. The residue was resuspended in 200 ml of water, neutralized with 1 N HCl, and the volume reduced to approximately 100 ml by evaporation. Upon cooling, a white precipitate of crude product was filtered off, washed twice with 20 ml of cold water, and dried to give analytically pure O6-benzyl-guanosine (1.971 g, 5.24 mmol, 93%) as a white solid.
1H NMR (Me2SO-d6),Preparation of N2,3-Etheno-O6-benzyl-GMP (6)-- Syntheses of compounds 6 and 10 were based on the method of Kusmierek et al. (32). In brief, O6-benzyl-cGMP (3) (137.1 mg, 300 µmol) was mixed with sodium acetate, bromoacetaldehyde, and ethanol and stirred in darkness at 37 °C for 7 days. Analytical HPLC indicated 95% conversion of the starting material and a major product signal (70%) of a compound with enhanced hydrophobicity. Evaporation of all volatile components under vacuum gave a yellow residue, which was purified using preparative HPLC (solvent: 30% methanol, 70% water, 40 mM TEAF (pH 7.0)). The sample was freeze-dried three times and neutralized with 0.1 N NaOH to yield 116 µmol (37%) analytically pure N2,3-etheno-O6-benzyl-GMP (6) (>98.5% purity).
1H NMR (Me2SO-d6),Preparation of N2,3-Etheno-cGMP (10)-- Compound 6 (80 µmol) was added to 10 ml of 70% methanol (in water) in the presence of 10% palladium on charcoal (50 mg); the sample was completely hydrogenated after 40 min at room temperature, as observed by analytical HPLC. The catalyst was filtered off, extracted 3 times with 70% methanol, and then extracted 3 times with toluene/methanol/water (5:5:1, v/v/v). The solvents were evaporated under vacuum at 20 °C and purified by preparative HPLC (solvent: 15% methanol, 85% water, 20 mM TEAF (pH 7.0)) to yield 52 µmol of 10 (97% purity). A purity higher than 99.3% with no detectable cGMP contamination was obtained after two additional chromatographic separations under identical conditions. Freeze-drying and neutralization with 0.01 N NaOH gave 27.5 µmol (34%) product.
1H NMR (Me2SO-d6),Analytic Section-- 1H and 31P NMR, as well as FAB-MS analyses, were used to confirm the structure of each cGMP analog. 1H and 31P NMR spectra (internal standard Me4Si and external H3PO4 85%, respectively) were recorded with a Bruker WH-360 spectrometer. Mass spectra were obtained with a Finnigan MAT spectrometer (model 8222) in the FAB mode and glycerol or nitrobenzyl alcohol as matrix. UV spectra were recorded with a Milton Roy Spectronic 1204 photometer in aqueous buffer at pH 7.0.
Photoreceptor PDE Preparation--
Rod outer segments (ROS) from
bullfrog (Rana catesbiana) retinas were purified on a
Percoll gradient in total darkness as described previously (33).
Briefly, ROS were isolated from frog retinas by gentle shaking and
purified on a discontinuous gradient containing 60, 30, and 5% Percoll
in a Ringer's solution consisting of (in mM): 105 NaCl,
2.0 MgCl2, 2.0 KCl, 1.0 CaCl2, and 10 HEPES, pH
of 7.5. Purified ROS were washed once in Ringer's to remove Percoll
and resuspended in Buffer A, containing (in mM): 77 KCl, 35 NaCl, 2.0 MgCl2, 1.0 CaCl2, 10 HEPES, and 1.2 EGTA (pCa 300 nM), pH 7.5; the ionic composition of Buffer
A partially mimicked that of frog ROS cytosol. The ROS suspension was
homogenized 12 times with a Potter-Elvehjem tissue grinder to
completely disrupt cellular morphology. Endogenous cGMP initially bound
to PDE would interfere with the cGMP binding assay; this problem was
avoided by incubating homogenized ROS suspensions at room temperature for 30 min, during which time >95% of the bound cGMP dissociated from
PDE and was hydrolyzed by the PDE activity in the homogenate (33). The
ROS extract was stored in the dark at 4 °C until use. Binding of
cGMP to photoreceptor PDE was determined without further purification
of the protein, since it has been previously demonstrated that PDE is
the only high affinity cGMP binding protein in frog ROS extracts (33).
The concentration of PDE holoenzyme was determined by measuring the
rhodopsin concentration by difference spectrophotometry (34) and
knowledge of the molar ratio of PDE to rhodopsin (0.003 mol of PDE
holoenzyme per mol of rhodopsin). The subunit stoichiometry of the PDE
holoenzyme in our frog ROS homogenates is assumed to be
2 based on the following considerations. 1) When
saturating levels of [3H]cGMP (1 µM) were
used in binding assays as controls, we routinely observed 1.8-2.1 mol
of cGMP bound per mol of PDE holoenzyme, in agreement with our previous
measurements of the binding site density of frog PDE (11, 33). Since
PDE which lacks P
subunits cannot bind cGMP at detectable levels
(11, 12), we infer that
90% of the PDE molecules must have bound
P
subunits. 2) Measurements of the PDE hydrolytic activity of frog
ROS homogenates (12 nM PDE) show <3% of the activity of
fully activated PDE (induced by either transducin activation or limited
trypsin proteolysis of the P
subunit). This too indicates that
>97% of the PDE catalytic subunits is associated with P
.
Filter Binding Assay-- The extent of equilibrium binding of [3H]cGMP in the presence of varying levels of cyclic nucleotide analogs was determined with a modification of a previously used filter binding assay (33). Portions of homogenized ROS (4 µM rhodopsin, 12 nM PDE holoenzyme) were mixed with 60 nM [3H]cGMP (6.64 × 1016 dpm/mol; prepared in Buffer A) which had been premixed with the indicated concentration of a cyclic nucleotide analog. This concentration of [3H]cGMP was chosen such that ~50% of the high affinity noncatalytic sites on PDE would be occupied in the absence of competing nucleotides. Within 5 min after the addition of 60 nM [3H]cGMP, ~90% of the maximum extent of binding was observed; no change in the extent of binding was observed between 10 and 60 min of incubation. The approach to equilibrium was not delayed when the competing nucleotide analog was added at the same time as cGMP.
After incubation at 24 °C for 15 min, samples were applied to a 96-well Multiscreen filter plate prewet with Buffer A, and the filters were rinsed with 300 µl of the same solution. The filters were punched out directly into scintillation vials, mixed with 100 µl of water, vortexed, and then mixed with 3 ml of scintillation fluid, and radioactivity was determined in a scintillation counter. To determine the extent of nonspecific entrapment of [3H]cGMP by the filters, ROS were mixed with [3H]cGMP containing 1 mM nonradioactive cGMP and treated as above. Less than 1% of the radioactivity was nonspecifically retained in the presence of ROS membranes; similar levels of nonspecific binding were observed when [3H]cGMP alone was passed through the filter and rinsed. Data analysis of the nucleotide displacement curves was performed assuming a single class of noninteracting sites to which cGMP and the nucleotide analogs both bound in a competitive fashion. The data analysis programs Sigmaplot and RADLIG (37) each provided similar values for the binding parameters. The Ki value was calculated from the IC50 value obtained from the displacement curve using the equation (38), Ki = IC50(1 + KD/[cGMP]), where the dissociation constant, KD, for cGMP binding to PDE is 60 nM (11, 33). The Ki values presented in Tables I-V represent the mean ± S.D. for three or four independent experiments for each analog. For all pairwise comparisons of cGMP analogs discussed in the text, the Ki values were determined to be statistically significant at the p < 0.005 level of significance using a paired t test. ![]() |
RESULTS AND DISCUSSION |
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We chose a set of chemically modified cGMP analogs (Fig.
1) to probe in detail the structural
requirements for cGMP binding to the noncatalytic cGMP binding sites of
frog rod PDE. Since the rod PDE consists of two non-identical and
catalytic subunits, we first addressed the question of whether we
could distinguish two distinct noncatalytic binding sites in our
experiments. Previous equilibrium binding data at sub-micromolar cGMP
concentrations have provided no evidence for two distinct classes of
high affinity sites in nonactivated PDE (11, 33). In addition, the
determination of the Ki for cIMP was carried out at
several different [3H]cGMP concentrations that resulted
in 0.2, 0.4, 1.0, or >1.8 mol cGMP bound per mol of PDE holoenzyme in
the absence of the cGMP analog; no significant difference was found in
the Ki value as a function of the extent of
occupancy by cGMP.4
Furthermore, in those instances where the [3H]cGMP
concentration was high enough to saturate all high affinity sites on
PDE, addition of cGMP analogs never displayed complex displacement
behavior. These observations support the notion that the two
noncatalytic binding sites on non-activated PDE holoenzyme cannot be
distinguished by the equilibrium binding measurements performed in this
study. In addition, binding of [3H]cGMP to the catalytic
sites of PDE can been ruled out for two reasons as follows: 1) the low
concentration of [3H]cGMP used was 1000-fold lower
than the KM of the enzyme, and 2) the
inclusion of 1 mM zaprinast (a competitive inhibitor of
cGMP hydrolysis) effectively blocked [3H]cGMP binding and
catalysis at the active site of PDE.
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Substitutions in the N-1/C-6 Region of the Purine Ring-- We first tested the ability of cGMP analogs with alterations in the C-6 and/or the N-1 positions of the cGMP molecule (Fig. 1B) to bind to the noncatalytic sites of photoreceptor PDE, since these atoms differ between cGMP and cAMP. Purified frog ROS extracts were incubated with 60 nM [3H]cGMP and various concentrations of a cGMP analog. After the binding reaction had reached equilibrium, the extent to which [3H]cGMP was displaced by the analog was quantitated by a filter binding assay. Fig. 2 shows typical results for several analogs substituted at the N-1 or the C-6 positions of the purine ring; displacement of [3H]cGMP by nonradioactive cGMP is also included for comparison. We found that 6-mercapto-cGMP (1) was able to bind to the noncatalytic sites nearly as well as cGMP itself, as judged by its ability to prevent [3H]cGMP binding at low concentrations (Table I). This suggests that the electronegativity of the oxygen attached to position 6 in cGMP stabilizes binding to the binding pocket of PDE and demonstrates that the mercapto group retains the ability to interact with the binding pocket. Although compound 1 can potentially exist in both a keto and enol tautomeric form (Fig. 1B, inset), existing evidence suggests that thio-substituted nucleosides exist almost exclusively in the keto (thio-oxo) form (39).
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Pyrimidine Nucleotides-- We examined whether cyclic nucleotides that contained a pyrimidine ring instead of a purine ring could occupy the binding pocket of the photoreceptor PDE. Neither thymidine 3',5'-cyclic monophosphate, uridine 3',5'-cyclic monophosphate, nor cytidine 3',5'-cyclic monophosphate were found to compete with [3H]cGMP for binding to PDE at concentrations up to 5 mM (data not shown). We conclude that there is an absolute requirement for the purine ring structure in order for a cyclic nucleotide to bind to the noncatalytic site of photoreceptor PDE.
Substitutions in the C-2/N-3 Region of the Purine Ring-- Since the amino group at the C-2 position is of primary importance in discriminating cAMP from cGMP in other binding proteins, it was important to determine the factors involved in stabilizing the preference for cGMP binding over cAMP in photoreceptor PDE. Nineteen cGMP derivatives modified at the C-2 position (Fig. 1C) were tested; of these, compounds 10 and 19 also resulted in changes at the N-3 position. Analysis of the binding results of cGMP analogs substituted at the C-2 position can most easily be done by categorizing the compounds according to H bonding potential, bulkiness, and the functional group of the substituent.
In the first group, several compounds were tested, having a formal loss of H bonding donor potential in position 2. Replacement of the 2-NH2 group with hydrogen (cIMP; 8) caused the least (11-fold) decrease in binding (Table II). This indicates that the interaction between the amino group and the binding pocket is not essential for high affinity binding and is consistent with other regions of the cGMP molecule providing the crucial stabilizing interactions with the binding pocket. Note that the dipole size and the angle of cGMP compared with cIMP are extremely similar; the angle differs less than 2° (41). Therefore, cIMP mimics cGMP with respect to electron density and localization within the purine ring. Nonetheless, there remains strong evidence that the presence of a hydrogen atom at the N2 position helps to stabilize binding of cGMP analogs to the noncatalytic site. Substitution of both hydrogens at the N2 position with methyl groups (N2-(CH3)2-cGMP; 26) results in a compound that is not tolerated by the binding pocket. However, N2-monomethyl-cGMP (9) retains H bonding potential and binds only 30-fold less well than the parent cGMP molecule. The 70,000-fold difference in binding between compounds 26 and 9 can be attributed to two factors as follows: 1) the total loss of H bonding donor capacity, and/or 2) the possibility that steric constraints from a second methyl group cannot be tolerated. The fact that the dimethyl analog (26) and 2-piperidino-cIMP (25) are both completely unable to bind (Table II) is also likely to be due to a combination of the following: (1) spatial constraints by the binding pocket in the immediate vicinity of the amino group of cGMP, and (2) a lack of hydrogen bond formation between the binding pocket and an N2 hydrogen. Both cXMP (19) and 2-Cl-cIMP (21) bound relatively poorly to the noncatalytic binding pocket of PDE (Table II). In both cases, the replacement of the NH2 group with an electronegative atom results in a 220- or 280-fold reduction in binding affinity, respectively. We conclude from these results that the presence of one hydrogen atom at the N2 position permits a favorable interaction with the binding pocket. In the absence of this interaction cyclic nucleotide binding can still occur, albeit with reduced affinity. Another group of 2-substituted cGMP analogs varying in the chain length and the type of functional group were synthesized to determine whether spatial factors, polarity, or the type of charge of the cGMP derivative affected the ability of these analogs to bind to the PDE noncatalytic binding pocket. For the case of three comparisons of short versus longer chain length substituents at the C-2 position (24 versus 15, 17 versus 11, and 20 versus 16), the longer alkyl group demonstrated a statistically significant higher binding affinity than the shorter alkyl chain. The moderate binding of N2-phenyl-cGMP (12), N2-biphenyl-cGMP (13), and N2-benzyl-cGMP (18) demonstrates that overall bulkiness is not the limiting factor for tight binding at the C-2 position; these three compounds are bulky but still retain their capacity for H bonding. The fact that methyl, ethyl, phenyl, biphenyl, and hexyl substituents at position 2 all bound with moderate affinity indicates that this region of the binding pocket is not spatially constrained, at least in one of the directions occupied by the two N2 hydrogen atoms in the cGMP molecule. Different functional groups were also introduced at the N2 position to test the effects of changing the charge and/or polarity in this region of the binding pocket. Replacing a hydrogen atom at N2 with a methyl group (9) caused only a 30-fold decrease in binding affinity. In contrast, N2-(5-carboxy-pentyl)-cGMP (22) and N2-(2-carboxy-ethyl)-cGMP (23) bound with a 300-340-fold reduction in affinity compared with cGMP. Carboxyl-containing derivatives 22 and 23 behaved similarly to electronegative analogs (i.e. 19 and 21) in terms of their relative binding affinity. In contrast, cGMP analogs containing a positively charged amino group (11 and 17) or a methoxy group (14) were better tolerated than other functional groups tested. It appears that either a negative charge or an electronegative atom in this region of the binding pocket destabilizes binding. Two derivatives were tested that altered the N-3 position in addition to changing the C-2 substituent. N2,3-etheno-cGMP (10) contains a five-member ring joining positions C-2 and N-3 yet binds relatively well to the binding pocket of PDE. The reduction in binding affinity of this compound is comparable in magnitude to other analogs with similar alterations only at C-2, suggesting that the N-3 position does not specifically interact with the binding pocket. cXMP (19) results in protonation of N-3 in addition to replacement of the 2-NH2 group with an oxo group, but in this case it is likely that the introduction of the electronegative oxygen, resulting in the loss of H bonding donor potential, may be the primary factor in the binding behavior of this analog. In summary, there is no evidence to indicate that the N-3 position forms an interaction with the noncatalytic binding pocket of PDE. We conclude that the C-2 position of the purine ring is a significant but secondary determinant for binding specificity of cGMP to noncatalytic sites on photoreceptor PDE. The presence of at least one hydrogen atom at N2 can stabilize cyclic nucleotide binding but is not an absolute requirement. The binding pocket tolerates a variety of substitutions reasonably well, which suggests that this region of the binding site is not spatially constrained. The neighboring N-3 position does not play a role in stabilizing nucleotide binding to the noncatalytic site of PDE.Substitutions to the N-7 and C-8 Positions of the Purine Ring-- In contrast to some other cGMP binding proteins in which substitutions at the C-8 position result in a higher binding affinity than cGMP itself (e.g. cGMP-dependent protein kinases (15, 42, 43) and the cGMP-gated ion channel (17, 19)), almost all substitutions to this position of the purine ring significantly destabilize cGMP binding to photoreceptor PDE noncatalytic sites (Table III).
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Substitutions in the Cyclic Phosphate Ring-- Seven derivatives of cGMP with modifications in the cyclic phosphate portion of the molecule were tested for their interaction at the noncatalytic binding site of PDE (Table IV). Two compounds, guanosine-3',5'-cyclic monophosphorodimethylamidate (Rp isomer) (42) and 5'-GMP (43), were completely unable to bind. For compound 42, a combination of steric factors, loss of potential ionic or hydrogen bonding interactions, and/or an increase in hydrophobicity in this region may account for its inability to occupy the binding pocket. In the case of 5'-GMP, no displacement of [3H]cGMP could be detected when the 5'-GMP was raised to 50 mM (106-fold discrimination), demonstrating that the cyclic phosphate structure is absolutely essential for nucleotide binding to the noncatalytic site.
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Substitutions in the Ribose Moiety--
Three different positions
of the ribose ring were examined for the possibility of specific
interactions with the noncatalytic binding pocket
(Table V). Replacing an oxygen at the
3'-position of the ribose ring with a secondary amino group
(3'-NH-cGMP, 44) had no effect on the binding affinity,
whereas a similar substitution at the 5'-position (5'-NH-cGMP,
45) reduced binding affinity 11-fold. Introducing an amino
group at the 5'-position may reduce the favorable ionic or hydrogen
bonding interaction of the axial exocyclic oxygen with the binding
pocket. This notion is consistent with the results of the previous
section with thio-substituted analogs. This difference in binding
affinity between 44 and 45 is also observed for
the cGMP-stimulated PDE from bovine adrenal tissue (22), whereas the
cGMP-dependent protein kinase type I (15) and the
cGMP-stimulated PDE from Dictyostelium discoideum (44) bound
both nucleotides with similar affinities.
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Evidence Favoring cGMP Binding to the Noncatalytic Site in the
Anti-conformation--
The orientation of the purine base relative to
the ribose sugar is determined by the torsion angle about the
glycosidic bond, with the main conformations referred to as
syn or anti (39). Both crystallographic (45) and
computational studies (46) support the idea that cGMP prefers the
syn-conformation over the anti-conformation.
Current evidence suggests that the formation of an intramolecular H
bond between the amino group on the C-2 position and the axial
phosphate oxygen atom contributes to stabilizing the
syn-form of cGMP compared with the
anti-conformation (20, 39, 40).
Comparison of the Noncatalytic and Catalytic cGMP Sites on Photoreceptor PDE-- A comparison of this study with the effects of cGMP analogs on the catalytic site of bovine rod photoreceptor PDE (20) clearly highlights the much greater degree of nucleotide discrimination at the noncatalytic site. Several analogs bind nearly as well as cGMP to the catalytic site, including 2'-deoxy-cGMP (46), cIMP (8), 8-Br-cGMP (29), 6-SH-cGMP (1), and 3'-NH-cGMP (44). Of this group of analogs, only 1, 8, and 44 show a similar relative affinity for the noncatalytic site. Compounds 46 and 29 bind with 27- and 220-fold lower affinity to the noncatalytic site than cGMP, respectively (20). In marked contrast to the inability of cAMP to bind at the noncatalytic cGMP binding sites on PDE (Table I), the KM for cAMP hydrolysis is only 20-fold greater than the KM for cGMP breakdown (51).
In terms of individual positions of the cGMP molecule, the catalytic (20) and noncatalytic sites of the photoreceptor PDE both appear to use the C-6 oxo group to interact with a hydrogen bond donor in the binding pocket. The N-1 position of the purine ring is also involved in stabilizing cGMP binding at both catalytic and noncatalytic sites. In other portions of the cGMP molecule, however, the two binding sites exhibit different properties. 1) Binding of cGMP to the catalytic site is not significantly stabilized by the amino group at the C-2 position (20), in contrast to the noncatalytic site where the majority of analogs tested bound >100-fold less well than cGMP (Table II). 2) Substitutions at the 8-position have little effect on the ability of analogs to bind to the catalytic site (20), except in the case of 8-OH-cGMP (36) which may be exerting its effects via protonation of the N-7 position (Fig. 1D, inset) or via changes in the electron density distribution of the purine ring. In contrast, almost all substitutions at the C-8 position adversely affect nucleotide binding to the noncatalytic site (Table III). 3) Substitutions at the 2'-position on the ribose ring have only minor (<2-fold) effects on catalytic site binding (20), whereas identical cGMP derivatives have 30-60-fold lowered affinity to the noncatalytic sites (Table V). In summary, the catalytic site of the bovine rod photoreceptor PDE stabilizes cGMP binding via favorable interactions at N-1, C-6, and N-7 on the purine ring. It may bind cGMP in the syn-conformation (20). The C-2, C-8, and C-2' positions do not appear necessary for cGMP binding to the catalytic site, whereas binding to the noncatalytic site is stabilized by interactions of these positions of the cGMP molecule with the binding pocket.cGMP Interactions with other Noncatalytic PDE Binding Sites-- In addition to the photoreceptor PDE, both the cGMP-stimulated PDE (PDE2) and the cGMP-specific PDE (PDE5) have noncatalytic cGMP binding sites that have been probed with cGMP derivatives (9, 22, 24). Qualitatively, there is good agreement among the three different noncatalytic cGMP binding sites regarding structural requirements for cGMP binding. Specifically, PDE2, PDE5, and the photoreceptor PDE all share favorable interactions between the noncatalytic binding pocket and the C-6, N-1, C-2, and C'-2 positions, as well as poorly tolerating substitutions at the C-8 position. However, the photoreceptor PDE shows a great deal more discrimination for cGMP over other nucleotides than the other PDEs examined to date. For example, cAMP is able to bind approximately 50-100-fold less well to the noncatalytic sites of PDE2 (22, 23) and PDE5 (52, 53) compared with the ~106 level of discrimination of cAMP from cGMP at the photoreceptor noncatalytic binding site. In addition, cPUMP (5) (22), 8-OH-cGMP (36) (9), and 8-CH3-cGMP (30) (24) all bind considerably less well to the photoreceptor PDE than to PDE2 or PDE5. There is also a notable lack of stereospecificity in the binding of (Rp)- (37) and (Sp)-cGMPS (38) to the photoreceptor enzyme (Table IV; see also Ref. 54) compared with PDE2 (22).
Because cGMP binding assays have been performed under a variety of conditions, direct comparisons of the intrinsic binding affinity of cGMP are difficult. Nonetheless, the photoreceptor PDE is likely to have more favorable binding interactions between the noncatalytic site and the cGMP molecule than PDE2 or PDE5. This can be inferred from the value of the dissociation constant for cGMP observed for amphibian rod photoreceptor PDE (60 nM (11)), mammalian cone (10 nM (54)), or rod photoreceptor PDE (sub-nanomolar (55)). In contrast, PDE2 and PDE5 have dissociation constantsConclusions-- The principal finding of this paper is that the noncatalytic cGMP binding pocket on the photoreceptor PDE interacts in a highly specific manner with the purine ring (principally at C-6/N-1, as well as at C-2 and C-8), the ribose ring (at the 2'-position), and with the cyclic phosphate ring of cGMP (Fig. 3). Furthermore, current evidence favors binding of cGMP in the anti-conformation to the binding pocket. Although the general binding motifs are similar to those of the PDE2 and PDE5 noncatalytic binding sites, the photoreceptor enzyme discriminates cyclic nucleotides more precisely and binds cGMP with higher intrinsic affinity than do the other cGMP-binding PDEs. In comparison to the catalytic site of the photoreceptor PDE (Beltman et al. (20)), the noncatalytic binding site interacts at more distinct sites with the cGMP molecule, leading to higher affinity interactions and more precise discrimination of other cyclic nucleotides.
A limitation to this approach of determining the structural features of the noncatalytic binding pocket is that a particular cGMP analog might contain a chemical group that interacts favorably with the binding pocket in a way that does not occur with cGMP itself. In this case, the apparent binding affinity of the analog would not provide information about cGMP binding interactions with the noncatalytic binding pocket. However, our use of several chemical modifications at each tested position of the cGMP molecule reduces the likelihood of misinterpreting the effects of a single substitution in terms of cGMP binding interactions with the noncatalytic binding site on PDE. Ultimately, structural information on the identity of the individual amino acids in the noncatalytic binding pocket that interact with each distinct region of the cGMP molecule must await functional expression and site-directed mutagenesis of the photoreceptor PDE, as has been initiated with PDE5 (53, 56, 57). ![]() |
FOOTNOTES |
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* This work was supported by National Institutes of Health Grant EY-05798 (to R. H. C.), by Deutsche Forschungsgemeinschaft Grant Ja 246/6-1 (to B. J.), and the Fonds der Chemischen Industrie, Germany (to B. J.). This paper is Scientific Contribution no. 1970 from the New Hampshire Agricultural Experiment Station.The costs of publication of this article were defrayed in part by the payment of page charges. The 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: Dept. of Biochemistry and Molecular Biology, University of New Hampshire, 46 College Rd., Durham, NH 03824-2617. Tel.: 603-862-2458; E-mail: Rick.Cote{at}unh.edu.
1 The abbreviations used are: PDE, phosphodiesterase; ROS, rod outer segments; (Rp)-cGMPS, guanosine 3',5'-cyclic monophosphorothioate (Rp isomer); (Sp)-cGMPS, guanosine 3',5'-cyclic monophosphorothioate (Sp isomer); cPUMP, purine 3',5'-cyclic monophosphate; TEAF, triethylammonium formate; PDE2, cGMP-stimulated PDE; PDE5, cGMP-binding, cGMP-specific PDE; HPLC, high pressure liquid chromatography; FAB-MS, fast atom bombardment mass spectroscopy.
2 B. Jastorff, unpublished results.
3 M. R. D'Amours and R. H. Cote, manuscript in preparation.
4 S. Thapar and R. H. Cote, unpublished observations.
5 F. Schwede, unpublished observations.
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