Identification of the Amino Acid Sequences Responsible for High Affinity Activation of cGMP Kinase Ialpha *

(Received for publication, November 11, 1996, and in revised form, February 11, 1997)

Peter Ruth Dagger , Alexander Pfeifer , Simone Kamm , Peter Klatt , Wolfgang R. G. Dostmann and Franz Hofmann

From the Institut für Pharmakologie und Toxikologie der Technische Universität München, Biedersteiner Straße 29, D-80802 München, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The cGMP-dependent protein kinases (cGK) Ialpha and Ibeta have identical cGMP binding sites and catalytic domains. However, differences in their first 100 amino acids result in 15-fold different activation constants for cGMP. We constructed chimeras to identify those amino acid sequences that contribute to the high affinity cGK Ialpha and low affinity cGK Ibeta phenotype. The cGK Ialpha /Ibeta chimeras contained permutations of six amino-terminal regions (S1-S6) including the leucine zipper (S2), the autoinhibitory domain (S4), and the hinge domain (S5, S6). The exchange of S2 along with S4 switched the phenotype from cGK Ialpha to cGK Ibeta and vice versa, suggesting that the domains with the highest homology between the two isozymes determine their affinity for cGMP. The high affinity cGK Ialpha phenotype was also obtained by a specific substitution within the hinge domain. Chimeras with the sequence of cGK Ialpha in S5 and cGK Ibeta in S6 were activated at up to 6-fold lower cGMP concentrations than cGK Ialpha . Based on the activation constants of all chimeras constructed, empirical weighting factors have been calculated that quantitatively describe the contribution of the individual amino-terminal domains S1-S6 to the high affinity cGK Ialpha phenotype.


INTRODUCTION

The amino terminus of the cGK1 regulates several important functions such as the dimerization of the two cGK subunits, the inhibition of the catalytic center in the non-active enzyme, the affinity of the binding sites for cGMP, and the concentration of cGMP required for activation of the enzyme (Refs. 1-5; for reviews see Refs. 6-8). The amino acid sequences responsible for these multiple functions of the amino terminus have been partially identified. The leucine zipper, a sequence of heptad repeats of leucines and isoleucines forming an alpha -helical structure at the first part of the amino terminus (9, 10), has been identified as the dimerization domain. Removal of the leucine zipper along with the autoinhibitory domain (residues 1-78) of cGK Ialpha results in a permanently active and monomeric enzyme, which still binds cGMP to its two binding sites but with lower affinity than the native enzyme (11). The removal of the leucine zipper in cGK Ibeta (residues 1-62) results in a monomeric cGK that is still activated by cGMP (2), suggesting that the autoinhibitory domain resides in a sequence that is carboxyl-terminal to the leucine zipper. The autoinhibitory sequences contain the main in vitro autophosphorylation sites of cGK Ialpha (12) and cGK Ibeta (13).

To date it has remained unclear which amino acid sequences of the amino terminus regulate the binding affinity for cGMP (1, 3). The two amino-terminal splicing forms of the cGK type I, Ialpha and Ibeta , which have been identified by cDNA cloning (14, 15) and protein purification (16, 17), differ only in their first 89 and 104 amino-terminal residues, respectively. Although these amino termini do not contain the cGMP binding sites, the two isozymes differ 15-fold in their affinity for cGMP. Cyclic GMP binds to a high and a low affinity site with Kd values of 10 and 150 nM in cGK Ialpha (3, 18), but to two low affinity sites with Kd values of 150 nM in cGK Ibeta (3). These differences in cGMP binding of the two isozymes are also reflected in the dissociation kinetics. Cyclic GMP dissociates with a fast and a slow rate from cGK Ialpha , but only with a fast rate from cGK Ibeta . The decreased binding affinity of the cGK Ibeta results in a 15-fold increase in the cGMP concentration needed for half-maximal activation when compared with cGK Ialpha (3, 19). To identify those amino acid sequences within the amino terminus that determine the affinity of the isozymes for cGMP, chimeras of cGK Ialpha and cGK Ibeta were constructed. The analysis of these chimeras shows that the leucine zipper and the autoinhibitory domain mainly affect the phenotype.


EXPERIMENTAL PROCEDURES

Materials

[Ser21]protein kinase inhibitor-(14-22) substrate peptide and protein kinase inhibitor-(5-24) amide were synthesized as described (20). The expression vector pMT3 was obtained from R. Kaufmann, Genetics Institute, Cambridge, MA.

Cloning of a Full-length Clone of cGK Ibeta from Bovine Testis

An oligo(dT)-primed, size-fractionated cDNA library was constructed in the pcDNAII vector. Colonies (3 × 105) were screened with a 980-base pair cDNA fragment (nucleotides 1635-2616) from mouse cGK II (21). Hybridization was performed under low stringency (50 °C). A clone with an insert of 2.5 kilobase pairs was identified as cGK Ibeta . This clone contained the complete coding region of cGK Ibeta and confirmed the sequence composed of partial clones (14).

Construction of Recombinant cGK Ibeta Baculovirus

The cGK Ibeta cDNA containing the consensus sequence for optimal expression in Sf9 cells was ligated into pFastBac1 yielding the bacmid transfer vector pFB1/cGKIbeta . Transformation of pFB1/cGKIbeta into Max Efficiency DH10Bac cells, identification of recombinant clones, and isolation of the recombinant bacmid DNA were performed according to the instructions of the Bac-to-Bac baculovirus expression kit (Life Technologies, Inc.). Recombinant bacmid DNA was transfected into Sf9 cells. Recombinant viruses were identified by their ability to direct the expression of cGK Ibeta . Recombinant virus was amplified without further purification and viral titer estimated by end point dilution.

Expression and Purification of Recombinant cGK Ibeta

Suspension cultures (1.3 × 1010 Sf9 cells/7.2 liters) were infected at a multiplicity of infection of 10. After 72 h, cells were harvested by centrifugation, washed twice with serum-free TC-100 medium, resuspended in buffer A (20 mM Tris/HCl, pH 7.4, containing 100 mM NaCl, 2.5 mM DL-dithiothreithol, 2.5 mM benzamidine, 0.1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin), and stored at -80 °C. Cells were lysed by freeze/thawing, homogenized, and centrifuged. The supernatant was loaded onto an cAMP affinity column packed with cAMP-agarose (Sigma, catalog no. A7775) equilibrated with buffer A. The column was washed with buffer A prior to elution of bound cGK Ibeta with buffer A supplemented with 100 µM cGMP. The eluate was dialyzed against 20 mM TES buffer (pH 7.1), containing 2.5 mM DL-dithiothreithol, 0.1 mM sodium azide, and 1 µg/ml leupeptin. The purified protein was concentrated to ~4 mg/ml using Macrosep-10 concentrators (Pall Filtron), made up 50% (v/v) in glycerol and stored at -20 °C. The cGK Ialpha was expressed (22) and purified (23) as described.

Construction of Chimeric cGMP Kinases and Expression in COS Cells

The coding DNA sequence of cGK Ialpha (nucleotides -6 to 2120) and cGK Ibeta (nucleotides -23 to 2165) were constructed into the eukaryotic expression vector pMT3 (24). For constructing chimeras, replacement sequences containing amino-terminal domains of cGK Ialpha and cGK Ibeta with the boundaries as given in Fig. 2 were generated by standard polymerase chain reaction overlap extension techniques. All chimeras exhibited the Koczak consensus sequence ACC (nucleotides -3 to -1) prior to the ATG start codon. COS-7 cells were transfected, and cytosolic proteins containing recombinant cGKs were extracted as described previously (3).


Fig. 2. Alignment of the cGK Ialpha and cGK Ibeta amino termini and the domains used for construction of chimeras. The bovine cGK Ialpha (residues 1-89) and cGK Ibeta (residues 1-104) amino termini and a segmental pictogram of amino-terminal domains in cGK Ialpha and cGK Ibeta are shown. The common sequences of cGK Ialpha and cGK Ibeta start from Ser90 and Ser105, respectively, and include the cGMP binding domains A and B, the ATP and the substrate binding sites, and the carboxyl-terminal domain. Identical amino acids are in bold letters and indicated by vertical lines. Conservatively substituted amino acids are indicated by dashed lines. The amino terminus is subdivided into the six domains S1-S6. The first two repeats of the leucine zipper of cGK Ibeta showed no homology to cGK Ialpha and were therefore assigned to S1. L/I zipper stands for leucine/isoleucine zipper.
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Ka Determination

The activity of the expressed Ialpha , Ibeta , and chimeric cGK was measured as described (3). Ka values were obtained by a nonlinear fit of the data according to the Hill equation.

Equilibrium Binding of cGMP and Determination of Dissociation Rate Constants

Equilibrium binding of [3H]cGMP to the expressed cGK proteins was carried out as described (3). Binding of [3H]cGMP to sites A and B was determined as described (1). Calculation of the Kd values and the maximal binding capacities was performed by the LIGAND program (25). The stoichiometry of cGMP binding was calculated by dividing the maximal binding capacity obtained from Scatchard analysis by the amount of cGK present in the cytosolic extract from COS cells. The concentration of cGK in COS cell extracts was determined by the phosphotransferase activity using the specific activity of 2.4 ng of pure cGK added to an identical amount of cytosolic extract from control COS cells. The 2.4 ng of cGK yielded a specific activity of 4 µmol of phosphate transferred/min/mg of pure cGK. Dissociation rate constants were measured as described (3).

Calculation of Weighting Factors

Weighting factors were obtained by an iterative procedure using chimeras 1-19 and optimized by one-dimensional interpolation.

Statistics

Data are means ± S.E. of 5-17 (Ka) and 3-5 (k-1(fast), k-1(slow)) determinations from at least three different expressions. Statistical significance was determined by Student's t test for paired and unpaired data. A p value less than 0.05 was considered to be significant.


RESULTS

Cloning and Basic Properties of Purified cGK Ibeta and cGK Ialpha

A full-length cDNA for the cGK Ibeta was cloned from bovine testis that confirmed the previously published cDNA sequence of bovine cGK Ibeta deduced from partial clones (14). The cloning of a full-length cDNA excluded the possibility that the cGK Ibeta differed from the cGK Ialpha isozyme in regions other than the amino-terminal domain. In contrast, the previously published human cGK Ibeta sequence (15) differed from the bovine full-length clone at Lys280 and Asn290, which were replaced in the human cGK Ibeta by Thr280 and Ser290, respectively.

To demonstrate that the differences in cGMP binding and activation between cGK Ialpha and cGK Ibeta (3) were intrinsic properties of the isozymes, cGK Ialpha and cGK Ibeta were expressed in Sf9 cells, purified to homogeneity, and subsequently characterized. The phosphotransferase activity of cGK Ialpha was stimulated 7-fold by cGMP with apparent Ka and Vmax values of 59 nM and 6.2 µmol/min × mg, respectively (Fig. 1). The phosphotransferase activity of cGK Ibeta was stimulated 35-fold with apparent Ka and Vmax values of 1210 nM and 7.6 µmol/min × mg, respectively. The dissociation kinetics of cGMP from the cGK Ialpha clearly defined a fast dissociation rate from a low affinity site and a slow dissociation rate from a high affinity site (Fig. 1). In contrast, cGK Ibeta exhibited only fast dissociation from two low affinity sites. The corresponding k-1 values were 6.2 and 0.009 min-1 for cGK Ialpha and 5.3 min-1 for cGK Ibeta . Equilibrium binding experiments indicated that cGK Ialpha and cGK Ibeta bound 1.8 and 1.6 mol of cGMP/mol of subunit, respectively. The apparent Kd values were 98 and 7 nM for cGK Ialpha and 172 nM for cGK Ibeta . These results suggest that the binding and catalytic constants of pure cGK Ialpha and cGK Ibeta expressed in Sf9 cells were similar to those measured in the cytosolic extract of COS cells (3).


Fig. 1. Phosphotransferase activity and dissociation kinetic of purified recombinant cGK Ialpha and cGK Ibeta . Phosphotransferase activity (left) and dissociation rate (right) was determined as described under "Experimental Procedures" using 40 ng (phosphotransferase experiments) and 1 µg (dissociation experiments) of purified enzymes.
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Alignment and Subdivision of the cGK Ialpha and cGK Ibeta Amino Termini

To identify the structural basis of their 15-20-fold differences in the Ka values, the amino-terminal domains of cGK Ialpha and cGK Ibeta were aligned and subdivided into six domains, S1-S6 (Fig. 2): S1, the extreme amino-terminal sequence; S2, the leucine zipper containing 5 heptad repeats of leucines; S3, the short linker connecting the leucine zipper and the autoinhibitory domain; S4, the autoinhibitory domain containing the substrate-like sequence PRT59TR with the autophosphorylation site (Thr59) of cGK Ialpha (12) and the putative pseudosubstrate sequence KRQAISAE of cGK Ibeta (26); and S5, the first part, and S6, the second part of the hinge domain, which links the amino terminus to the other domains of the cGK. The hinge domain was subdivided since proteolysis at Arg78/Gln79 led to a monomeric and active cGK Ialpha , which had different cGMP binding properties than the intact cGK Ialpha (11), suggesting that the sequences amino-terminal from Gln79 considerably influence the binding properties of the enzyme.

Expression of cGK Chimeras in COS Cells

For the construction of chimeras, the six domains with the boundaries defined above were exchanged between cGK Ialpha and cGK Ibeta . Expression vectors carrying the coding sequence of cGK chimeras or that of cGK Ialpha and cGK Ibeta were transfected into COS cells. The chimeric and wild type cGK proteins were identified in the soluble extracts of transfected cells by an antibody directed against a carboxyl-terminal sequence of the enzyme (20). The mutant and wild type cGK had apparent Mr values of 75 kDa and were expressed at concentrations between 0.5 and 4.5 µg/mg of soluble protein, respectively, as assessed by their maximal cGMP binding capacities. All of the chimeras expressed comigrated with the pure cGK Ialpha and cGK Ibeta in 5-15% sucrose density gradient centrifugation with s20w values between 7.2 and 7.8, suggesting that they were dimers. Subsequently, the chimeras were characterized by the cGMP concentration needed for half-maximal activation (Ka) and the dissociation rate constants (k-1) of cGMP from the high and low affinity site.

The Leucine Zipper and the Autoinhibitory Domain Predominantly Affect the High Affinity cGK Ialpha and the Low Affinity cGK Ibeta Phenotype

The Ka values for cGMP of the cGK Ialpha and cGK Ibeta expressed in COS cells were 77 and 1748 nM, respectively (Fig. 3). Almost identical values were obtained with the pure isozymes (Fig. 1). Cyclic GMP dissociated with a slow rate (k-1 = 0.008 min-1) and a fast rate (k-1 = 6.5 min-1) from the high and low affinity site of the cGK Ialpha (Fig. 3). In contrast, only fast dissociation from the two low affinity sites was observed for cGK Ibeta (k-1 = 5.0). The two cGMP binding sites of cGK Ibeta could not be distinguished kinetically. Studying several chimeras with randomized permutations in the six amino-terminal domains, we identified the sequences necessary to generate the respective phenotype. Chimeras containing the sequence of cGK Ibeta in S2 and S4 (chimeras 1-5) exhibited Ka values between 580 and 1136 nM. These constants were only 1.5-3-fold lower than the Ka value of cGK Ibeta . The cGK Ibeta phenotype of chimeras 1 to 5 was further confirmed by monophasic dissociation with rate constants between 4.4 and 10 min-1. Substitutions of cGK Ialpha sequence in S1, S3, S5, and S6, alone or in combination, did only marginally affect the kinetic constants of chimeras 1-5. Conversely, chimeras 6-9 containing the cGK Ialpha sequence in S2 and S4 had Ka values between 112 and 169 nM. These values were only 1.4- and 2.2-fold above that of cGK Ialpha (Fig. 3). In agreement with the cGK Ialpha -like activation constants, these chimeras showed biphasic dissociation kinetics with a fast and a slow component (Fig. 3). The presence of cGK Ibeta substitutions in S1, S3, S5, and S6 did not substantially alter the high affinity cGK Ialpha phenotype of these chimeras. These results suggested that the leucine zipper along with the autoinhibitory domain were the main structural elements which determine the affinity of the cGK isozymes. Interestingly, these two domains are very homologous between cGK Ialpha and cGK Ibeta with 28 identical, 4 conservatively substituted, and 22 different amino acids (Fig. 2), suggesting that 29% and 25% of the total residues of the amino terminus (cGK Ialpha  = 89 and cGK Ibeta  = 104 amino acids) were sufficient to generate the high affinity cGK Ialpha and the low affinity cGK Ibeta phenotype, respectively.


Fig. 3. Activation constants and dissociation rate constants of cGK chimeras. The left part shows schematically the composition of the chimeras, where sequences from cGK Ialpha and cGK Ibeta are indicated by bullet  and down-triangle, respectively. The Ka values and the dissociation rate constants from the low (k-1(fast)) and high affinity binding site (k-1(slow)) were determined as described under "Experimental Procedures." The Ka values are means ± S.E. of 5-17 independent determinations from at least three transfection experiments. The k-1 values are means from at least two transfection experiments with the number of determinations given in parenthesis. Con stands for construct.
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Equilibrium Binding of cGMP to cGK Chimeras

Chimera 9 and 5 were exemplarily selected to measure the equilibrium binding of cGMP, because they fulfilled the minimal requirements for the high affinity cGK Ialpha and low affinity cGK Ibeta phenotype, respectively. Both chimeras bound about 4 mol of cGMP/mol of holoenzyme (Fig. 4). As expected, chimera 9 bound cGMP to sites 1 and 2 with apparent Kd values of 4.3 nM and 439 nM and a stoichiometry of 2.4 and 1.4 mol of cGMP bound/mol of holoenzyme, respectively. The dissociation induced by the addition of a 1000-fold excess of unlabeled cGMP yielded two rates of 0.008 and 6.5 min-1. Chimera 5 bound 4 mol of cGMP/mol of holoenzyme with an apparent Kd of 195 nM. A distinct high affinity site was not detected. These results suggested that chimera 5 contained two low affinity binding sites. This finding was confirmed by monophasic dissociation of cGMP with a rate constant of 4.8 min-1. These values are not different from the wild type enzymes and indicate, therefore, that S2 and S4 determine the high affinity cGK Ialpha and low affinity cGK Ibeta phenotype.


Fig. 4. Equilibrium binding and dissociation kinetic of chimeras 9 and 5 exhibiting the minimal requirements for the high affinity cGK Ialpha (upper) and low affinity cGK Ibeta (lower) phenotype. Left, binding of cGMP was determined in aliquots containing 0.79 pmol (chimera 5) and 3.43 pmol (chimera 9) of cGMP kinase holoenzyme directly before (bullet ) and 1 min after the addition of 1 mM cGMP (black-triangle). The binding of cGMP to site B (open circle ) was calculated by subtracting binding to site A (black-triangle) from the total specific binding (bullet ). The inset shows the same data as a Scatchard plot fitted by the LIGAND program (see "Experimental Procedures."). Right, cytosolic extracts containing 1.25 pmol of chimera 9 or 1.25 pmol of chimera 5 were incubated with 0.2 µM [3H]cGMP for 60 min at 4 °C. The exchange of bound [3H]cGMP was initiated by the addition of 1 mM unlabeled cGMP.
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Chimeras of Intermediate Phenotype

The results above did not definitely rule out that the cGK Ialpha or cGK Ibeta phenotype would also be obtained by the substitution of S2 or S4 alone. Chimeras 10-17 with substitution in only one domain exhibited an intermediate phenotype with Ka values of about 300 nM. These values were roughly 5-fold higher and lower than the values for cGK Ialpha and cGK Ibeta , respectively (Fig. 3). This shift in the Ka values was also reflected in the accelerated dissociation of cGMP from the high affinity site in most of these chimeras. However, dissociation was never monophasic as observed for chimeras exhibiting the cGK Ibeta phenotype. In addition to the effect on the high affinity site, chimeras 10-13 showed also a slight, statistically not significant deceleration of dissociation from the low affinity site. The rate constants of chimera 14 are disconcordant with those of the other chimeras in the group. The Ka of 363 nM is probably caused by a decrease in the association rate. These results obtained with different substitutions in S2 and S4 confirmed the observation that only the combined exchange of both domains alter the phenotype from cGK Ialpha to cGK Ibeta and vice versa.

The Linker Sequence (S3) between S2 and S4 Has a Reverse Effect on the Phenotype

Exceptions from the above rule were observed with some chimeras. Chimeras 18 and 19 containing the sequences of cGK Ibeta in S2, S5, and S6 also exhibited the low affinity phenotype with Ka values of 1 µM, although the sequence of cGK Ialpha was present in the autoinhibitory domain S4 (Fig. 3). The structural basis of this phenomenon was limited to the short linker sequence between S2 and S4. This linker varied in length between cGK Ialpha and cGK Ibeta (Fig. 2). Apparently, the cGK Ialpha linker exerted this paradoxical effect since it facilitated the expression of the low affinity cGK Ibeta phenotype (Fig. 3). In fact, the exchange of the linker sequence from cGK Ialpha to cGK Ibeta decreased 3-fold the Ka from 1 µM in chimeras 18 and 19 to about 380 nM in chimeras 12 and 13. These chimeras suggested that the expression of the respective phenotype was strengthened by inserting the linker sequence of the other cGK isozyme.

Chimeras Exhibiting a cGK Ialpha Phenotype with Increased Affinity

Similar as found for the low affinity cGK Ibeta phenotype, chimeras were identified that express the high affinity cGK Ialpha phenotype without fulfilling the requirements in S2 and S4. Chimera 20 contained the sequence of cGK Ialpha in S1-S5 and that of cGK Ibeta in S6 but exhibited a 6-fold lower Ka value than cGK Ialpha itself (Fig. 3). Even further cGK Ibeta substitutions additional to S6 in S1, S2, and S3 (chimera 21) or in S4 (chimera 22) yielded cGK enzymes, which were stimulated at significantly lower cGMP concentrations (p < 0.03) than cGK Ialpha . Obviously, a specific constellation, i.e. the combination of cGK Ialpha substitution in S5 and that of cGK Ibeta in S6, resulted in a cGK Ialpha phenotype with increased affinity for cGMP (Fig. 3). The increased affinity of chimera 20 was apparently caused by slowing the dissociation of cGMP from the high affinity site. In contrast, dissociation of cGMP in chimera 21 was accelerated 5-fold instead of being decelerated when compared with cGK Ialpha . The high affinity of this chimera was due to a faster association rate for cGMP. Whereas association of cGMP included a fast (k+1 approx  0.05 min-1·nM-1) and a slow component (k+1 = 0.00042 min-1·nM-1) in the cGK Ialpha , only the fast component was observed with chimera 21 resulting in equilibrium binding within 30 s after addition of cGMP. Taken together, the combination of cGK Ialpha substitution in S5 and that of cGK Ibeta in S6 enhanced the Ka decreasing effect of cGK Ialpha substitutions in S2 and S4. Even the effect of cGK Ialpha sequence in one domain alone, either S2 or S4, exceeded that of cGK Ialpha sequence in both domains as indicated by the significantly lower Ka value of chimera 21 and 22 when compared with the Ka of cGK Ialpha . These results suggest that the flexibility and anchoring function of the hinge domain increased the interaction of S2 and S4 with the cGMP binding domain under the condition described above. This effect was possibly mediated by altering the position of the the amino-terminal domain relative to the other domains in the cGK holoenzyme structure.

Weighting Factors Predict the Impact of Individual Amino-terminal Domains on the High Affinity cGK Ialpha Phenotype

The contribution of each of the 6 amino-terminal domains, S1-S6, to induce the high affinity cGK Ialpha phenotype could be best assessed by assigning arbitrary weighting factors to the individual domains of cGK Ialpha (Fig. 5). The weighting factors were obtained by an iterative procedure. The factors for the leucine zipper and the autoinhibitory domain were found to be most influential with values of 0.2 and 0.3, respectively, thereby confirming the importance of these domains for the high affinity cGK Ialpha phenotype. On the other hand, the individual effect of the extreme amino-terminal sequence (S1) and the second part of the hinge domain (S6) were marginal with factors of 0.9 and 0.8, respectively. The linker sequence (S3) contributed with the factor 1.8 to the phenotype indicating that it acted in a reverse manner, i.e. cGK Ialpha substitution therein increased Ka 1.8-fold instead of decreasing it. The weighting factors allowed the estimation of the Ka values for most of the chimeras constructed with considerable accuracy (Fig. 5). To obtain the Ka value of any chimera, the Ka value of cGK Ibeta (= 1748 nM) was multiplied with the individual factors for S1 to S6 of each cGK Ialpha and cGK Ibeta domain. With three exceptions, the ratio between the Ka values predicted and the Ka values actually measured varied between 0.70 and 1.28 with a mean value of 1.03 ± 0.05 suggesting that the factors reliably described the impact of the individual amino-terminal domains on the high affinity cGK Ialpha phenotype. This arbitrary equation, however, failed to describe the Ka decreasing effect of the specific constellation having cGK Ialpha substitution in S5 and that of cGK Ibeta in S6. The specific requirements in S5 and S6 suggested that this effect depended rather on synergistic than on individual contribution of the two domains. This idea was supported by the finding that the reverse substitution, i.e. substitution of cGK Ibeta in S5 and that of cGK Ialpha in S6 did not generate a "super" cGK Ibeta enzyme (see chimeras 1, 6, 11, and 15). Thus, the dramatic Ka decreasing effect observed with chimeras 20, 21, and 22 could not be covered by the empirical equation.


Fig. 5. Weighting factors describing the impact of amino-terminal domains S1-S6 on the high affinity cGK Ialpha phenotype. Upper, the weighting factors for the domains S1-S6 of cGK Ialpha and cGK Ibeta . The factors were calculated as described under "Experimental Procedures." Middle, the empirical equation to determine the Ka value of any chimera by the use of the factors S1-S6. Lower, the Ka values determined by the equation are compared with those obtained experimentally. Ka pre/Ka indicates the ratio of the predicted and experimental values. For further details see Fig. 3.
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Chimeras Constructed to Test the Validity of the Empirical Equation

Of the remaining possibilities for permutations, we selected chimeras 23, 24, and 25 to test the validity of the equation. Chimera 23 was constructed to show that also large portions of the cGK Ibeta sequence (in this case almost 70%) were able to generate the cGK Ialpha phenotype when the Ka increasing effect of cGK Ibeta substitution in S2 was partially neutralized by the reverse effect of cGK Ibeta substitution in S3. The Ka of 166 nM was similar to that of cGK Ialpha . In contrast, chimera 10 with cGK Ibeta substitutions in S2 but not in S3 had an intermediate Ka of 305 nM (Fig. 6). Furthermore, chimera 23 demonstrated that the weighting factors contributed independently to the Ka, even when clustered. Chimera 24 was constructed to show that cGK Ibeta substitution in S4 alone did not produce an intermediate phenotype when all other domains contained cGK Ialpha substitutions. This result was exactly predicted by the equation. The replacement of the cGK Ialpha substitutions in S3 and S1 in this chimera yielded chimera 25. The exchange decreased the Ka as expected, although the proportion of cGK Ibeta sequence increased. These results demonstrate that the empirical equation can be used to describe the individual impacts of amino-terminal domains on the high affinity cGK Ialpha phenotype.


Fig. 6. Chimeras constructed to test the validity of the empirical equation. See Figs. 3 and 5 for further details.
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DISCUSSION

The present study demonstrates that the leucine zipper and the autoinhibitory domain determine the affinity of cGK Ialpha and cGK Ibeta for cGMP. These results assign a new role for the two domains. Up to now the leucine zipper and the autoinhibitory domain were thought to mediate holoenzyme formation and to inhibit phosphotransferase activity in the absence of cGMP, respectively. The regulatory effect of the leucine zipper (S2) and the autoinhibitory domain (S4) is mediated indirectly, since the primary structure of the binding domains are identical in cGK Ialpha and cGK Ibeta . The indirect effect mainly concerns the binding site which represents the high affinity site in cGK Ialpha . Recent evidence suggests that this is the amino-terminal site A (27). The presence of cGK Ialpha substitutions in S2 and S4 confers high affinity for cGMP to site A, whereas the presence of cGK Ibeta substitutions herein confers low affinity to this site. This interpretation is supported by the dissociation rate constants which indicated that the S2 and S4 substitution affected mainly the slow dissociation rate.

High affinity binding to site A in cGK Ialpha depends on cooperative interaction of site A and B (3, 4, 28, 29). The dissociation from site A of cGK Ialpha obtained by dilution of the cGK/cGMP complex is about 100-fold faster than that observed by addition of an excess of unlabeled cGMP (4). For cGK Ibeta , however, the rate constants are similar, regardless whether dissociation has been induced by addition of excess unlabeled cGMP or by dilution of the cGK/cGMP complex (3). It has therefore been concluded that cooperativity is diminished or even abolished in cGK Ibeta . This study identifies S2 and S4 as the sequences that allow cooperativity between the binding site A and B. S2 and S4 enable high cooperativity when derived from cGK Ialpha and low cooperativity when derived from cGK Ibeta as demonstrated by the dissociation kinetics. The involvement of the autoinhibitory domain in regulating cooperativity was postulated previously. Autophosphorylation of the amino terminus of the purified lung cGK (>= 90% cGK Ialpha ) decreases the cooperativity between sites A and B by accelerating the dissociation from site A (4, 28).

The ability of S2 and S4 to affect cooperativity between cGMP binding site A and B implies that these amino-terminal domains have to be included in any structural model of the cGK and its binding domains. Only the conformation of the cGK induced by S2 and S4 results in the appropriate affinity of the cGMP binding sites. The structure of the RIalpha subunit of cAMP kinase provides evidence that an interdomain hydrophobic interaction region carries out the communication between the cAMP binding domains A and B in the cAMP kinase (30). This interdomain region allows that structural alterations in domain B, the site that is initially occupied by cAMP during activation of the kinase, are transmitted to site A. This interdomain region is therefore assumed to play a central role in the cooperativity between the two cAMP binding sites in cAMP kinase. Assuming similar tertiary structure of the cyclic nucleotide binding domains in cGK and cAMP kinase, it is conceivable that S2 and S4 interfere with this interdomain region in the cGK, thereby affecting the cooperativity between the binding sites A and B.

While the leucine zipper and the autoinhibitory domain control holoenzyme formation and inhibition of catalytic activity individually, synergistic interactions of both domains are required to develop their full regulatory potential on cGMP binding. The involvement of the two domains in regulating affinity for cGMP is intriguing, as both the S2 and S4 domain represent the amino-terminal sequences with highest homology between cGK Ialpha and cGK Ibeta . Thus, the interaction of the amino terminus with the cGMP binding domains is apparently based on a conserved structure.

Non-homologous sequences of the cGK Ialpha and cGK Ibeta amino termini are less important for the regulation of binding affinity. For example, the linker sequence (S3) between the leucine zipper and the autoinhibitory domain, which is variable in length between cGK Ialpha and cGK Ibeta , is not crucial for producing the cGK Ialpha or cGK Ibeta phenotype. Moreover, this linker even exerts a reverse effect as it significantly decreases affinity when it derives from cGK Ialpha and increases affinity when it derives from cGK Ibeta . Specific residues in the linker may contribute to this effect. Other non-homologous amino-terminal domains, such as S1 and S6, only negligibly affect the affinity of binding site A. The domain S5, however, which has been regarded as a part of the hinge domain, still exerts a considerable, but less pronounced effect than S2 and S4. Its contribution to the phenotype suggest that it may be attributed structurally and functionally to the preceding autoinhibitory domain.

The molecular basis underlying the effect of the leucine zipper and the autoinhibitory domain is unclear at the moment. The nature of their interactions with the binding site may depend on the net charge of the leucine zipper. Both the cGK Ialpha and cGK Ibeta zipper are interspersed with positive and negative amino acid residues. The negative charges predominate in the cGK Ibeta zipper and the positive charges in the cGK Ialpha zipper (Fig. 2). Thus, the electrostatic interactions resulting from a positioning of the zipper near the cGMP binding domain would be different in cGK Ialpha and cGK Ibeta . The leucine zipper is thought to form an alpha -helix that allows the dimerization of two cGK subunits (2, 9-11). The absolute length of the leucine zipper and, thus, the length of the putative helix are not crucial for the interaction with the binding site, since two additional helical turns provided by S1 of cGK Ibeta do only marginally affect the high or low affinity cGK phenotype. The additional need for the autoinhibitory domain (S4) may also be explained by structural interactions. Autoinhibition of cGK Ialpha is achieved by a substrate-like sequence including the autophosphorylation site -Thr59- (12), and that of cGK Ibeta is achieved by the autoinhibitory pseudosubstrate sequence -75KRQAI- (31). It is conceivable that this unique features of cGK Ialpha and cGK Ibeta have influence on the interaction between the autoinhibitory and catalytic domain. As both domains are located on the same polypeptide chain, having the cGMP binding domain in between, different interactions between autoinhibitory and catalytic domain also differently affect the structure of the binding pockets. Consequently, the affinity for cGMP may be increased and decreased through the autoinhibitory domains of cGK Ialpha and cGK Ibeta , respectively.

The alteration of the primary structure in the various chimeras changed the structural context of the autoinhibitory domain (S4). Nevertheless, all of the chimeras exhibited less than 10% basal activity, regardless whether they contained the S4 from cGK Ialpha or cGK Ibeta . This suggests that both S4 domains included the essential sequence necessary for autoinhibition. Only a cGK Ialpha deletion mutant, which lacked the sequence Thr59-Thr60-Arg61 of S4 had a 75% basal activity,2 thereby confirming that the in vitro autophosphorylated Thr59 (12) is essential for autoinhibition of cGK Ialpha . The in vitro autophosphorylated Ser64 of cGK Ibeta (13), however, is apparently not involved in autoinhibition. This site resides in S3 of cGK Ibeta and is not present in S3 of cGK Ialpha . The exchange of S3 from cGK Ibeta by that from cGK Ialpha does not yield a cGK with increased basal activity.

Finally, the identification of specific amino acid sequences that regulate the affinity for cGMP may help to find analogues sequences in other cyclic nucleotide regulated proteins. A specific alpha -helical segment that regulates the affinity of the cGMP binding site has already been characterized in cGMP-regulated ion channels (32). This segment shows considerable homology to part of the leucine zipper of the cGK.


FOOTNOTES

*   This work was supported by grants from the Deutsche Forschungsgemeinschaft and Fond der Chemischen Industrie.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) Y08961[GenBank].


Dagger    To whom correspondence should be addressed. Tel.: 49-89-4140-3285; Fax: 49-89-4140-3261; E-mail: ruth{at}ipt.med.tu-muenchen.de.
1   The abbreviations used are: cGK, cGMP-dependent protein kinase; COS cells, African green monkey kidney cells; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; S1-S6; amino-terminal domains harboring the amino acid sequence of the extreme amino terminus (S1), the leucine zipper (S2), the linker between the leucine zipper and autoinhibitory domain (S3), the autoinhibitory domain (S4), the first (S5) and second part (S6) of the hinge domain.
2   P. Ruth, unpublished results.

ACKNOWLEDGEMENTS

We thank L. Koblitz and Stefan Beck for technical help.


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