Interlobe communication in multiple calcium-binding site mutants of

Protein Science (1996), 5:468-477. Cambridge University Press. Printed in the USA. Copyright 0 1996 The Protein Society

Interlobe communication in multiple calcium-binding site mutants of Drosophila calmodulin

.. - ." . .- ~ ~~

POUSHALI MUKHERJEA,' JOHN E MAUNE, AND KATHY BECKINGHAM Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005-1892

(RECEIVED November 20, 1995; ACCEPTED December 21, 1995)

Abstract

We have generated mutants of Drosophila calmodulin in which pairs of calcium-binding sites are mutated so as to prevent calcium binding. In all sites, the mutation involves replacement of the -Z position glutamate residue with glutamine. Mutants inactivated in both N-terminal sites (B12Q) or both C-terminal sites (B34Q), and two mutants with one N- and one C-terminal site inactivated (B13Q and B24Q) were generated. The quadruple mutant with all four sites mutated was also studied. UV-difference spectroscopy and near-UV CD were used to examine the influence of these mutations upon the single tyrosine (Tyr-138) of the protein. These studies uncovered four situations in which Tyr-138 in the C-terminal lobe responds to a change in the calcium-binding properties of the N-terminal lobe. Further, they suggest that N-terminal calcium-binding events contribute strongly to the aberrant behavior of Tyr-138 seen in mutants with a single functional C-terminal calcium-binding site. The data also indicate that loss of calcium binding at site 1 adjusts the aberrant conformation of Tyr-138 produced by mutation of site 3 toward the wild-type structure. However, activation studies for skeletal muscle myosin light chain kinase (SK-MLCK) established that all of the multiple binding site mutants are poor activators of SK-MLCK. Thus, globally, the calcium-induced conformation of B13Q is not closer to wild type than that of either the site 1 or the site 3 mutant. The positioning of Tyr-138 within the crystal structure of calmodulin suggests that effects of the N-terminal lobe on this residue may be mediated via changes to the central linker region of the protein.

Keywords: calcium-binding proteins; circular dichroism; skeletal muscle myosin light chain kinase; UV-difference spectroscopy

The calcium-binding protein calmodulin is widely distributed in eukaryotes and plays a central role in mediating intracellular calcium signaling. The structure of the calcium-saturated (holo) form of the protein is relatively well understood. Crystal struc- tures for the holo forms of mammalian and Drosophila calmodulins have been determined (Babu et al., 1988; Taylor et al., 1991). These two calmodulins differ by only three conservative amino acid differences (Smith et al., 1987) and the two struc- tures are very similar. Both are dumbbell-shaped molecules with two globular terminal lobes, each containing a pair of the calcium-binding sites. The central region separating the termi-

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Reprint requests to: K. Beckingham, Department Biochemistry and Cell Biology, MS140, Rice University, 6100 Main Street, Houston Texas, 77005-1892; e-mail: [email protected]. ' Present address: Department of Neuroscience and Cell Biology, UMDNJ-RW Johnson Medical School, Piscataway, New Jersey 08854- 5635.

Abbreviations: EGTA, [ethylenebis(oxyethylenenitrilo)]tetra-acetic acid; Hepes, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid; IPTG, isopropyl-thio-P-D galactoside; MOPS, (3-[N-morpholino]pro- pane-sulfonic acid; PEG, polyethylene glycol; SAXS, small angle X-ray scattering; SK-MLCK, skeletal muscle myosin light chain kinase.

nal lobes is an a-helix of about eight turns. Small angle X-ray scattering analysis (Heidorn & Trewhella, 1988) and NMR (Bar- bat0 et al., 1992) studies indicate that this overall conformation is maintained in solution, but also indicate that part of the central region is unstructured rather that helical.

Less is known about the conformation of the calcium-free (apo) form of calmodulin and hence of the conformational changes induced by calcium binding. SAXS analysis has indicated that the terminal lobes of the protein are maintained in the absence of calcium, but are closer together by about 5 A (Seaton et al., 1985; Heidorn & Trewhella, 1988). Nevertheless, biophysical and biochemical studies with both the intact protein and the separated N- and C-terminal halves have suggested that the calcium-induced conformational changes in the two terminal lobes are independent of one another (Forsen et al., 1986; Mar- tin & Bayley, 1986). Similarly, the model for apo-calmodulin proposed by Strynadka and James (1988) predicted that calcium- induced events in each of the terminal lobes would occur inde- pendently. The recent NMR determinations of the solution structure for apo-calmodulin (Kuboniwa et al., 1995; Zhang et al., 1995) have largely confirmed the Strynadka and James model and reinforce the view that calcium-induced events in the two halves of the molecule are independent phenomena.

468

Multiple CaZi binding site mutants of calmodulin 469

We have used site-directed mutagenesis of Drosophila calmodulin to investigate the contribution of the individual “E-F hands” (Kretsinger & Nockolds, 1973) to overall calcium binding and calcium-induced conformational changes in the protein. Ini- tially, two series of mutants were generated and studied (Maune et al., 1992b). In each mutant, the conserved glutamic acid residue at the -Z coordination position of the calcium-binding sites (Kretsinger & Nockolds, 1973) was mutated in one site. In the four proteins of the Q series, the -Z residue was mutated to glutamine. Direct calcium-binding studies demonstrated that, in each site, the Q mutation effectively prevents calcium binding, such that under the quasi-physiological conditions used for binding studies, calcium binding at the mutated site was undetect- able (Maune et al., 1992b).

Surprisingly, calcium binding and conformational studies of these single binding site mutants (Martin et al., 1992; Maune et al., 1992a, 1992b) provided some evidence for effects of N-terminal mutations upon calcium-induced conformational changes to the single tyrosine of the protein (Tyr-138) located in the C-terminal lobe. Some of the most striking effects on this residue were detected when one of the C-terminal sites was mutated. However, given the presence of at least one functional calcium-binding site in the C-terminus of these mutants, it was impossible to determine whether these effects originated from N-terminal binding or aberrant C-terminal binding. We have investigated these phenomena further through the use of calmodulin mutants in which two of the calcium-binding sites are disabled. The E to Q mutations of the -Z coordinate described above, with their known effects on calcium binding, were used to create these mutants. The mutant with both N-terminal binding sites mutated and the mutant with both C-terminal sites mutated were generated to allow study of calmodulins in which calcium binding was primarily to only one terminal lobe of the protein. Using our previous nomenclature (Maune et al. 1992b), these two calmodulins have been termed B12Q and B34Q, indicating that the E to Q mutation is present in sites 1 and 2, or sites 3 and 4, respectively (see Table 1). In addition, two proteins in which one N-terminal and one C-terminal site contain

Table 1. Multiple calcium-binding site mutants of calmodulin”

Mutant name Residues mutated Sites mutated

Bl2Q

~~~~ ~ ~ . ~

”

~~ .~ ~

E31 -+ Q Sites 1 , 2 E67 + Q

B13Q

B24Q

B34Q

B 1234Q

E31 + Q Sites I , 3 E104 + Q

E67 + Q Sites 2, 4 E140 + Q

E104 + Q Sites 3, 4 E140 -+ Q

E31 + Sites 1 , 2, 3 , 4 E67 -+ Q

E104 + Q El40 + Q

~” .”

a In all mutants, the residues mutated are the conserved glutamates found at the - Z position of the calmodulin Ca2+-binding sites.

the Q mutation have been examined. Of the four possible mutants of this class, those with (1) site 1 and site 3 mutated, and (2) site 2 and site 4 mutated were chosen for study. Previous work (Martin et al., 1992; Maune et al., 1992a, 1992b; Starovas- nik et al., 1992; Gao et al., 1993; Mukherjea & Beckingham, 1993) indicates these two mutants, B13Q and B24Q respectively, represent the combinations of binding site mutations with (1) the mildest effects on calcium binding and conformational change (the B13Q mutant) and (2) the strongest effects on these properties (the B24Q mutant). The mutant calmodulin with all four calcium-binding sites mutated (B1234Q) was also generated and studied.

UV-difference spectroscopy and near-UV CD (Maune et al., 1992a, 1992b) have been used to monitor conformational changes to Tyr-138. In addition, the ability of these mutants to activate one of the better characterized target enzymes of calmodulin, skeletal muscle myosin light chain kinase, has been examined. The findings provide strong evidence that events in the N-terminal lobe of calmodulin are detected in the C-terminal lobe, as monitored by Tyr-138. Mechanisms whereby such interlobe communication could occur are considered in the light of current knowledge on the positioning of Tyr-138 and the calcium-induced changes to this residue.

Results

Protein purification

The mutant calmodulins studied here are detailed in Table 1. Each has two or more calcium-binding sites containing the E to Q mutation at the -Z calcium coordination position. In previous studies (Maune et al., 1992b), it was found that mutant calmodulins carrying this E to Q mutation in a single calcium-binding site could be purified by phenyl-Sepharose affinity chromatography in the presence of high salt. This purification procedure relies upon the induction of hydrophobic surfaces on calmodulin in response to calcium binding. However, no conditions could be found under which calmodulins with more than one mutant binding site would bind to phenyl-Sepharose. A more conventional procedure, based on that of Newton et al. (1988) and involving ammonium sulfate fractionation, DEAE-Sephacel chromatography, and HPLC anion exchange chromatography, was therefore developed for purification of these proteins (see the Materials and methods). Drosophila calmodulin contains no tryptophan, nine phenylalanines, and a single tyrosine residue. As a result, the ratio of the tyrosine peak at 279 nm and the shortest wavelength absorbance peak for phenylalanine at 253 nm is a extremely sensitive measure of protein purity. For pure wild- type calmodulin in the presence of calcium, the 279/253-nm ratio is less than 1.00. All mutant calmodulins were therefore purified until spectra revealed 279/253-nm ratios lower than 1 .OO. SDS-PAGE was used to confirm the purity of the proteins. Extinction coefficients for the five purified proteins were determined in the presence and absence of calcium and are shown in Table 2.

Con formational change during calcium titration

The single tyrosine (Tyr-138) of Drosophila calmodulin responds to calcium binding to the protein and thus can be used to monitor conformational changes to its environment in the fourth

470

Table 2. Molar extinction coefficients for wild-type and mutant calmodulins at the tyrosine absorbance maxima

~~~ ~~~~~ ~

~~ ~~~~~ ~~~

~ ~ ~~ ~~~~

~ ~~ ~ ~~~ ~~

Extinction coefficient X,,,,,. (k0.3 nm) ~~~~~~~~~ ~~ ~ ~

Calmodulin +Ca2+ +EGTA +Ca2+ +EGTA

WT 1.578 1,874 278.9 278.9 B12Q 1.722 1,860 279.9 278.3 B13Q 1,688 1,878 278.4 278.9 B24Q 1,720 1,848 277.7 277.3 B34Q 1,799 1,946 279.6 279.3 B 1234Q 1,799 1,900 278.6 278.6

~~~~ ~ ~

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calcium-binding domain of the protein. We have used UV-difference spectroscopy and near-UV CD to monitor changes to Tyr-138 during titrations at high protein concentration (100 pM) with progressive addition of equivalents of calcium. Under these conditions, the wild-type protein shows stoichiometric calcium binding, with the higher-affinity sites being occupied first and all changes to Tyr-138 are completed after addition of four equivalents of calcium. For the mutant proteins, however, two further factors influence the titration behavior. Although the Q mutation used here effectively eliminates calcium binding at the mutated site under the conditions studied originally (Maune et al., 1992b), NMR analysis has shown that, under high protein concentrations, binding at some mutated sites is detectable late in calcium titrations (Starovasnik et al., 1992). In addition, the presence of the Q mutation in some binding sites was shown to lower calcium affinity at some of the residual functional sites on the protein (Maune et al., 1992b). Thus, as a result of these two phenomena, spectral changes to some of the mutant proteins continue at levels of calcium higher than those nominally required to fill all remaining functional calcium-binding sites on the protein. These factors are considered in discussing the conformational changes seen during calcium titration of the mutant proteins.

UV-difference spectroscopy

In previous work, UV-difference spectra produced for wild-type Drosophila calmodulin during calcium titration were shown to contain two negative peaks corresponding to changes in absorbance at the peak and shoulder for tyrosine absorbance (279 and 286 nm) (Maune et al., 1992b). A negative difference spectrum in the region 279-287 nm is usually interpreted as increased ex- posure of tyrosine residues to the solvent (Donovan, 1973). However, the ratio of the 279- and 286-nm peaks for calmodulin is unusual in that, unlike other proteins, the 279-nm peak gives stronger differential absorbance than the 286-nm shoulder. During calcium titration, the major changes to these two peaks occur during the addition of the first two equivalents of calcium (see Fig. IA), indicating that they are induced by binding to the high-affinity C-terminal sites. Findings for calmodulins containing mutations in single calcium-binding sites reinforced this interpretation (Maune et al., 1992b). Thus, for mutants to site 1, the unusual ratio of the 279/286-nm UV-difference spectrum peaks is maintained and the response of these

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Equivalents Ca2+ Fig. 1. Changes to the UV-difference spectra peaks of wild-type and mutant calmodulins during titration with calcium. Changes in A€ associated with the two tyrosine peaks at 279 nm (0) and 286 nm (.) of the UV-difference spectra are shown as a function of equivalents of calcium added per calmodulin. Titrations for wild-type protein, B3Q, and B4Q have been published previously (Maune et al., 1992b). Where error bars are not visible, they were smaller than the symbols used for the data points.

peaks is quantitatively similar to that of the wild type in the initial phases of the calcium titration.

The single B2Q mutant was not examined previously and was investigated here in order to aid with interpretation of multiple mutants containing a mutation to site 2. As can be seen from Figure ID and a comparison to Figure 6B of Maune et al. (1992b), the response of B2Q is very similar to that of BlQ. Changes very like those for the wild-type protein are seen over the addition of the first two equivalents of calcium and the only detectable difference is that the small incremental increases in both the 279-nm and 286-nm peak intensities seen for the wild- type protein upon addition of the third and fourth aliquots of calcium are not detected for B2Q.

Given that mutation of either site 1 or site 2 has essentially no effect on the early response of Tyr-138, the double B12Q mutant might be expected to behave similarly. As shown in Fig- ure IC, however, this is not the case. Unexpectedly, the entire calcium-induced change in the environment of Tyr-138 is com-

Muitipie Ca2+ binding site mutants of calmodulin 47 1

pleted upon the addition of one equivalent of calcium as opposed to two. Data for up to 10 additions of calcium are shown in Figure IC, but addition of up to 16 equivalents produced no further changes (data not shown). The overall responses of the 279- and 286-nm peaks and the 279/286-nm peak ratio are also somewhat reduced in the B12Q mutant as compared to BlQ and B2Q (compare Fig. ID and G).

Maune et al. (1992b) showed that mutation of either site in the C-terminus produces a response that dramatically alters the UV-difference changes seen during calcium titration. The 279/286-nm peak ratio, the intensity of the peaks, and behavior of these peaks during titration are all very different from the wild-type protein. These titrations for B3Q and B4Q have been repeated to provide controls for the double mutants investigated in this study and are shown in Figure IB and C. As can be seen, B3Q shows a biphasic response, whereas the response of B4Q is essentially monophasic. The relative affinities of the three nonmutated calcium-binding sites of B3Q and B4Q are closer than those of the wild-type protein (Maune et al., 1992b), and NMR experiments indicate that they fill more or less simultaneously (Starovasnik et al., 1992). Thus, it was impossible to determine which calcium-binding events produced the dramatically altered behavior of Tyr-138 seen in these two mutants. The calcium affinities of the residual sites were consistent, however, with the interpretation offered by Maune et al. (1992b) that the initial phase of the B3Q titration represents changes due to the unusual situation of calcium binding at site 4 in the absence of binding at site 3, and that the following recovery phase represented occupancy of the mutated site 3 itself. For B4Q, the changes were consistent with the entire titration curve representing the occupancy of the partner site, site 3. However, for both mutants, the possibility that some of the dramatic changes seen result from binding at the N-terminal sites remained open.

Examination of the UV-difference changes in mutant B34Q, in which both C-terminal sites are mutated, addresses the question of whether calcium binding at the residual C-terminal site is indeed required for any aspect of the responses seen in B3Q and B4Q. As shown in Figure IH, the changes during addition of the first four equivalents of calcium to this mutant are very limited and do not show the unusual 279/286-nm ratio charac- teristics seen for the B3Q and B4Q responses. A comparison to Figure 1 J shows that the behavior of B34Q early in the titration is not significantly different from that of the quadruple mutant in which all four calcium-binding sites are mutated. Thus, in the absence of any functional calcium-binding sites in the C-terminus, calcium binding to the N-terminus alone does not produce changes in the environment of Tyr-138 similar to those seen for B3Q and B4Q. Calcium binding at the residual C-terminal site is therefore at least a component of the B3Q and B4Q responses.

The B13Q and B24Q mutants each contain a single N-terminal and a single C-terminal calcium-binding site mutation. These mutants thus allow a different question to be posed in relation to the unusual UV-difference titration curves seen for Tyr-138 in B3Q and B4Q. Is there any effect of calcium binding at the N-terminal sites on the behavior of Tyr-138 when one of the C-terminal sites is functional? For the mutant pair B13Q and B3Q, the UV-difference changes during calcium titration are shown in Figure 1B and E. As can be seen, the UV-difference calcium titrations are strikingly different. Surprisingly, the behavior of the 279/286-nm peaks in the B13Q double mutant is more similar to the wild-type titration than that of the B3Q mu-

tant (compare Fig. 1A and E). Although the relative intensities of the 279- and 286-nm absorbance values are not completely restored to the wild-type situation, in other aspects the B13Q mutant UV-difference titration curve is more like that of the wild-type protein than that of any other mutant studied. These findings thus suggest that calcium binding at site 1 contributes strongly to the aberrant behavior of Tyr-138 in B3Q and that loss of binding at this site has a “corrective” effect on the environment of this residue.

A comparison of the B4Q mutant with the B24Q mutant addresses a similar question concerning the contribution of binding at site 2 to the unusual UV-differences changes seen when site 4 is mutated. As shown from a comparison of Figure IC and F, loss of calcium binding at site 2 strongly modifies the behavior of Tyr-138, but in this case, most of the response of the tyrosine is lost and minimal changes in the 279/286-nm peaks are seen during the calcium titration. Thus, calcium binding at site 2 appears to be a significant component of the aberrant Tyr- 138 response of mutant B4Q. From a comparison of Figure I F with Figures 1H and 1 J, it can be seen that the response of the tyrosine in B24Q mutant is as poor as that in the B34Q and B1234Q mutants, suggesting that mutation of sites 2 and 4 has at least as deleterious an effect on the overall responsiveness of Tyr-138 as does mutation of sites 3 and 4.

Near- U V CD

The near-UV CD signal at 280 nm (AE280) provides different information on the conformation of the single tyrosine in Dro- sophila calmodulin, probably reflecting the rotational freedom of its side-chain moiety. In a previous study (Maune et al., 1992a), Atzso values for the wild-type protein and mutants carrying the single Q mutants were determined in the absence of calcium (apo At28o) and in a IO-fold molar excess of calcium (holo AeZso). In the absence of calcium, the wild-type protein shows a strong positive AezSo (3.2) that changes to an even higher negative ALt280 value upon calcium saturation (-5.00). This indicates a conformational change for the tyrosine side chain from one restricted conformation to a second restrained position upon calcium binding. Examination of At280 during calcium titration demonstrated that these changes were largely completed after addition of two equivalents of cakium, suggesting, as for the aspect of conformation examined by UV-difference spectroscopy, that binding to the C-terminal sites largely governs this change in the environment of Tyr-138.

As for UV-difference spectroscopy, the calcium-induced changes to Atzso seen for N-terminal site mutants largely reinforced this idea. Thus, the overall change in A L ~ ~ ~ ~ for mutants BlQ and B2Q is similar to that seen for the wild-type protein, although the A6280 value for holo B2Q is somewhat greater than that of the wild-type protein (Table 3; Fig. 2) . Given that mutant B12Q behaved differently with respect to UV-difference titrations than either mutant BlQ or B2Q alone (see above), it was of interest to determine whether it would dem- onstrate differences with respect to A6280. As shown in Figure 2, the values for the apo and holo forms of B12Q are not strikingly different from wild type or the individual BlQ (Fig. 2; Maune et al., 1992a) and B2Q mutations. However, a significant difference is that changes to ALtz80 do not saturate until between two and four equivalents of calcium are added, whereas for BlQ and B2Q, changes are completed after addition of two

412 P . Mukherjea et al.

Table 3. Near-UV CD properties of Drosophila calmodulin calcium-binding site mutants

Calmodulin

Wild-type B2Q B3Q

~~ ." .~

8124 B13Q B24Q B34Q B 1234Q

At280 (Ca2+-free)

3.17 2.88 2.36 2.03 1.86 0.83 I .52 0.095

~~

(+ lo equivalents Ca2+) Ac2s0

-5.02 -6.50 -3.1 1 -4.44 -4.54 - 1.34

~

1.17 -0.80

equivalents of calcium. This suggests that when both N-terminal sites are mutated, the affinity of one or both sites in the C-terminus is decreased significantly.

Mutation of either C-terminal site was shown previously to affect the &280 signal severely, again indicating a dominant role for C-terminal binding in the Tyr-138 At280 signal. B3Q nevertheless gave a change to a final negative k 2 8 0 value of -1.35, whereas for B4Q the decrease in &280 was so low as to retain a final positive value (+ 1.05). In repeating studies of the single mutants as controls for the experiments described here, we found our values to be in good agreement with those of Maune et al. (1992a), although our holo h 2 8 0 value for mutant B3Q is somewhat more negative than previously reported (Table 3).

The role of the C-terminal sites in generating the At280

change to Tyr-138 is most clearly demonstrated by the double C-terminal site mutant B34Q. Calcium-induced changes to At280 are almost nonexistent (Fig. 2), and, in addition, the At180

41 WT 8 2 4 B3Q B4Q B12Q B34Q E l 3 0 8 2 4 4 B1234Q e h

Fig. 2. Calcium-induced changes to the near-UV CD signal (Atzso) for wild-type and calcium-binding site mutant calmodulins. Values for AtZIO in the absence of calcium and in the presence of 10 equivalents of calcium are shown for all mutants. For the double and quadruple calcium-binding site mutants, values at 2 and 4 equivalents of calcium are also shown. For clarity, error bars for these values are not shown, but they were comparable to, or smaller than, those shown for the 10 equivalents of calcium values. Data for the wild-type protein and the single mutants BlQ, B3Q, and B4Q have been published previously (Maune et al., 1992a). Measurements for most of these proteins were repeated as part of this study, and proved to be very similar to the previously reported values (see text).

value for the apo form of this protein is significantly reduced. Thus, mutation of the two C-terminal calcium-binding sites alters the initial environment of Tyr-138 and prevents almost entirely the conformational change detected by in the wild- type protein.

Interestingly, for the aspect of conformational change measured by At280 , the response of the quadruple mutant B1234Q is noticeably different from that of the B34Q mutant (see Fig. 2). Strikingly, the apo At280 value is almost zero, whereas for the B34Q mutant, as noted above, a relatively strong positive value is maintained. A further difference between B1234Q and B34Q is that a greater change toward a more negative At280 is seen upon addition of calcium to B1234Q with a total change of 0.9 as opposed to 0.35 for the B34Q mutant (Table 3). These differences indicate a contribution from calcium binding in the N-terminal domain to the positioning of Tyr-138 and demon- strate that two aspects of the conformation of Tyr-138 are altered when this binding is lost. These are (1) loss of the restricted rotational conformation, seen in the apo form of the B34Q mutant and (2) gain of some ability to move into a conformation showing greater rotational restriction upon calcium binding.

Examination of the At28() changes for the B13Q and B24Q double mutants again allows the effects of mutating one of the N-terminal sites upon the altered responses seen for mutants with one functional C-terminal site to be assessed. The apo Atzso for B13Q is similar to that of B3Q (Fig. 2), but surprisingly the overall change in At2*,, upon calcium binding is increased significantly relative to B3Q. This value is closer to the wild-type value (see Table 3; Fig. 2) and is close to that for the BlQ mutation alone (see Fig. 3 of Maune et al., 1992a). Thus, as found in the UV-difference experiments described above, mutation of site 1 and site 3 appears to adjust the conformational change experienced by Tyr-138 toward that seen for the wild- type protein.

Comparison of the apo and holo At280 values for B24Q with those for B4Q alone reveals that mutation of site 2 also modifies the response of its homologous mutation in the C-terminal domain. The apo value is decreased strongly relative to the B4Q value, to a At28o that is the second lowest recorded among this set of mutants (Table 3; Fig. 2). However, the overall change in Atzxo upon calcium saturation is approximately the same as that seen for the B4Q mutation, that is, a change of about -2 At28o units (Table 3).

SK-MLCK activation

In a previous study (Gao et al., 1993), the ability of the single site Q mutations to activate SK-MLCK was examined. For this enzyme, BlQ proved to be poorest activator, followed by B2Q, then B3Q, with B4Q showing the best activation capacity. Given the findings described above, it was of interest to examine the capacity of these multiple calcium-binding site mutants to activate SK-MLCK. The activation curves are shown in Figure 3 and the derived kinetic parameters listed in Table 4. Data for wild- type calmodulin and the BlQ and B3Q mutants, generated as controls for these experiments, are shown. Although the K,,., values derived for our data are somewhat higher than those of Gao et al., qualitatively the activation abilities of BlQ and B3Q relative to the wild-type protein are as determined previously (compare Table 4 with Table 1 of Gao et al., 1993).

Multiple Ca2+ binding site mutants of calmodulin 473

120

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60 -

40 - G 0

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40

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- 20

-

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log [Calmodulin] M

Fig. 3. Activation of SK-MLCK by multiple calcium-binding site mutants of calmodulin. Activation is normalized as percentage maximal activation for the wild-type protein.

All of the double calcium-binding site mutants proved to be poor activators of SK-MLCK (Fig. 3), considerably worse than any of the single-site mutants examined previously. The B12Q mutant proved to be the best activator of the group (Fig. 3A), but even for this mutant, the KO,, was shifted to the micromo-

Table 4. Kinetic parameters for activation of SK-MLCK by calcium-binding site mutantsa

Calmodulin Koc, (nM) 070 Maximal activation

Wild-type 1.66 B l Q 43. I 93Q 33.4 B13Q 2,236.8 B 12Q 1,311.7 B34Q N D ~ B1234Q N D ~

100 48 89 48

ND 8' 8'

a KO,, is the concentration of calmodulin required for half-maximal activation of the enzyme. VO Maximal activation is the Vmax of a given mutant expressed as a percentage of V,,,, for the wild-type protein.

ND, not determined. Activity at 1,000 nM mutant protein.

lar range- approximately 1,000-fold higher than that of the wild-type protein (see Table 4). A K,,., could not be determined for the B34Q and B1234Q mutants because, even at micromo- lar concentrations, they showed negligible activity. The findings of Gao et al. (1993) demonstrated that mutation of either N-terminal calcium-binding site was more detrimental to activation of SK-MLCK than mutation of either of the C-terminal sites. Thus, the finding that the B34Q mutation is a much worse activator of the enzyme than the B12Q mutant would not have been predicted from those studies.

The UV-difference spectra and near-UV CD analyses described above indicate that the conformation of Tyr-138 in mutant B13Q mutant is closer to that of the wild-type protein than that of the B3Q mutation alone, suggesting that loss of calcium binding at site 1 "corrects" somewhat the conformational prob- lems produced by the B3Q mutation in the region of this residue. Thus, it was of interest to compare the ability of the B13Q mutant to activate SK-MLCK with those of the BlQ and B3Q mutants individually. As can be seen from Figure 3B and Ta- ble 4, the B13Q is a much worse activator of SK-MLCK than either B3Q or BlQ alone. Thus, at least in terms of this enzyme, the two mutations appear to act additively to debilitate the enzyme.

Discussion

Evidence for interdomain interactions

Previous studies with single calcium-binding site mutants suggested that calcium-binding events in the lobe of calmodulin containing Tyr-138 (that is, the C-terminal lobe) play a major role in determining conformation in the environment of Tyr-138 (Maune et al., 1992a, 1992b). The present study reinforces that interpretation. Thus, the near-UV CD response of Tyr-138 in B34Q is the smallest among all the mutants studied and similarly the UV-difference titration changes are minimal. However, from both the UV-difference and near-UV CD data, four comparisons provide striking evidence for effects of events in the N-terminus upon the environment of Tyr-138. These comparisons are: (1) the effects of the double N-terminal mutant B12Q upon Tyr-138 compared to those of the single-site N-terminal mutants; (2) the effects of the quadruple mutant B1234Q compared to the those of the C-terminal double mutant B34Q; (3) the effects of the B13Q mutant compared to those of the B3Q mutant; and (4) the effects of the B24Q mutation compared to those of the B4Q mutation.

We consider each of these comparisons below. In offering in- terpretations of the effects seen, two assumptions concerning the behavior of the mutants proteins have been made. The first is that the mutated sites, if occupied at all during titrations, will fill after any remaining nonmutated functional sites on the protein. This assumption is founded on the known effects of the E to Q mutation in each of the binding sites as determined in previous studies (Maune et al., 1992a, 1992b; Starovasnik et al., 1992). The second assumption is that the conformation of the apo form of the mutant proteins is very similar to that of the wild-type protein, but that loss of calcium binding at a partic- ular site results in loss of those components of the calcium- induced conformational changes normally associated with calcium binding at those sites. This assumption is supported by far- UV CD studies of these mutant proteins (Mukherjea, 1995). For

414 P. Mukherjea et ai.

all four double mutants studied here, the UV CD signal at 222 nm for the apo form is very similar to wild type, but the calcium-induced change is diminished. For the quadruple mutant, there is a very limited calcium-induced change and the apo 222-nm signal is approximately 80% of the wild-type value. Thus, for this mutant, far-UV CD indicates changes to the apo structure that are relevant to our observations (see below).

B12Q versus BlQ, B2Q, and wild-type calmodulin The UV-difference data revealed an unexpected difference be-

tween mutant B12Q and the single-site mutants BlQ and B2Q and the wild-type protein; the aspect of conformational change at Tyr-138 monitored by this parameter is completed upon addition of one equivalent of calcium instead of two. This surpris- ing finding requires consideration of why the UV-difference spectral changes to Tyr-138 for the wild-type protein require addition of two equivalents of calcium. One obvious possibility is that calcium binding to both C-terminal sites affects Tyr-138. Our findings for mutants B3Q, B4Q, and B34Q clearly support this argument. If only one of the C-terminal sites could affect Tyr-138, the titration curve for B34Q would resemble that of either B3Q or B4Q, as opposed the observed result, which is that the Tyr-138 response is essentially eliminated. However, even if calcium binding to a single C-terminal site were responsible for the Tyr-138 UV-difference changes, they would still occur over addition of two equivalents of calcium, because coopera- tivity in the C-terminus (Linse et al., 1991) produces overlap- ping occupancy of the two sites on individual molecules, and the critical site would not be occupied on all calmodulin molecules until this point. Thus, for mutation of the two N-terminal sites to produce a change in Tyr-138 that requires addition of only one equivalent of calcium implies, at the very least, that coop- erativity in the C-terminus is altered and occupancy of one C-terminal site lags behind its partner. The near-UV CD data for B12Q provide some support for this interpretation. As dis- cussed in the Results, the change to A t 2 ~ o to B12Q is not complete upon addition of one equivalent of calcium, but rather takes addition of between two and four equivalents of calcium to saturate. This finding indicates that the calcium affinity of at least one C-terminal site has been reduced.

Whatever the explanation, some interdomain interaction must be evoked to explain the effects of the B12Q mutant upon Tyr- 138. However, it is important to recognize that the data for this interaction (and for all the other interdomain interactions dis- cussed here) can be interpreted as evidence for interdomain communication before or after mutational changes. Thus, the wild-type behavior of Tyr-138 may reflect an interaction of the N- and C-terminal domains that is lost upon mutation of the N-terminal sites or , alternatively, the interaction is newly induced as a result of mutating these sites.

B34Q versus B1234Q For the comparison of B1234Q with B34Q, the near-UV CD

data provide evidence for a difference in the behavior of Tyr- 138 and, thus, for a role for N-terminal events upon conformation in the vicinity of this residue. Although for B1234Q the magnitude of the near-UV CD change to Tyr-138 is somewhat greater than that seen for B34Q, the more significant difference between them is seen in the AeZso values for the apo forms of the two proteins. The A€,,, value of close to zero for B1234Q

suggests that, after mutation of all four calcium-binding sites, Tyr-138 is essentially unrestrained in this protein. In contrast, in the apo form of B34Q mutant, the residue gives a significant AtrRo value. This suggests a role for the wild-type N-terminus in providing structure in the environment of Tyr-138. As noted above, the far-UV CD signal for the apo form of B1234Q is diminished relative to the wild-type apo signal.

B13Q versus B3Q In mutant B3Q, the behavior of Tyr-138 is markedly affected

(Maune et al., 1992a, 1992b). The studies presented here show that mutating site 1 in the N-terminus strongly modifies the responses of Tyr-138 associated with the B3Q mutation, produc- ing near-UV CD and UV-difference changes that are more similar to the wild-type behavior. The striking UV-difference spectral changes seen in B3Q were interpreted previously as orig- inating entirely from anomalous events in the C-terminus. How- ever, our discovery here that loss of binding at site l dramatically modifies those changes leads to a new interpretation of the B3Q UV-difference spectra. It suggests that the aberrant behavior of the B3Q mutant during addition of the first two aliquots of calcium is produced by generation of one or more calmodulin conformers not normally seen in the wild-type titration in which the remaining C-terminal site (site 4) and one N-terminal site are occupied. Given that the B13Q mutation, a protein in which calcium binding is primarily to sites 2 and 4, gives UV-difference titration changes for Tyr-138 similar to the wild-type protein, it would be predicted that the unusual species with sites 1 and 4 occupied is the main source of the aberrant behavior for Tyr- 138 seen during titration of B3Q rather than the species with sites 2 and 4 occupied.

B24Q versus B4Q A similar logic can be evoked to explain the differences be-

tween the B4Q mutant and the B24Q mutant. That is, the aberrant behavior of Tyr-138 in B4Q can be viewed as arising from unusual conformers in which site 3 and one of the N-terminal sites is occupied rather than from events restricted to the C-terminus. The finding that the UV-difference response of Tyr- 138 is essentially lost in B24Q suggests that a species with calcium bound at site 2 plays a prominent role in determining the spectral properties of the B4Q mutant.

A comparison of the UV-difference spectral properties of mutant B13Q (in which sites 2 and 4 are the functional binding sites) with mutant B24Q (in which sites 1 and 3 are the active sites) permits an evaluation of the efficacy of these two pairs of sites in affecting conformation at Tyr-138. In B13Q, Tyr-138 responds promptly, giving a calcium titration similar to the wild- type protein. In B24Q, the response of Tyr-138 is minimal. These findings reinforce previous indications (see the Introduction) that calcium binding at sites 2 and 4 influences calcium-induced conformational change more strongly than calcium-binding at sites 1 and 3.

Activation of SK-MLCK by the multiple mutants

Although the UV-difference and near-UV CD data described above suggest that the local environment of Tyr 138 is closer to wild type in mutant B13Q than in the single mutant B3Q, the activation data for SM-MLCK indicate that the overall cal-


cium-bound conformation of B13Q is less like that of the wild- type protein than that of either mutation alone. The previous studies of enzyme activation by individual calcium-binding site mutants of calmodulin (Gao et al., 1993) predict that the effects of preventing calcium binding at two sites on the protein would be cumulative. Thus, given that loss of calcium binding and the associated calcium-induced conformational change at any single binding site of calmodulin significantly decrease the ability of the protein to activate several target enzymes, it is reasonable to expect that preventing two components of the calcium- induced change would be more deleterious than preventing one.

Although all of the multiple mutants examined are poor activators of SK-MLCK, the double mutant with both N-terminal binding sites inactivated emerged as the best activator of the set. The double C-terminal mutant and the quadruple site mutant showed the lowest activity. These rankings would not have been predicted from the previous studies of SK-MLCK activation by the single calcium-binding site mutants (Gao et al., 1993), because mutants BlQ and B2Q both proved to be worse activators than B3Q or B4Q. However, studies with the related enzyme, smooth muscle MLCK (VanBerkum & Means, 1991), suggest that interaction of the SK-MLCK target binding region with the C-terminal lobe of calmodulin is the initiating event in enzyme activation, and thus that complete loss of calcium- induced conformational change in this half of calmodulin might be expected to have a more deleterious effect on enzyme activation.

Analysis in terms of the known properties of Tyr-138

In order to understand how changes in the calcium-binding properties of the N-terminus of calmodulin may affect the Tyr- 138 in the C-terminus, it will be necessary to understand in de- tail the calcium-induced structural changes to this residue. NMR studies of both wild type (Seamon, 1980) and single calcium- binding site mutants (Starovasnik, 1992) indicate that Tyr-138 can respond to at least three different processes during calcium titration. Time-resolved fluorescence anisotropy (Torok et al., 1992) has indicated that side-chain movement of Tyr-138 be- comes more restricted upon calcium binding. Recently, incorporation of Tyr-138 into the exiting a-helix of site 4 has been shown to be a calcium-induced event (Kuboniwa et al., 1995)

One possible mechanism whereby N-terminal calcium binding might affect Tyr-138 is by direct interaction between the two terminal lobes of the protein. However, although the two lobes can approach more closely in solution than indicated by the crystal structure (Seaton et al., 1985; Heidorn & Trewhella, 1988), no NOE interactions are detected between side chains located in opposing terminal lobes of calmodulin in either the apo- or the holo- form of the protein (Ikuraet al., 1991). Therefore, on the time scale detected by NMR, no residues of the N-terminus are within 5 A of any C-terminal residues, suggesting that direct interaction between the two terminal lobes is unlikely.

The alternative route for N-terminal events to influence Tyr- 138 would be through the central linker region of the protein. Available data for Tyr-138 support this interpretation. The hydroxyl group of Tyr-138 has an unusually high pK that is un- affected by calcium binding (Richman & Klee, 1978, 1979). This suggests that Tyr-138 is buried within the hydrophobic core of calmodulin and remains so upon calcium saturation. In the crystal structure of holo-calmodulin, Tyr-138 is clearly part of an

organized system of hydrophobic residues, but, unexpectedly, the hydroxyl group of Tyr-138 forms a hydrogen bond with glutamate 82- one of three adjacent glutamates in the continuous central helix of the crystallized form of the protein (Babu et al., 1988). In solution, however, NMR studies indicate that the secondary structure of the central linker region is in dynamic flux and is influenced by calcium binding. Thus, in the apo form, residues 76-81 appear to adopt a helical conformation approximately one third of the time (Kuboniwa et al., 1995), whereas there is no evidence for any helical structure in this region of holo-calmodulin (Barbato et al., 1992; Kuboniwa et al., 1995). It seems possible therefore that, in solution, the hydrogen bond between Tyr-138 and Glu-82 is a dynamic feature of the protein, that could be influenced by calcium-binding events in the N-terminal lobe of the protein.

This idea that information concerning events in the N-terminus might be transmitted to Tyr-138 via the central linker region is given some validity by recent findings for the crystal structure of calmodulin with one molecule of trifluoperazine bound to the C-terminal lobe (Cook et al., 1994). Surprisingly, this binding event is sufficient to contort the central linker region and N-terminal lobe into the bent conformation seen for calmodulin with a target peptide bound to both the N- and C-terminal halves of the protein. Thus, an event primarily in one lobe of calmodulin can produce conformational changes in the central linker region of the protein.

Materials and methods

Molecular cloning

Generation of calmodulin cDNA constructs for wild-type Dro- sophila calmodulin and the single-site mutants BlQ, B2Q, B3Q, and B4Q has been described previously (Maune et al., 1992b). The double mutants used here were prepared by combining restriction fragments derived from the appropriate single-site mutant constructs. Thus, (1) the double mutants B12Q and B13Q were generated by replacing the Pst I-Sal I fragment of BlQ with the equivalent fragments of B2Q and B3Q, respectively; and (2) the double mutants B24Q and B34Q were generated by replacing the Nco I-EcoR V fragment of B4Q with the equivalent fragments of B2Q and B3Q, respectively. The quadruple mutant was generated by ligation of the BamH I-Fok I fragment of the B12Q mutant and the Fok I-EcoR I fragment of the B34Q mutant into the BamH I - EcoR I sites of vector pEMBL8’.

All multiple mutant constructs were sequenced throughout the entire calmodulin coding region prior to transfer to an expression vector. In some cases, proteins were expressed from the vector POTS Nco 12, as described previously (Maune et al., 1992b). For some mutants, the vector PET 15b (Novagen) was used. In these cases, mutant Nco I-Sal I cDNA fragments were trans- ferred to the Nco I and Xho I sites of PET 15b.

Protein expression and purification

Wild-type Drosophila calmodulin and calmodulins containing single binding site mutants were purified as described previously using affinity chromatography on phenyl-Sepharose (Maune et al., 1992b). For mutants expressed from PET 15b constructs, the DE3 pLysS host cells were grown in 2 L of “terrific broth” (Tartof & Hobbs, 1987) at 30 “C to earlylmid log phase and then

476 P. Mukherjea et al.

protein expression was induced by addition of IPTG to 30 pM followed by incubation at 30 "C for a further 16-20 h. Phenyl- Sepharose chromatography could not be used for the multiple binding site mutants and therefore an alternative purification scheme was devised, based on that of Newton et al. (1988). The start point for the purification was a 100,ooO-g supernatant prepared after French press disruption of the bacterial suspensions as described previously (Maune et al., 1992b). Ammonium sulfate fractionation was used as the first purification step. The pellet formed between 60 and 80% ammonium sulfate saturation was dialyzed against 10 mM histidine buffer, H 5.6, containing 1 mM CaCl, (Buffer H). After determination of the protein concentration of the solubilized dialysate (Bio-Rad protein assay), it was adjusted to approximately 10 mg/mL protein, loaded onto a DEAE-Sephacel column at 4 "C, and eluted with a linear salt gradient of 0-0.4 M NaCl, in Buffer H. Fractions containing calmodulin were identified by SDS-PAGE, pooled, and dialyzed against Buffer H containing 20% PEG (15,000- 20,000 MW) or Aquacide (Calbiochem) to concentrate the protein. After a further protein concentration determination, the dialysate was adjusted to approximately 2 mg/mL protein for HPLC on an anion exchange column (AX300, 250x 10 mM). For each HPLC run, about 20 mg of protein were used and eluted with a 0-1.5 M NaCl gradient in Buffer H. Fractions containing calmodulin were identified by SDS-PAGE, pooled, and dialyzed against 20 mM Tris-HC1, pH 7.0, 1 mM CaCI,, again containing PEG or Aquacide. HPLC on the AX300 resin was repeated, again using a 0-1.5 M NaCl gradient and the purity of the calmodulin-containing fractions was assessed by SDS- PAGE and absorbance in the 200-300-nm range. After these four purification steps, all of the multiple mutants proteins were purified to homogeneity, although yields were low (approximately 20 mg protein per 14 L of bacterial culture).

Decalcification and desalting of proteins

Buffer exchange and desalting of proteins was achieved either by repeated passage over (3-25 Sephadex (PD-10 columns, Phar- macia) or by extensive dialysis (Spectra-por tubing, MW cut-off 6,000-8,000). Proteins were decalcified by Chelex 100 treatment, as described previously (Maune et al., 1992b), immediately before use and after exchange into the appropriate buffer.

U V extinction coefficient determinations

Like wild-type calmodulin, all of the multiple mutants studied here contain no tryptophan or cysteine, a single tyrosine, and nine phenylalanine residues. Extinction coefficients at the absorbance maximum for the tyrosine peak (at approximately 279 nm) could thus be determined using a 9:l mixture of the N-acetyl methyl esters of phenylalanine and tyrosine, as described previously (Maune et al., 1992b).

UV-difference spectroscopy

UV absorbance spectra were collected on a Shimadzu UV- 2101-PC spectrophotometer during sequential addition of equivalents of CaCl, to decalcified proteins at 100 pM concentration in 1 0 0 mM KCI, 10 mM Hepes, pH 7.6. After incremental addition of four equivalents of CaCl,, six equivalents were added, followed by a further six equivalents (16 equivalents total). Ap-

propriate buffers were used for baseline determinations and all spectra were corrected for dilution and baseline effects. Data were processed using the UV2101/3101 Kinetics software package.

Near- U V CD

CD spectra were recorded at 22 "C using a Jasco 5-600 spectro- polarimeter, with a sampling interval of 0.1 nm. Near-UV CD spectra (250-310 nm) were recorded using a pathlength of 10 mm. Mutant proteins at 100 pM concentration in 20 mM Hepes/KOH buffer, p H 7.6, were used for the experiments and spectra were recorded for the initial decalcified samples and after addition of 2, 4, and 10 equivalents of CaCl,. At least three scans were averaged for each spectrum, with baseline corrections for the buffer, light scattering (as measured at 310-350 nm), and protein dilution. Data processing of the ASCII files was performed using the program PS plot of Micro-cal. As in previous work (Maune et al., 1992a), data are presented in terms of the mean residue CD, Acaso, on the basis of a mean residue weight of 112.7.

SK-MLCK assays

SK-MLCK activity was assayed by incorporation of 32P into the regulatory light chain largely as described previously (Gao et al., 1993). The reaction mixture (50 pL final volume) consisted of 50 mM MOPS, pH 7.0, 100 pM CaCl,, 1 mM dithiothreitol, 30pM regulatory light chain, 5 nM SK-MLCK, and 1 mM y3*P ATP (200-250 cpm/pmol). Calmodulin concentrations of 0.05 nM-1 pM were used for the wild-type protein and 1 .OO nM-5 pM for the mutant calmodulins. Activation was normalized as per- cent activation for the wild-type protein (20 pmol of 32P/ midmg).

Acknowledgments

This work was supported by the following grants to K.B.: NIH grant GM49155, grant 003604-028 of the Advanced Research/Advanced Tech- nology Program of the Texas Higher Education Board, and Welch Foun- dation Grant C-1119. We thank Drs. James Stull and Beatrice Clack for their generous help with the myosin light chain kinase assays described here. We are grateful to Drs. John Olson and Fred Rudolph and the members of their laboratories for helpful discussions and the use of many facilities. Spectropolarimetry was performed on an instrument belong- ing to the Institute of Biosciences and Technology of the Texas A&M University. We thank Jacquelynn Larson for her help with the use of that instrument. We appreciate the helpful suggestions of our colleagues Bernard Andruss, Dr. Richard Atkinson, and Dr. Heidi Nelson, after initial reading of the manuscript.

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Interlobe communication in multiple calcium-binding site mutants of