Topographical Mapping of Calmodulin-Target Enzyme Interaction ...

Vol. 264, No. 4, Issue of February 5, pp. 2373-2378, 1989 Printed in U.S.A .

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1989 by The American Society for Biochemistry and Molecular Biology, Inc.

Topographical Mapping of Calmodulin-Target Enzyme Interaction Domains* (Received for publication, June 14, 1988)

Dennis M. MannSg and Thomas C. Vanaman7 From the $Graduate Center for Toxicology and the llDepartment of Biochemistry, Albert B. Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40536

Calmodulin derivatives, specifically biotinylated in domains I and 111, were synthesized to address the structures of calmodulin necessary for binding to its target enzymes in active conformations. By binding avidin to these biotinylated calmodulins, the role of specific sequences of the calmodulin molecule in target enzyme interactions could then be evaluated. The role of domain I in these interactions was assessed by biotinylation of Cys-27 of wheat germ calmodulin with N-ethylmaleimidobiotin. This modification did not affect the ability of this calmodulin to activate 3’-5’cyclic pucleotide phosphodiesterase (PDE) or human erythrocyte Ca2+-M&+ATPase. The addition of avidin to form a stable calmodulin-avidin complex also did not affectactivation.Bovinetestes calmodulin was biotinylated on Lys-94 by calcium-dependent reaction with N-hydroxysuccinimido ester-biotin at pH 6.0. This derivative was used to probe the Ca+”binding region of domain 111. The incorporation of biotin at Lys-94 of bovine calmodulin did not affect calmodulin activation of PDE. However, compared to unmodified calmodulin, a 4-fold higher concentration of this derivative was required to fully activate the ATPase. The addition of excess avidin to this derivative abolished all activation for both PDE and the ATPase. Sites of modification were determined by sequence analysis of labeled peptides.

1-77 and 78-148), which retain the ability to bind to phenothiazine affinity columns in a calcium-dependent manner. The COOH-terminal (residues 78-148) half can competitively inhibit stimulation of PDE‘ by whole calmodulin (4). Both the 78-148 COOH-terminal half moleculeand aminor trypsin fragment containing residues 1-106 retain the ability to stimulate the ATPase, although when compared to intact calmodulin, much greater amounts arerequired (5). These fragments, coupled to Sephrose 4B, have been used to evaluate the ability of different target enzymes to recognize different domains of calmodulin (6). A wide variety of hydrophobic compounds have been reported to inhibit calmodulin stimulation of target enzymes, including antipyschotics (7), antineoplastic agents (8), and small peptides (9-10). Attempts to map binding sites for these compounds have led to the use of reactive phenothiazine derivatives to produce specifically modifiedcalmodulin derivatives. Calmodulin modified with norchlorpromazine isothiocyanate to a stoichiometry of 1:l has been reported to retain the ability to bind to PDE but does not give activation (11). Through amino acid analysis of modified peptides, this calmodulin has been suggested to be modifiedspecifically at Lys75 (12). Recently, calmodulin modified with POS-TP, another reactive phenothiazine, was determined to be modified specifically at Lys-148. This modification did not affect the ability of the calmodulin to stimuIate PDE or the ATPase, whereas plant NAD kinase was not activated by this derivative (13). A fluorenyl-based spin label derivative of calmodulin, believed to be modifiedat Lys-148 and Lys-75, as determined by amino The interactions of the calcium-dependent regulatory pro- acid analysis of labeled peptides, is incapable of binding to or tein calmodulin (for reviews, see Refs. 1and 2) with its target stimulating PDE, although treatment of the derivative with enzymes are only poorly understood. Although the threebase to hydrolyze a portion of the adduct results in the dimensional crystal structure of calcium-bound calmodulin restoration of some activation (14). has been elucidated (3),the exact structuresinvolved in target The role of lysyl residues in the binding of calmodulin to enzyme binding and activation remain unclear. In the presskeletal muscle MLCK and calcineurin was assessed by aceence of calcium, the three-dimensional structure of the protein tylation in the presence of these enzymes. Lys-75 was deterresembles a highly asymmetric dog bone, with a long eightmined to be substantially protected from acetylation in the turn central a-helical segment (helix IV). The hydrophobic presence of both calcineurin (15) and MLCK (16).In contrast, “pocket” formed by helices I1 and I11 has been proposed to be Lys-77 was shown to be protected from acetylation in the involved in forming sites for Ca+’-dependent drug and target presence of calcineurin in a recent reportby Winkler and coenzyme binding (3). workers (17). Several approaches have been used to isolate and characThe synthesis of a calmodul’n gene has allowed generation terize specific areas of calmodulin necessary for target enzyme of a series of artificial calmodulins that resemble bovine testes binding and activation. Limited trypsin digestion of calmodcalmodulin with the exception of two missing posttranslaulin in the presence of calcium yields half molecules (residues tional modifications (18). Replacement of residues 81-84 * This work was supported by Research Grant NS-21868 from the (Glu-Glu-Glu) in the central helix (helix IV) with Lys-LysNational Institutes of Health. The costs of publication of this article were defrayed in part by the payment, of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. J To whom correspondence should be addressed: Dept. of Biochemistry, College of Medicine, University of Kentucky, Lexington, KY 40536.

The abbreviations used are: PDE, bovine brain 3’-5’-cyclic nucleotide phosphodiesterase; POS, TP-lO-(3-propinyloxysuccinimide) (2-trifluoromethyl)phenothiazine;MLCK, skeletal musclemyosin light chain kinase; NHS, N-hydroxysuccinimido ester; MES, 4-morpholineethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)] tetraacetic acid; PTH, phenylthiohydantoin.

2373

2374

Domains

InteractionCalmodulin

HCI, 0.8 M sodium chloride, pH 7.0. Separations were achieved using a gradient from 40-60% B increasing a t l%/min.Peaks of UVabsorbing materialwere collected manually andanalyzed as described in following sections. Relative amounts of native and modified cal(19). modulins were determined by manual integration of the UV elution To draw valid conclusions about the effects of specific profiles or by automated peak integration using a Hewlett-Packard modifications on the functional propertiesof calmodulin, the 3392A integrator. Determination of Sites of Biotinyylation-Sixty-five nmol of bovine site of modification mustbe unambiguously determined. Herein, we report the formation and functional characteriza- testes calmodulin were reacted with [3H]NHS-biotin(specific activity = 2.6 mCi/mmol, final concentration 150 p M ) in the reaction mixture tion of specifically biotinylated calmodulin derivatives. The described above. Fromthisreactionmixture, 14nmol of labeled use of biotin enables avidin,a large globularprotein withhigh calmodulin (specific activity = 3.0 mCi/mmol) were purified by reaffinity (Kd > lo-") for biotin (20) to be bound to calmodulin petitive HPLC using the DEAE-anion exchange system described at specified sites. The role of these sites in target enzyme previously, desalted over Sephadex G-50, and lyophilized. The tritiinteractions can then be assessed through quantitation of ated biotinylated calmodulin was digested with trypsin and the resulting peptideswere separated as described by Klevit and Vanaman functional effects. (30). The labeled peptides obtained were desalted using a reversedphase C-3 column with 0.1% trifluoroacetic acid in deionized water EXPERIMENTALPROCEDURES as bufferA and 0.05% trifluoroacetic acid, 88% acetonitrile, 12% deionized water as buffer B and a l%/min increasing gradient from Materials 10-50% B. Following lyophilization, thepeptides were sequenced Calmodulin was isolated from bovine testes according to the pro- using an Applied Biosystems Vapor-Phase Sequenator, as described cedure of Jamieson and Vanaman (21) or from wheat germ (Inter- by Hunkapillar andco-workers (31).The PTH-aminoacid derivatives nationalMulti Foods) by the procedure of Andersonet al. (22). obtained from the sequenatorwere identified and quantified usinga Calmodulin-deficient 3'-5'-cyclic nucleotide phosphodiesterase was Du Pont Bioseries PTH-amino acid column as described by Glajch partially purified from fresh bovine brain by the procedure of Klee and co-workers (32). For biotinylated wheat germ calmodulin, native and biotinylated and Krinks (23) and was activated 7-fold by saturating concentrations species were separated by repetitive HPLC on DEAE-Spherogel using of calmodulin. Calmodulin-sensitive Ca"-Mgf2 ATPase was isolated from human erythrocytes, reconstituted into phospholipid vesicles, the conditions described above. Pure biotinylated wheat germ calmodulin (as verifiedby nondenaturing gel electrophoresisinthe and assayedusingacoupledenzyme determination of inorganic phosphate according to the procedure of Niggli and co-workers (24). presence and absenceof excess avidin) was digested with trypsin and The enzyme was activated 6-fold by saturating calmodulin concen- the peptides separatedas described by Klevit and Vanaman (30). The trations.NHS-biotinand[3H]NHS-biotin (specific activity = 2.6 biotinylated peptide was identified by mixing the digest with avidinmCi/mmol) were synthesized using theprocedure of Bayer and Wil- agarose, centrifuging, and separating the peptides remaining in the chek (25) from biotin (Sigma) or [3H]biotin (Du Pont-New England supernatant as described above. The biotinylated peptide was obNuclear, specific activity = 50 mCi/mmol). Calmidazolium was pur- tained by difference between the chromatogram obtained from inchased fromBoehringer Mannheim. L-l-Tosylamido-2-phenylethyl jecting this sample and that obtainedfrom an identical sample that chloromethyl ketone-trypsin andsoybean trypsin inhibitor were pur- had not been mixed with avidin-agarose. Following desalting, the site chased from Worthington. HPLC grade acetonitrile was purchased of modification was determined by sequence analysis of the peptide capable of binding to avidinagarose as described above. from Burdick and Jackson. Aqueous HPLC buffers, prepared with Functional Assays of Modified CaEmodulins-Partially purified 3'Milli-Q water, were filtered through 0.2-pm millipore filters prior to use. HPLC columns (DEAE-TSK Spherogel2SW4 X 250 mm) were 5'-cyclic nucleotide phosphodiesterase was assayed for activation by purchasedfrom Altex. Exceptasnoted,allother chemicals and native DEAE-HPLC-purified calmodulin and the specifically modified calmodulins by the methodof Wallace et al. (33). The biotinylated reagents were purchased from Sigma. andnativecalmodulins were assayed for abilitytostimulatethe enzyme in the presence and absenceof excess (100 pg) avidin. Ca+*Methods Mg+'-ATPase was purified from human erythrocytes, reconstituted Reaction of Calmodulin with NHS-Biotin-Initial reactions were into phospholipid vesicles, and assayed for stimulation using a couperformed at pH 8.0 using the conditions outlined in the previous pled enzyme assay for the determination of nmoles of released inorganic phosphate, which were converted to percent of control (maxisection for reactionof nitrosoureas with calmodulin. As modification was found tooccur both rapidly and ina relatively calcium-independ- mal) activation (24). Lys-94 biotinyl calmodulin andCys-27 biotinyl ent manner, it was necessary to perform the modifications at pH 6.0 calmodulin were assayed and compared to the respective HPLCpurified native calmodulins for activation. The biotinylated derivato enhance monoadduct formation and calcium dependence. Reactives were also assayed in the presenceof excess (20 pg) avidin. tions were conducted a t room temperatureovernightin abuffer containing 30 mM MES, pH6.0, and 2 mM calcium chloride with 150 p~ NHS-biotin. NHS-Biotin was first dissolved in dimethylformRESULTS amideandaddedin 4 % of thetotalreaction volume. Calcium dependence was assessed by replacement of the calcium chloride with Modification With NHS-Biotin-Thecalcium-dependent 2 mM EGTA. Excess reagent was quenched with a 10-foldmolar modification of calmodulin at pH 6.0 by [3H]NHS-biotin is excess of free lysine. shown in Fig. 1,panel C. The major adduct formed in the Biotinylation of Wheat Germ Calmoduylin-From 2 kg of fresh wheat germ, 18 mg of pure calmodulin was obtained. The free thiol content presence of calcium (peak 2, panel C), was purified by multiple separations using DEAE-anion exchange chromatography. was estimated to be greater than 90%by carboxymethylation (26) followed by visual quantitation of Coomassie Blue-stained polyacryl- Although some modification was evident in the absence of amide gels run under nondenaturing conditions (27) and approxicalcium(Fig. 1, panel B ) , this represents only 12% of the mately 50% by reaction of wheat germ calmodulin with Ellmann's available calmodulin. Biotinylated calmodulin (peak 2) was reagent (28). The reason for this discrepancy is unclear. Wheat germ electrophoresed on a nondenaturing gel in the presence and calmodulin was modified using essentially the method of Riordan and Vallee (29) for the modification of cysteine with N-ethylmaleimide. absence of excess avidin (Fig. 2, lanes C and D ) . No calmodBriefly, N-ethylmaleimido-biotin, a t a final concentration of 1 mM ulin was observed in the biotinylated calmodulin plus avidin was dissolved in dimethylsulfoxide (Burdick and Jackson), and addedlane. Following purification, thisderivative was digestedwith in a minimum volume to a 100mM sodium phosphate, pH 7.0, solution trypsin and the resulting peptides separated as shownFig. in containing 2 mM EGTA and 20 ~ L Mwheat germcalmodulin. The 3. Two fractions containing radiolabel were obtained from mixture was incubated for 30 min at room temperature, and the this digest; the first contained a doublet eluting at 24.31 and reaction was stopped by addition of excess 8-mercaptoethanol. 25.41 min, and the second, a single peak eluting a t 33.58 min. HPLC Analysis and Purification of Modified Calmodulins-A of the radioactivity (6.3 DEAE-TSK spherogel2SW 4 X 250 mm was used for allseparations. The latter contained the majority Buffer A was 10 mM Tris-HCI, pH 7.0, and buffer B was 10 mM Tris- nmol, 3.0 mCi/mmol). The aminoacid sequenceof the peptide

Lysresulted in acalmodulin thatretaineditsabilityto stimulate PDE, had30% maximal ability to stimulatemyosin light chain kinase, but was unable to activate NAD kinase

Domains InteractionCalmodulin

2375

a

n

V I

O

n

f 20 i IO

20

ELUTION TIME

40 (rnin)

20

40

FIG. 1. Calcium-dependent modification of 10 PM calmodulin by 150 PM NHS-biotin. Reactions were performed as described under “ExperimentalProcedures.” A, the failure of C-3-reversedphase HPLC to resolvebiotinylated and native calmodulins. C, the samesample resolved by DEAE-anion exchange HPLC. Peak 1 is in the position of unmodified calmodulin and peak 2 is the main derivative peak. For A and C, the modification was done in the presence of 2 mM calcium chloride. B, the modification was conducted in 2 mM EGTA and resolved using DEAE-anion exchangeHPLC.

A

B

C

D

FIG. 2. Nondenaturing polyacrylamide (15%) gel electrophoresis of biotinylated HPLC-purified calmodulins in the presence and absence of avidin. Lane A is 10 pg of HPLC-purified Cys-2720 pg of avidin. Lane B is the same biotinylated calmodulin mixed with without avidin. Lane amount of biotinylated calmodulin electrophoresed C is 10 pg of Lys-94-biotinylated calmodulinand 20 pg of avidin, and Lane D is the Lys-94-biotinylated calmodulin alone.

eluting at 33.58 min (Val-91-Arg-105)is shown in Table I, A. The majority of the radioactivity was recovered in cycle 4. Liquid scintillation analysis of the elution profile obtained from PTH analysis of cycle 5 (Asp-95) indicated that no radioactivity was present in the PTH-Asppeak (not shown), whereas correlation of the radioactivity in thebiotinyl-PTHLys peak indicated this compound to have a specific activity

I

I

10

20

30

40

FRACTION NUMBER

FIG. 3. The peptide map resulting from complete tryptic digestion of 14 nmol of anion exchange HPLC-purifiedNHS-biotinylated calmodulin(Fig. lC,peakZ).One-min (1.5 ml) fractions were collected and assayed for radioactivity.The histogram in the top panel shows the total amount of radioactivity contained in the two labeled fractions. The first contained a doublet withpeaks eluting at 29.34 and 29.77 min, and thesecond a single peakeluting at 33.53 min.

of 3.0mCi/mmol, which is in good agreement with the specific activity obtained for the original modified calmodulin. Further separation of the doublet using reversed-phase C-3 chromatography as described aboveshowed the radiolabel to be exclusively in the component eluting a t 24.31 min (2.8 nmol, 3.0 mCi/mmol). The sequence of this peptide was shown to be identical to thatdescribed above in that the radiolabel was detected only at thecycle releasing PTH-Lys-94. This tryptic peptide is commonly found at two different elution times (34). Biotinylation of Plant Calmodulin at Cys-27-The separation of native and biotinylated wheat germ calmodulin is shown in Fig. 4. The original reaction mixture is shown in panel A, Fig. 4. Peak 1represents unmodified plant calmodulin and peak 2, the biotinylated species. Peak 2 was collectedand reinjected using the same separation system as described for the separation of NHS-biotin-modified mammalian calmodulin and, as shown in panel C, was free of unmodified plant calmodulin. This preparation was shown to be pure biotinylated plant calmodulin by nondenaturing gel electrophoresis in thepresence and absence of avidin (Fig. 2, lanesA and B). Following trypsin digestion, the biotin-containing peptide was isolated as shown in Fig. 5. The arrow points to thepeak eluting a t 33.44 min that was absorbed to avidin agarose. This peak was collected as a discrete fraction in subsequent injectionsandthe peptide desalted using C-3 reversed-phase HPLC as described previously. The biotinylated peptide was sequenced, and thebiotin was determined to be on Cys-27 by altered retention timeof biotinyl-PTH-Cys-27 (8.4 min) compared to unmodified PTH-Cys-27 (3.5 min). The sequence of the biotinylated peptide is shown in Table I, B. Functional Effects of Lysine Modification-Both biotinylated calmodulin derivatives were tested forability to stimulate enzymes in the presence and absence of excess avidin. The modification itself did not affect the ability of Lys-94 biotinyl calmodulin to stimulate PDE (Fig. 6A). The addition of excess avidin to the enzyme assay, while having no effect on the ability of unmodified calmodulin to stimulate the enzyme, completely abolished all stimulatory activity by Lys-94-biotinylated calmodulin for PDE (Fig. 5A). In the presence of excess avidin, 100 ng of this calmodulin had no effect on the activation of PDE by unmodified calmodulin (not shown). The stimulation of the ATPase by this derivative is more complex. The biotinylation itself required addition of 4-fold higher concentrations of Lys-94-biotinylated calmodulin to reach maximal stimulation when compared to ATPase stimulation by unmodified calmodulin (Fig. 6B).The addition of excess avidin to the assay system also abolished all stimula-

Domains InteractionCalmodulin

2376

TABLE I Sequence of [3H1NHS-biotin-labeledpeptide no.

Cycle

1

2

3

4

5

6

7

8

9 1 0 1 1 1 2 1 3 1 4 1 5

Expected sequence (91-104)” Pmol PTHaa* detected

Val-Phe-Asp-Lys-Asp-Gly-Asn-Tyr-Ile-Ser-Ala-Ala-Glu-Leu-Arg

cpm/cycle‘

------------1600-230---------------------------------

521 520

60 231 55

99

41

87

102

ND 114 81

70

81

ND

Seauence of N-ethvlmaleimide-biotinvlatedDeDtide no. Cycle Sequence (15-31 ) nmol PTHaa detectede

1

2

3

4

5

6

7

8

9

1 0 1 1 1 2 1 3 1 4 1 5

Glu-Ala-Phe-Ser-Leu-Phe-Asp-Lys-Asp-Gly-Asp-Gly-Cys-Ile-Thr 2.9 3.5

3.9 0.2 3.5 3.0 0.9 2.5 1.1 1.1

1.2 1.5 ND

1.5 0.4

Taken from the known sequence of mammalian calmodulin (34). nmol of each PTH amino acid derivative recovered/sequencer cycle, as outlined under “Experimental Procedures.” ND means none detected. For each sequencer cycle, an aliquot was assayed for radioactivity by liquid scintillation spectrometry. The numbers listed refer to counts/min above background. The known sequence of plant calmodulin from residues 15-31 (41). e The nmol of PTH amino acid present in each cycle, as quantitated using a Waters Data Module. For Cys-27, none of this PTH amino acid was recovered in the sequence of this biotinylated peptide as the modification changes the retention time of PTH-Cys.

I

10

I

I

20 30 ELUTION TIME ( m l n )

40

FIG. 5. The trypsin digestionof biotinylated plantcalmoddin is shown in panel A. The effect of incubating the biotinylated plant calmodulin digest with avidin agarose is shown in p a n e l B. The armw indicates the missing peptide in p a n e l B, which eluted at 33.44 min in p a n e l A. DISCUSSION

The studies presented here address three distinct questions concerning the structure and function of calmodulin. First, IO 20 the specificity of lysine modification with a relatively hydrophilic reagent, NHS-biotin, is addressed. Previously, reactive ELUTION TIME (mln) FIG. 4. DEAE-HPLC separation of N-ethylmaleimide-biotin- hydrophobic phenothiazine derivatives have been employed ylated plant calmoddin. In p a n e l A, the original reactionmixture was (11-14) in these experiments. Second, the effect of specific injected and the peaks collected manually.Peak 1elutes in the position biotinylation of 2 residues in different regions of calmodulin of unmodified plant calmodulin. Peak 2 was shown to contain biotin by on its functional properties, and third, the role of spatially nondenaturing gel electrophoresis in the presence and absence of excess distinct sequences of calmodulin in target enzyme activation, avidin. In p a n e l B, peak 2 from p a n e l A was reinjected using the same are addressed. separation conditions. Peaks 1 and 2 were again collected as discrete The reactivity of specific lysyl residues toward modification fractions. In p a n e l C, peak 2, p a n e l B, was separated by DEAE-HPLC, by hydrophobic and/or aromatic amine-directed reagents has and peak 2 was collected and used for functional assays. been studied in some detail. Reactive derivatives of the tricyclic phenothiazines, such as norchlorpromazine isothiocytory activity by Lys-94-biotinylated calmodulin (Fig. 6B). Biotinylated wheat germ calmodulin also was tested for anate ( l l ) , and a fluorenyl-based spin labeling reagent (14), activation of PDE and Ca+’-Mg+’ ATPase. Modification did preferentially modify Lys-75, and to a lesser extent Lys-148 not affect the activation of PDE or the ATPase by this in a calcium-dependent manner, whereas POS-TP, another derivative compared to the stimulation obtained with intact reactive phenothiazine derivative, preferentially modifies wheat germ calmodulin (Fig. 7, A and B, respectively) or with Lys-148 (13). Aliphatic cyclic reagents, such as cyclohexyl bovine testes calmodulin (not shown). The addition of excess isocyanate and its 4-methyl derivative also give specific, calavidin to these enzyme assays also did not affect the stimu- cium-dependent modification of Lys-75. The more hydrophilic 4-hydroxy and 4-carboxy cyclohexyl isocyanates do not lation of either enzyme (Fig. 7).

Calmodulin Interaction Domains

" I

10

100 CALMODULIN CONC InM)

2377

J

1000

FIG. 6. The functionaleffects of using Lys-94-biotinylatedcalmodulin inthe presence and absence of excess avidin to activate PDE and theATPase. A, the activationof PDE by native calmodulin

, I

10 CALMODULIN CONC

I

100 (nM)

FIG. 7. Thefunctional effects of using Cys-27-biotinylated activate PDE and theATPase in the presence (closed triangles), Lys-94-biotinylated calmodulin (closed triangles), and plant calmodulin to Lys-94-biotinylated calmodulin in the presence of 100 pg of avidin. % and absence of excess avidin. A, the activation of PDE by native plant calmodulin (open circles), Cys-27-biotinylated plant calmodulin in Control PDE Activity refers to nmol cAMP hydrolyzed/min/mg PDE, converted to percent of maximal activationof the enzyme by saturating the presence of 100 pg avidin. % Control PDE Activity refers to nmole of to percent of maximal concentrations of unmodified calmodulin. B, the activation of the AT- cAMP hydrolyzed/min/mg PDE as converted activation of the enzyme by saturating concentrations of unmodified Pase by native calmodulin (closed circles), by Lys-94-biotinylated calmodulin inthe absence (closed triangles) and presence of 20 pg avidin. % plant calmodulin. B, the activation of the ATPase by unmodified plant the Control ATPase Activity refers to nmoles of inorganic phosphate released calmodulin (open triangles), Cys-27-biotinylated plant calmodulin in as converted to maximal release bysaturating concentrationsof unmod- absence (open circles) and presence (closed squares) of 20pg avidin. % ified calmodulin. Each point representsthe average of three determina- ControlATPase Activityrefers to nmoles of inorganic phosphate released as converted to maximal release by saturating amounts of unmodified tions. plant calrnodulin. Each point represents the average of three determimodify calmodulin at all, which also implicates hydrophobic nations. interaction in the direction of calcium-dependent modification a t Lys-75 (35). Trace labeling studies with acetic anhy- cyclohexyl isocyanate in that 7-fold higher amounts of this dride also demonstrate that the reactivity of the Lys-75 side derivative were required formaximal activationof PDE, while chain is highly calcium-dependent compared with otherlysyl the activation of the ATPase was unaffected (35). Additionally, modification of Lys-148 with POS-TP did not affect the residues. The second most reactive residue is Lys-94 (36). ability of calmodulin toactivate PDE (13).Therefore,in Sequence analysis of NHS-biotinylated mammalian calmodulin has clearly demonstrated that this hydrophobic re- contrast to a previous report suggesting that acetylation of any lysyl residue is sufficient to reduce the affinityof calmodagentreactspreferentiallywith Lys-94 a t p H 6.0 in the specific lysyl ulin for a target enzyme (15),it seems clear that presence of calcium, althoughsomecalcium-independent modification was observed. This is in contrast to themodifi- residues are involved in the interaction of calmodulin with cation of Lys-75, in that modification with acetic anhydride different target enzymes. Since these modifications destroy of the lysyl residues, is 25-fold greater in the presence of calcium (36) and com- the positive charge on the t-amino group pletely absent when reacted withcyclohexyl isocyanate in the it seems likely that the charges on Lys-94 and Lys-75 are presence of a calcium chelator (35). It should be noted that important in the activationof the ATPase andPDE, respechydrophobic NHS ester derivativesof azidosalicylate, used in tively. Alternatively, the possibility that the chemical modithe preparation of photoaffinity probes, modify Lys-75 spe- fication itself causes gross structural changes in calmodulin cifically in a calcium-dependent manner (37). Thus, the ability cannot be overlooked, although the ability of Lys-94-biotinof NHS-biotin to modify Lys-94 is not due to the chemical ylated calmodulin to activate PDE indistinguishably from properties of the acylatingmoiety. unmodified calmodulin, argues against this interpretation. The modification of mammalian calmodulin on Lys-94 The addition of avidin to the Lys-94-biotinylatedcalmodproduces differential effects on the activationof PDE and the ulin to form a ternary complex completely blocks activation ATPase. The activation of PDE is unaffected, whereas 4-fold of both PDE and the ATPase. The avidin molecule would be higher concentrations of the Lys-94 biotinylated derivative expected to occlude sequencesin the domain I11 calciumarerequired for maximalactivation of theATPase.The binding loop. Due to steric hindrance, access to the PDE opposite effect was observed for modification of Lys-75 with interaction regions in the Lys-75 area(35) would also be

2378

Inter

Calmodulin

blocked. The incorporation of avidin at domain I on Cys-27 of plant calmodulin had no effect on the activation of either enzyme, thus indicating that thisregion is not involved in the interactions between calmodulin and these enzymes. Recently, the role of the centralhelix in structure-function relationships in calmodulin has been examined using genetically modified calmodulins. Introduction of cysteinyl residues at Gln-3 and Thr-146followed bycross-linking has indicated that the centralhelix can act as a flexible tether between the two globular lobes of calmodulin. In addition, these studies have shown that the helix itself need not be intact for the cross-linked calmodulin to activate MLCK (38). The functional effects of the incorporation of avidin into domains I and I11of calmodulin maybe interpreted in light of this information. The domain I derivative did not affect activation because the ability of the two globular domains to interact as the central helix folded was not compromised by the presence of avidin on Cys-27. Alternatively, the complete blockade of activation by the incorporation of avidin onto Lys-94 was possibly due to prevention of the folding of calmodulin and contact between the two globular domains. Unfortunately, these studies cannot differentiate between simple steric hindrance and interference with proper folding mechanisms. Mutations along the centralhelix produce calmodulins that are capable of maximal activation of calcineurin and Caf2 ATPase, whereas cyclic guanosine monophosphate phosphodiesterase and MLCK required higher concentrations of these modified calmodulins for maximal activation (39). These data are in good agreement with our observations that PDE and the ATPase interact with different (Lys-75and Lys-94 areas, respectively) regions. Several models for calmodulin function have been proposed. An electrostatic model based on a computer-predicted structure of calmodulin has postulated that these interactions and the sequestering of hydrophobic residues are the primary driving forces of the interactions of calmodulin with basic amphilphilic peptides (40).The datadiscussed herein are not inconsistent with this model. The modification of chargecontaining lysyl residues, while reducing the apparent initial affinity for interaction, does not prevent maximal activation if sufficient concentrations of modified calmodulin are employed. This suggests that theelectrostatic binding mediated by these residues, while not of primary import in the two types of calmodulin-target enzyme interactions described, still plays some kind of regulatory role. REFERENCES 1. Klee, C. B., and Vanaman, T. C. (1982) Adu. Protein Chem. 3 5 , 213-303 2. Manalan, A.E., and Klee, C. B. (1984) Adu. Cyclic Nucleotide Protein Phosphorylation Res. 1 8 , 228-278 3. Babu, Y. S., Sack, J. S., Greenhough, T. J., Bugg, C. E., Means, A. R., and Cook, W. J. (1985) Nature 315,37-40 4. Newton, D. L., Oldewurtel, M. D., Krinks, M. H., Shiloach, J., and Klee, C. B. (1984) J. Biol. Chem. 259,4419-4426 5. Guerini, D., Krebs, J., and Carafoli, E.(1984) J. Biol. Chen. 2 5 9 , 15172-15177 6. Ni, W-C., and Klee, C. B. (1985) J. Biol. Chern. 260,6794-6981 7. Levin, R. M., and Weiss, B. (1977) Mol. Phurmacol. 13,690-697

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