THE J O U R N A L OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry I and Molecular Biology, Inc

Vol. 263, No. 32, Issue of November 15, pp. 17023-17029,1388 Printed in U.S.A.

Investigation of the Mechanism of Calcium Binding to Calmodulin USE OF AN ISOFUNCTIONAL MUTANT WITH A TRYPTOPHAN INTRODUCED BY SITE-DIRECTED MUTAGENESIS*

(Received for publication, May 11, 1988)

Marie-Claude KilhofferS, Daniel M. Roberts§nII, Abiodun 0. Adibi$**, D. Martin Wattersonsll, and Jacques Haiech§S$$§ From the $Universite Louis Pasteur, Laboratoire de Biophysique, UA Centre National de la Recherche Scientifique 491 Faculte de Pharmacie, B. P. 10 67048 Strasbourg, France, the §Department of Pharmacology, Vanderbilt University, and Wuboratory of Cellular Molecular Physiology, Howard Hughes Medical Institute, Nashville, Tennessee 37232, and the $$Centre de Recherches de Biochimie Macromoleculaire de Centre National de la Recherche Scientifique, B. P. 5051, 34033 Montpellier, France

A mutant calmodulin, in which phenylalanine 99 of calcium binding site I11 was changed to a tryptophan by using cassette-based, site-directed mutagenesis, has been used to analyze the mechanism of calcium binding. The combined study of direct calcium binding, modifi- cation of tryptophan fluorescence properties upon cal- cium binding, and terbium titration allows some dis- crimination among proposed mechanisms of cation binding to calmodulin. Calmodulin appears to have six cation binding sites, four of which are selective for calcium, that seem to be coupled. Under a given set of conditions, these calcium-selective sites are not iden- tical. In addition to providing insight into the mecha- nisms of calcium modulation of calmodulin, these stud- ies demonstrate the feasibility of using isofunctional, tryptophan-containing mutants of proteins to gain in- sight into protein-ligand interaction.

Calmodulin is a calcium binding protein involved in calcium signal management in all eukaryotic cells (Van Eldik et al., 1982; Cox et al., 1984). In order for calmodulin to serve as a transducer of calcium signals, it must reversibly bind calcium, undergo conformational changes, and properly transduce these alterations in calmodulin structure to the calmodulin binding proteins. Therefore, calcium binding and calcium- induced conformational changes are the first steps in the process of calmodulin activation of enzymes. A thorough knowledge of these processes is required for a complete un- derstanding of how a calcium signal is transduced into a biological response.

Calcium binding to calmodulin has been studied by gel filtration, ultrafiltration, flow dialysis, equilibrium dialysis, calcium titration using calcium-sensitive dyes or a calcium electrode (for a review, see Kilhoffer et al., 1983). Calmodulin possesses four calcium sites and two to four other low affinity cation sites (Milos et al., 1986). The binding properties are

* This research was supported in part by National Institutes of Health Grant GM30861 (to D. M. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Knoxville, T N 37996-0840. I( Present address: Dept. of Biochemistry, University of Tennessee,

Nashville, TN 37203. ** Present address: Dept. of Biology, Tennessee State University,

To whom correspondence should be addressed Dept. of Phar- macology, Vanderbilt University School of Medicine, Nashville, TN 37232.

dependent on pH, ionic strength, and the concentration of other cations (Kilhoffer et al., 1983).

In general, the binding mechanism of a protein with four ligand sites can be described by 15 independent microscopic binding constants and coupling factors (Klotz, 1985). For example, a given site, S1, with an intrinsic binding constant, K,, would have an intrinsic binding constant W1 when one of the other three sites is occupied; the coupling factor, C1, between the site S1 and the other site is described by the ratio K1/Kl. If this ratio is 1, this means that the occupancy of the other site does not modify the affinity of site S1. Similarly, if the ratio is greater than 1, then the affinity of site S1 is increased upon occupancy of the second site; if the ratio is less than 1, then the affinity of site S1 is decreased. Thus, for a protein with four ligand binding sites, there will be 11 distinct coupling factors plus four intrinsic binding constants for a total of 15 independent mathematical terms to describe the ligand binding.

Specific to calmodulin, the Scatchard representation of data from previous binding experiments was a straight line (Potter et al., 1977; Burger et al., 1984). Based on these representa- tions, it was thought that calmodulin had four independent calcium sites characterized by four intrinsic constants of equal magnitude. In this latter case, any mathematical treatments of the system would set all coupling factors, which attempt to describe interactions among the sites, equal to unity. Later, a more detailed study of calcium binding to calmodulin by Crouch and Klee (1980) indicated a positive cooperativity between at least the first two sites occupied. In this case, at least one coupling factor is necessary to describe the binding mechanisms. Subsequently, a study of calcium binding to calmodulin under various conditions (different magnesium and potassium concentrations) demonstrated that calcium binding activity varied, depending on the conditions (Haiech et al., 1981). A minimum of three coupling constants were needed to describe the system in mathematical terms under these various conditions. Thus, as more is learned about cation binding properties, increasingly complex models have been proposed to explain how these cation binding properties are related to calmodulin’s physiological roles.

A methodological limitation in attempts to verify experi- mentally various models of cation binding by calmodulin is the fact that one cannot readily decouple the calcium binding, per se, from the calcium-induced conformational changes. For example, calcium-induced conformational changes have been monitored by a variety of techniques such as nuclear magnetic resonance (NMR) spectrometry, ultraviolet fluorescence spec- troscopy, ultraviolet circular dichroism spectroscopy, and vol-

17023

17024 Calcium Binding to Calmodulin

ume change (for a review, see Forsen et al., 1986; Krebs, 1981). Based on the observations that the environment of residues located in the carboxyl-terminal part of the molecule was altered under conditions where two calcium ions should be bound to the protein, it was concluded that calmodulin pos- sesses two high affinity sites and two low affinity sites. Ini- tially, this conclusion appeared not to be in complete agree- ment with data obtained from calcium binding studies, but the assumption that binding between the different sites is coupled allowed full agreement between the two sets of data (Haiech et al., 1981; Kilhoffer et al., 1983; Wang, 1985). However, the exact nature of this coupling is not yet fully understood and has not been described in mathematical terms. Thus, it is imperative that new approaches be devel- oped in order to allow attempts at further definition of the process of calcium binding to calmodulin if we are to gain increased insight into how a calcium signal is transduced into a biological response.

The availability of an efficient site-specific mutagenesis and protein engineering system (Roberts et al., 1985; 1986a; Craig et al., 1987) for calmodulin allows the opportunity to generate engineered calmodulins in order to begin to address the problems of describing the coupling between the four calcium binding sites. Because calmodulins do not contain tryptophan residues, it might be especially useful if an iso- functional derivative of calmodulin could be made in which strategically placed tryptophans could be used as a reporter group of a local environment. In this paper, we report the design, the production, and the use of a calmodulin mutant with a tryptophan at residue 99 (calcium binding site 111) in order to discriminate among some of the different calcium binding mechanisms which have been proposed.

MATERIALS AND METHODS’

RESULTS’ AND DISCUSSION

Design, Construction, Production, and Characterization of VU-9 Calmodulin

Because calmodulin does not contain tryptophan residues, which are useful spectroscopic probes in some proteins (for a review, see Demchenko, 1986), attempts were made to select a potential site for introducing a tryptophan reporter group for calcium binding. However, the introduction of the reporter group should not drastically modify the ion binding properties or activator properties of calmodulin. Therefore, we utilized the known primary structures of several calcium binding proteins and an algorithm (Haiech and Sallantin, 1985) that uses logical rules to analyze for structural features within 27- residue segments of amino acid sequence. This analysis indi- cated that if residue 99 were a tryptophan, the local environ- ment of the third calcium binding loop would not be altered drastically. Moreover, the amino acid residue at this position varies among calmodulins from different species (for a review, see Roberts et al., 1986133, and the side chain appears to be exposed to the solvent (Babu et al., 1985). Thus, the fact that tryptophan is a bulkier residue may not have a major effect on the structure of the protein. In order to introduce this change, oligonucleotide cassette mutagenesis was used (see “Materials and Methods”, in Miniprint, and Figs. 1 and 2). The approach has been de-

l Portions of this paper (including “Materials and Methods” and part of “Results”) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

6 50 M ~ C A T G G C E A T C A G C T G A C T G A C G A G C A G A A A G A G

a A C C G A C T A G T C C G I ‘ C X A G C G A C I T A 4 A T I T C T C

n a d q l t d e q i a e f k e

51 101 G C m C L % T C A A A G A C G C T G A C G G M C C A T C A ~ C C A A A G A G C G A A ? S G A G A G A C M A C T G l - l T C C A ” ~ C

a f s l f d k d g d g t i t t k e

102 152 C T C G G C A C ~ A ~ C A G C ~ ~ C C ~ ~ G ~ M ~ G G A G C C C G G A A C ~ ~ C l T C G A C l T G A C

l g t v m r s l g q n p t e a e l

153 203 C A ~ C A ~ ~ M ~ G T C ~ ~ M C G G C A C C A T C ~ ~ a C ~ a A ~ M ~ C l T C A G ~ ~ ~ C ~ ~ C ~ A G ~ ~

q d n i n e v d a d g n g t i d f

204 2 54 C c G G A A m C T G A A C ~ G A T G G C G C G ~ G A ~ G A ~ ~ ~ ~ ~ G ~ G G C m A A A G A ~ A C C G ~ ~ ~ A ~ ~ ~ ~ G A G A ~

p e f l n l n a r k n k d t d s e

255 ####################*************** G A G G A A ~ G A G G c m c ~ c G A C A A A G A ~ A A c G G l T G G ~ C m G A ~ C I C C G G A A G G C A ~ G ~ ~ G C C A T I C

++++++++ e e l k e a f r v f d k d g n g v

n n n n n * n ~ n h n r l * * * * * n n ~ ~ ~ n ~ ~ n

***** 356 A T C T C G G C C G ~ ~ ( T T G ~ C A ~ A T ~ ~ M C ~ ~ ~ G C ~ T A G A G C C G G C ~ ~ ~ ~ C A G T G C A A T A ~ ~ ~ ~ C C ~ ~ C G ~ +++++++++ i s a a e l r h v n t n l g e k l

357 407 A ~ ~ ~ ~ ~ ~ T G A ~ ~ C ~ G ~ C ~ C ~ T ~ G A C T G A ~ G C l T C l T C ~ A ~ M G ~ C l T ~ ~ G C A ~ A C ~ ~ G

t d e e v d e n i r e a d v d g d

408 455 GGCCAGGJTMCTACGAAGAGlTCGlTCAGGTTATGATCGCTMGTAG C ~ C ~ ~ ~ m ~ ~ A G T G C A A ~ C A G T G C A A T A ~ A C C G

g q v n y e e f v q v ~ r a k

FIG. 1. Primary structure of gene coding for VU-9 calmod- ulin. The double-stranded structure of the mutant VU-9 calmodulin and the amino acid sequence (single letter code) generated from translation of the coding strand are shown. Protein coding starts at nucleotide 6, but the engineered protein starts at the alanine, amino acid residue 2. The symbols denote the individual oligonucleotides used to construct the cassette, as described under “Materials and Methods”; * = nucleotides from W99.1; + = nucleotides from W99.2; # = nucleotides from Stul.1; A = nucleotides from Stul.2.

scribed previously (Roberts, 1985, 1986a, 1987; Craig et al., 1987; Lukas et al., 1987) and involved the use of a synthetic calmodulin gene designed for cassette-based, site-specific mu- tagenesis while the gene is resident in a combination ampli- fication/expression vector. The details of the construction and characterization are given under “Materials and Meth-

Calcium Binding to Calmodulin 17025

ods.” The resultant protein has the expected properties in terms of amino acid composition, amino acid sequence change, and spectroscopic properties (Fig. 1, Tables I and 11, and “Materials and Methods”). In addition, VU-9 calmodulin has enzyme activator activities that are indistinguishable from VU-1 calmodulin (data not shown).

Comparison of Calcium Binding Properties of VU-1 and VU-9 Calmodulins

In order to determine if the tryptophan-containing calmod- ulin (VU-9 calmodulin) has cation binding properties similar to those of the fully active standard of comparison (VU-1 calmodulin), the calcium binding properties of the two pro- teins were examined under identical conditions and found to be indistinguishable. Ca2+ binding to VU-1 and VU-9 calmod- ulins was performed both in the absence and in the presence of 5 mM M$+ ions (Figs. 3 and 4). When binding data were not corrected for Ca2+ binding to the dialysis membrane, the

TABLE I Amino acid compositions

Amino acid

VU-9 cal- Peptide (SP-9) con- modulin taining mutation

ASP 25.2” (25)b 3.2” (3)b Glu 26.4 (26) 1.2 (1) Ser 3.7 (4) 1.1 (1) GlY 11.9 (11) 2.4 (2) His 1.0 (1) Arg 5.4 (5) 1.3 (1) Thr 9.4 (10) Ala 11.0 (11) 3.1 (3) Pro 2.1 (2) TYr 1.2 (1) Val 9.1 (9) 1.0 (1) Met 7.6 (8) Ile 5.9 (6) 1.0 (1) Leu 11.6 (11) Phe 7.9 (8) 2.0 (2) Lvs 8.6 (9) 0.9 (1)

a Molar ratios greater than 0.3 given. Values in parentheses are those expected from DNA sequence.

TABLE I1 Amino acid seauence annlvsis

VU-9 calmodulin” Peptide (SP-9)* containing mutation

Cycle Amino Amount Cy$ Amino Amount no. acid acid

pmol P m l 1 Ala 200 1 Ala 162 2 Asp 77 2 Phe 121 3 Gln 138 3 Arg 171 4 Leu 119 4 Val I7 5 Thr 95 5 Phe 84 6 Asp 52 6 Asp 70 7 Glu 65 7 Lys 64 8 Gln IO 8 Asp 52 9 Ile 64 9 G b 39

10 Ala 68 10 Asn 37 11 Glu 21 11 GlY 33 12 Phe 55 12 Trp 14 13 LYS 50 13 Ile 24 14 Glu 15 14 Ser 10 15 Ala 47 15 Ala 20

16 Ala 23 17 Glu 5

“Automated Edman degradation of 216 pmol as determined by

bAutomated Edman degradation of 218 pmol as determined by amino acid analysis.

amino acid analysis.

FIG. 2. Dot blot screen of clones for the presence of the tryptophan coding mutation. Oligonucleotide probe W99.1 was labeled with 32P and the labeled probe hybridized with a nitrocellulose filter containing plasmid samples from randomly selected colonies, done as described under “Materials and Methods.” After appropriate washes, the filter was dried and exposed to film. The relatively high efficiency of the mutagenesis is evident from the preponderance of positively hybridizing colonies. The plasmid containing the gene for VU-1 calmodulin was the negative control, and it does not show detectable hybridization under the same conditions.

U C 3 0 9 E

pCa

FIG. 3. Calcium binding to VU-1 in 60 mM Hepes buffer, pH 7.6, in the absence (a and c) and in the presence ( b and d ) of 6 mM M8’. Panels a and b represent the total amount of bound calcium in the presence (W) or absence (0) of VU-1. Panels c and d correspond to the corrected calcium binding isotherm of VU-1 ex- pressed in moles of calcium bound per mol of VU-1 as a function of pCa. Calcium binding experiments were performed as described under “Materials and Methods.” Protein concentrations were 5.2 X lo-’ M in the absence of magnesium and 2.6 X M in the presence of magnesium. Each experiment has been performed at least three times under different protein concentrations ranging from to lo-‘ M.

isotherms represented in Figs. 3a and 4a were obtained. These binding isotherms indicate the presence of two types of Ca2+- binding sites with different affinities for the ion. The sites with the higher affinity are saturable, whereas the low affinity sites remain unsaturated even at a free Ca2+ concentration of

M. When binding experiments were performed under the same conditions except for the absence of the protein, an isotherm corresponding to Ca2+ binding to the membrane could be obtained. Figs. 3, a and b, 4, a and b, show that Ca2+ binding to the membrane becomes significant for free Ca2+ concentration above lo-‘ M. Taking into account this nonse-

17026 Calcium Binding to Calmodulin

7 6 5 4 3 7 6 5 4 3 PC a

FIG. 4. Calcium binding to VU-9 calmodulin in 50 mM Hepes buffer, pH 7.5, in the absence (a and c) and in the presence ( b and d ) of 5 mM Mg2+. Panels a and b represent the total amount of bound calcium in the presence (m) or absence (0) of VU-9 calmodulin. Panels c and d correspond to the corrected calcium binding isotherm of VU-9 calmodulin expressed in moles of calcium bound per mol of VU-9 as a function of pCa. Calcium binding experiments were per- formed as described under “Materials and Methods.” Protein concen- trations were 8 X M in the absence of magnesium and 7.6 X M in the presence of magnesium. Each experiment has been performed at least three times under different protein concentrations ranging from to 10“ M.

lective binding, Ca2+ binding isotherms for VU-1 and VU-9 calmodulin can be obtained (Figs. 3, c and d; 4, c and d ) . In the absence of Mg”’, the isotherm plateaus at 6 mol of Ca2+/ mol of VU-9 calmodulin; whereas, in the presence of 5 mM M e , saturation is reached for 4 mol of Ca2+/mo1 of VU-9 calmodulin. When data were analyzed according to the Scat- chard equation, the subsequent graphical representation ap- pears to be linear (data not shown). The dissociation con- stants obtained from such analyses are 4.2 X 1O“j M and 3 X IOu5 M for VU-9 calmodulin in the absence and presence of 5 mM M e , respectively. Although it is possible that some positive cooperativity could be present between the two first sites, it is not possible to assess this possibility under the conditions used in this study.

The existence of six Ca2+ binding sites on VU-9 calmodulin is not unique to this calmodulin with a tryptophan residue in position 99, as seen from the results with VU-1 calmodulin (Fig. 3c). The location of the “extra” sites is so far unknown, but from terbium binding studies (see below) it can be as- sumed that each half of the calmodulin molecule contains one of the extra sites in addition to the two “EF hand” binding sites.

The number of calcium-selective sites in VU-1 and VU-9 calmodulins are decreased to four, with a 10-fold lower affin- ity, when the calcium binding analyses are done in the pres- ence of magnesium (Figs. 3d and 4d) . In addition, the calcium binding properties of VU-1 and VU-9 calmodulins in the presence or absence of 5 mM magnesium were indistinguish- able. Thus, it appears that VU-1 and VU-9 calmodulins possess four calcium-selective sites and two cation sites which do not discriminate between calcium and magnesium.

Regarding the possible application of these results to a generalized binding mechanism (see below), the determina-

tion of the mechanism of calcium binding to a protein with six cation sites is even more complex, because we must deal with at least 63 constants (6 intrinsic constants and 57 coupling factors). However, if we assume that the six sites are independent and equivalent (all the coupling factors are set to 1 and the six intrinsic dissociation constants are equal to 4.2 X M), and that the conformational changes are linked to specific site occupancy, then the change in fluorescence degree of polarization or fluorescence intensity of VU-9 cal- modulin as a function of calcium concentration should follow the calcium binding isotherm. As discussed below, this possi- bility was initially addressed by spectroscopic studies of VU- 9 calmodulin under different experimental conditions and found not to be the case.

Spectroscopic Properties of VU-9 Calmodulin as a Function of Calcium Binding

All calmodulins isolated from biological sources or engi- neered from recombinant DNA lack tryptophan, a potentially useful spectroscopic reporter group. As summarized above, VU-9 calmodulin mffers by only one amino acid residue from VU-1 calmodulin, the fully active standard of comparison, and this difference is a tryptophan residue located at position 99 in calcium binding domain 111. Further, VU-9 calmodulin has cation binding properties and enzyme activator activities that are indistinguishable from VU-1 calmodulin. Thus, VU- 9 calmodulin appears to be a potentially useful isofunctional derivative with an internal spectroscopic reporter group. Therefore, as a part of an initial set of studies to test the feasibility of using tryptophans introduced into the molecule as localized reporter groups, the changes in the fluorescence properties of VU-9 calmodulin as a function of Ca2+ were examined.

Fluorescence Emission-Fig. 5 represents the change in the fluorescence intensity at 348 nm during Ca2+ titration. The fluorescence intensity increases up to 3 Ca2+ bound/VU-9 calmodulin. Between 3 and 4 Ca2+/VU-9 calmodulin there is no change, then the intensity decreases again between 4 and 6 Ca2+/VU-9 calmodulin.

An interesting feature of the change in fluorescence inten- sity as a function of calcium concentration is the fact that it does not coincide with the calcium binding isotherm generated from the flow dialysis experiments. As shown in Fig. 6, the fluorescence intensity change occurs over a lower calcium

FIG. 5. Changes in VU-9 calmodulin parameters as a func- tion of calcium bound to the protein. W and 0 correspond, respectively, to the fluorescence polarization degree and fluorescence intensity at 348 nm. Calcium bound to VU-9 calmodulin was calcu- lated taking into account the calcium affinity constants obtained from calcium binding studies. Experiments were performed in 50 mM Hepes buffer, pH 7.5, with an excitation wavelength set at 295 nm. On the ordinate, changes are expressed as the percentage of the maximum change. Protein concentration was 3.3 X M for the experiment where fluorescence intensities were monitored and 2.2 X lo-’ M when degree of polarization changes were monitored.

Calcium Binding to Calmodulin 17027

1004 inert, whereas terbium has the convenient property of being highly luminescent, when it binds close to an aromatic residue

01-

previously characterized properties of terbium and calcium in a protein, as a result of energy transfer. Based on these

0 9 c

50- binding proteins, if terbium were bound to VU-9 calmodulin .- ?

7 6 5 4 3 within this distance (Babu et al., 1985) is the calcium binding

only if there were an energy transfer between the tryptophan K

luminescence monitored at 545 nm would be expected to occur - m -

and the excitation wavelength were set at 295 nm, the terbium I

P)

01 I I

at residue 99 and a terbium ion. A logical structural location

&a site 111. FIG. 6. Changes in V u - 9 calmodulin parameters as a func- As shown in Fig. 7, excitation of mixtures of terbium and

tion ofpCa. Legend is similar to that of Fig. 5, except that the curves VU-9 calmodulin at 295 nm results in emission peaks at 545 are represented as a function of pCa. In addition, changes are com- nm, indicating that energy transfer between tryptophan 99 pared to the calcium binding isotherm of vu-9 calmodulin taken and the terbium has taken place. ~l~~ evident from ~ i ~ . 7 is from Fig. 4c. the fact that most of the increase in terbium luminescence at

concentration range than does binding to the six cation sites. The fact that the change in the tryptophan emission proper- ties of VU-9 calmodulin does not follow the binding of calcium to the six sites suggests that the environment around residue 99 is altered early in the calcium binding mechanism (i.e. at low ratios of calcium to calmodulin). Clearly, one possible interpretation of these results in terms of mechanisms of calcium binding is that calcium binding site I11 is one of the first three calcium binding sites occupied. However, a poten- tially better indicator of the local environment is the degree of fluorescence polarization as a function of calcium concen- tration.

Change in the Degree of Fluorescence Polarization-The degree of polarization of tryptophan 99 in VU-9 calmodulin is equal to 0.152 in the absence of calcium and 0.192 in the presence of calcium. The change in p (the change as a per- centage of maximum polarization) as a function of calcium bound to the protein is shown in Fig. 5. The degree of polarization increases up to three calciums bound per mol of VU-9 calmodulin. Then, between four and six calciums bound, there is a slight decrease in p. As shown in Fig. 6, the larger change in polarization is being detected at a calcium concen- tration that corresponds to a point in the Ca2+ binding iso- therm where only a few sites (one or two sites) would be occupied by calcium.

Thus, both the change in intensity and degree of polariza- tion indicate that the change in the environment of residue 99 does not follow exactly the binding of Ca2+ to the six cation sites of VU-9 calmodulin, suggesting nonequivalence of cal- cium binding sites. Further, the change in both intensity and degree of polarization as a function of calcium concentration, under conditions where the cation sites have not been satu- rated by calcium, suggests that calcium binding site I11 is one of the first two sites occupied in the mechanism of calcium binding to calmodulin.

Terbium Binding to VU-9 Calmodulin A complementary approach to tryptophan fluorescence and

emission that can provide further insight is the analysis of terbium binding to calmodulin. Terbium has been shown to be a useful probe to investigate the cation binding sites of calcium binding proteins (for a review, see Prados et a i , 1974). Because of terbium’s ionic radius, coordination number (be- tween six and eight) and propensity for oxygen donor groups, this trivalent ion can replace calcium in many biological systems. Previous studies (Kilhoffer et al., 1980) have shown that terbium binds to mammalian calmodulin in a sequential and ordered manner. However, the relative order of sites occupied is different for Tb3+ and Ca2+ ions (Wang et al., 1984). In terms of spectroscopy, calcium is spectroscopically

545 nm starts at a molar ratio of 3 or higher, i.e. most of the increase appears to occur with the binding of 4-6 mol of terbium/mol of VU-9 calmodulin. Thus, the luminescence at 545 nm as a function of increasing terbium added to a fixed amount of VU-9 calmodulin indicates that there are at least six terbium binding sites on VU-9 calmodulin, similar to the six cation sites found in calcium binding studies.

Another similarity between terbium and calcium binding is the effect on tryptophan 99 fluorescence. When the first terbium is bound to VU-9 calmodulin, the emission maximum shifts from 348 to 341 nm. Then, between 1 and 3 mol of terbium/mol of VU-9 calmodulin, the intensity at the maxi- mum (341 nm) reaches a plateau. At 3 mol and greater of terbium/mol of protein, the tryptophan emission decreases as terbium luminescence increases. In contrast to calcium bind- ing, this dramatic decrease in tryptophan 99 emission at higher terbium/protein ratios was not observed during cal- cium titrations of VU-9 calmodulin. Quenching of chromo- phores (e.g. tryptophan or tyrosine) by terbium ions has been reported (Martin and Richardson, 1979) for certain calcium binding proteins, such as mammalian calmodulin and some parvalbumins. Although there currently is not a clear inter- pretation of this quenching of tryptophan fluorescence in proteins, this phenomenon might be a useful monitor of other interactions.

In terms of the relationship between calmodulin structure and the order of cation binding, previous studies (Wang et al.,

””

f E 50

c 2 75 ? -

- 0 ul - 25 0 +

- 0

0 1 2 3 4 5 6 7 Tb3+/VU-9(mol/mol)

FIG. 7. Change in the fluorescence intensity of tryptophan 99 at 348 nm (- - -) and in terbium luminescence at 645 nm (-) as a function of terbium added to VU-9 calmodulin. Terbium titrations were performed in the presence of various molar ratios of Ca2+ to VU-9 calmodulin. A, W, 0, 0, * correspond respec- tively, to 0, 1,2, 3, 4 calciums/mol of VU-9 calmodulin. The curve at 10 calciums/mol of VU-9 calmodulin was superimposable to that at four calciums. Excitation wavelength was set a t 297 nm, and emission was recorded between 290 and 600 nm. Intensity changes were ex- pressed as a percentage of the maximum. Experiments were per- formed in 50 mM Hepes buffer, pH 7.5, at protein concentrations varying between 1.2-1.5 X M.

17028 Calcium Binding to Calmodulin

1984) have suggested that terbium binds first to the cation sites in the amino-terminal half of calmodulin and calcium appears to bind first to the cation sites in the carboxyl terminal half of the molecule. Our results in which the energy transfer occurs only after the binding of three terbium ions are consistent with this interpretation. As discussed below and summarized in Fig. 7, our results with the terbium titra- tions at different ratios of calcium to VU-9 calmodulin are also consistent with this model.

Terbium and Calcium Binding to VU-9 Calmodulin In an attempt to learn more about the relationship between

terbium binding and different “bound calcium” states of cal- modulin, a terbium titration of VU-9 calmodulin was per- formed at different molar ratios of Ca2+ to VU-9 calmodulin. Specifically, terbium binding to VU-9 calmodulin was inves- tigated in the presence of 1, 2, 3, 4, and 10 mol of Ca2+/mol of VU-9 calmodulin. As shown in Fig. 7, terbium luminescence at 545 nm (solid lines) increases very slightly with the binding of the first three terbium ions, regardless of the ratio of calcium to VU-9 calmodulin. Overall, the titration of VU-9 calmodulin by terbium, as monitored by the luminescence at 545 nm, is changed very little by the presence of calcium except at the highest ratios (e.g. 2 4 mol of calcium/mol of calmodulin). In contrast, the quenching of tryptophan 99 emission by terbium (broken lines) is diminished as the cal- cium to calmodulin ratio is increased. The effect is different for the three phases of the curve. In the first phase, between 0 and 1 terbium/mol of calmodulin, there is a small but reproducible decrease in the fluorescence intensity of trypto- phan 99 when no calcium is added. This first phase of terbium quenching of tryptophan 99 is abolished with a calcium to calmodulin ratio of only one. In the second phase of the terbium quenching of tryptophan 99, between 1 and 3 ter- biums/mol of calmodulin, there is a plateau or slight increase in tryptophan emission. This phase of the curve is less affected by the ratio of calcium to calmodulin. In the third phase, between 3 and 6 terbiums/mol of calmodulin, there is a strong quenching of the tryptophan 99 emission that correlates with the increase in terbium luminescence. It is this third phase of almost dose-dependent quenching that is affected in a pro- gressive manner by the ratio of calcium to calmodulin (up to four calciums per calmodulin in Fig. 7). In fact, it is only this phase that shows a reproducible effect between 1 and 10 mol of calcium/mol of calmodulin.

In terms of models of calcium binding by calmodulin, these results are in agreement with models of unequal sites. If all of the calcium binding sites were of the same affinity, then addition of each additional calcium would affect the trypto- phan 99 emission in Fig. 7 in a manner similar to the first, and so on. This is clearly not the case, so it must be concluded that the sites are not equivalent. Further, the titration data in Fig. 7 are not readily explained by mechanistic models that assume there are two pairs of calcium binding sites in which each pair would bind calcium with positive cooperativity (in this type of model the “not all sites equal” criteria could be satisfied by having pairs of sites that differ in their affinity for calcium). Based on such a model, the first phase of terbium quenching of tryptophan 99 emission (dashed lines in Fig. 7) would be altered by the addition of at least 2 calciums/mol of calmodulin. Under the conditions used in this study, the modification follows the binding of only one calcium.

Conclusions The studies summarized here demonstrate the utility of

producing isofunctional mutant proteins into which trypto-

phan residues have been introduced as spectroscopic reporter groups. The analysis of VU-9 calmodulin, which has a tryp- tophan at residue 99 in the calcium binding site 111, provided insight into possible mechanisms of calcium binding to cal- modulin. The results from the analysis of calcium binding, fluorescence emission and degree of polarization, and terbium binding indicate that the calcium binding sites in calmodulin are not independent sites and do not have equal affinities for calcium ( i e . calmodulin does not appear to be the sum of four independent calcium binding sites or the sum of two inde- pendent pairs of sites). In structural terms, this indicates that the occupancy of one calcium site modifies the binding prop- erties of the other sites. In mathematical terms, this indicates that the coupling factors (Kilhoffer et al., 1983) between the different sites are not equal to 1. Overall, the results are consistent with a model in which there is binding of calcium to sites on calmodulin which have some degree of interaction, or “cross-talk.” In theory, additional regulation could be brought about through the interaction with calmodulin bind- ing proteins or post-translational modifications of calmodulin or the calmodulin binding protein. Based on the initial results presented here, the use of additional isofunctional mutant proteins with internal reporter groups, combined with other analyses, may provide insight into the structural basis of these functional couplings.

Acknowledgments-We thank Mdm. Scandellari (Laboratoire de Chimie Bacterienne, Marseille), Paul Matrisian, Augustine Smith, and Theodore Craig (Nashville) for their assistance. We also thank Cindy Reeder and Janis Elsner for their assistance with the prepa- ration of the paper. We are grateful to Alain Diab for automating the titration and binding experiments and to F. G. Prendergast for critical reading and extremely helpful comments.

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