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Investigating calmodulin-target sequence interactions using mutant proteins and synthetic target peptides
Investigating Calmodulin-Target Sequence Interactions Using Mutant Proteins and Synthetic Target Peptides Wendy A. Findlay, Stephen R. Martin, and Peter M. Bayley Division of Physical Biochemistry, National Institute for Medical Research, MiU Hill, London NW7 lAA, England, U.K.
I. Introduction Protein-protein interactions are important in the function and regulation of many biological pathways. Associations between proteins are often characterized by strong and extremely specific noncovalent interactions between complementary surfaces. One way of looking at the details of these interactions is to use peptides corresponding to the "interaction region" of one of the proteins to determine the minimum sequence needed for the interaction as well as the effect of changing individual residues. We report here on the use of this approach to study the interaction of calmodulin with a target sequence from skeletal muscle myosin light chain kinase (sk-MLCK). The strategy is to use a number of sequence variants of the peptide and site directed mutants of calmodulin. Calmodulin is a small ubiquitous calcium binding protein which regulates a variety of enzymes in several different metabolic pathways. Calmodulin interacts with many of its target proteins with very high affinity (K^ — nM), usually in a calcium specific manner. It also binds target sequence peptides derived from the calmodulin binding regions of many of these proteins with affinities close to those for the intact enzymes. Many target sequences are predicted to form basic amphipathic helices and this has been proposed as a common structural motif for calmodulin binding (1). In the solution structure of a complex of calmodulin with a 26-residue target peptide derived from the sequence of sk-MLCK, the two domains of TECHNIQUES IN PROTEIN CHEMISTRY VI Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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calmodulin surround the Ml3 peptide, which has adopted an oj-helical conformation (2). The peptide lies in a hydrophobic channel and the sidechains of Trp4 and Phel7 of the peptide appear to anchor the peptide to the two domains by fitting into the hydrophobic "pockets". Maune et al. (3) have produced a set of single site mutants of Drosophila melanogaster calmodulin, each of which has the conserved glutamic acid residue in the 12th position of one of the four calcium binding loops mutated to either glutamine or lysine. Mutations to sites 2 or 4 effectively eliminate calcium binding to the mutated site and cause structural changes in the protein (3, 4) as well as decreasing the ability of calmodulin to activate target enzymes (5). The availability of these mutant calmodulins and the choice of synthetic peptides derived from the sk-MLCK target sequence allows the manipulation of both components involved in the interaction. The 18residue sk-MLCK target sequence has 3 aromatic residues: Trp4, Phe8, and Phel7. In this work, we use peptides with tryptophan in either position 4 (WFF peptide) or position 17 (FFW peptide). Since the calmodulin itself has no tryptophan residues, we can use optical spectroscopy to monitor the interaction of a tryptophan in a specific position in the target sequence with an individual domain of calmodulin. We have studied binding of the two peptides to wildtype calmodulin and to the site 2 and site 4 mutants (B2Q, B2K, B4Q, and B4K) to see how mutations which effectively eliminate calcium binding to a particular site affect the interaction of the protein with the two target sequence analogues.
n . Materials and Methods Proteins and peptides - Drosophila melanogaster calmodulin and the various mutants expressed in E. coli were purified essentially as previously described (3). Peptides were synthesized on an Applied Biosystems 430A peptide synthesizer and purified by reverse phase HPLC on a CIS column (WFF peptide) or a C8 column (FFW peptide) and were provided with free carboxy and amino termini. All concentrations were determined spectrophotometrically using a calculated extinction coefficient of 5560 M"^ cm"^ at 280 nm for the peptides (2 Phe and 1 Trp) and published extinction coefficients for wildtype and the four mutant calmodulins (3).
Calmodulin-Target Sequence Interactions
403
Fluorescence and affinity measurements - Peptide in 25 mM Tris, 100 mM KCl and 1 mM CaCl2 at pH 7.5 and 30 C was titrated with a stock solution of calmodulin in UV transmitting plastic cuvettes since the peptides appear to bind to glass. Fluorescence titration spectra were recorded using a SPEX FluoroMax fluorescence spectrometer with excitation at 280 nm and emission scanned from 310 to 390 nm. The value of fluorescence intensity at 330nm was plotted as a function of calmodulin concentration and fitted using standard non-linear least squares methods (6) to obtain optimal values of the dissociation constant (K^) and the maximum fluorescence enhancement (F/FQ). The detection limit under our experimental conditions was 50 nM peptide and all quoted K,, values are the average of at least 3 independent determinations. Circular dichroism spectra - Spectra were recorded on a Jasco J-600 spectropolarimeter at room temperature. Far UV CD spectra (190 to 260 nm) of 7.5 fiM peptide:calmodulin complex in 25 mM Tris, 100 mM KCl and 1 mM CaCl2 were measured in a 0.1 cm path length cuvette. Near UV CD spectra (250 to 340 nm) of 20 /xM peptide:calmodulin complex in the same buffer were measured in a 1 cm path length cuvette.
n i . Results We have used two synthetic 18-residue peptides related to the target sequence of sk-MLCK to study their interaction with calmodulin in the presence of calcium. The WFF peptide (KKRWKKNFIAVSAANRFK) corresponds to residues 577 to 594 of rabbit sk-MLCK. In the FFW peptide (KKRFKKNFIAVSAANRWK) the W4 and F17 residues have been interchanged. Upon binding to the protein the tryptophan fluorescence emission maximum for each peptide is shifted from 356 nm to 334 nm as shown in Fig. lA, with an enhancement in fluorescence intensity at 330 nm of about 2.4-fold for the WFF peptide and 3-fold for the FFW peptide (Table 1). These results indicate that the Trp residue is in a hydrophobic environment when either peptide binds to the protein. By monitoring fluorescence intensity at 330 nm while titrating either peptide with calmodulin, we determined the affinities of calmodulin for the FFW peptide (Kd= 1.6 nM) and the WFF peptide (K^<0.2 nM). The affinity of calmodulin for the native sequence (WFF peptide) is at least an order of magnitude higher than that for the modified sequence (FFW peptide).
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We used near UV CD spectroscopy to obtain information about the steric environment of the Trp sidechain in each of the protein:peptide complexes. Figure IB shows the near-UV CD spectra of wild-type Drosophila calmodulin in the presence of calcium alone and in (1:1) complexes with the WFF and FFW peptides. Free calmodulin shows prominent bands at 262 and 268 nm, which derive from the nine Phe residues. The signal at longer wavelengths (X > 275 nm) derives from the single Tyr located at position 138 in the C-terminal domain. The free peptides show negligible circular dichroism in this wavelength range. The spectra of the complexes of calmodulin with the WFF or FFW peptide show clear evidence of a major contribution from the Trp residue in the peptide. Tryptophan model compounds (7) generally show two sharp bands (from I^ transitions), one at 289 - 294 nm and the second some seven nanometres to shorter wavelength, which generally has the same sign. Bands corresponding to the L. transitions usually occur at shorter wavelengths (265-275 nm) and show little fine structure. The Ae values for these bands are expected to lie in the range ± 3 M"^ cm"^ (7). The changes in the near UV CD spectrum upon binding of peptide conform to the general pattern described for Trp CD (indicating that there is little contribution from the two Phe residues in the peptides), but the two spectra differ significantly in both magnitude and sign. The large negative intensity of the Trp of the bound FFW peptide clearly indicates that the indole chromophore is strongly immobilized in an asymmetric environment. Based on the solution structure of the CaM:M13 CaM:WFF
280
300
320
340
360
wavelength (nm)
380
400
250
260
270
280
290
300 310
320
wavelength (nm)
Figure 1 - A) Fluorescence spectra of WFF and FFW peptides, free and bound to wildtype calmodulin, [peptide] = 200 nM, [CaM] = 200 nM in 25 mM Tris (pH 7.5), 100 mM KCl, and 1 mM CaClj. B) Near UV CD spectra of 20 /xM wildtype calmodulin alone and in (1:1) complex with WFF and FFW peptide (Ae is per mole calmodulin).
Calmodulin-Target Sequence Interactions
405
peptide complex (2) the Trp sidechain of the bound WFF peptide is also expected to be strongly immobilized. The weaker CD signal of the WFF Trp is diagnostic of lower asymmetry but not necessarily greater mobility. The asymmetry derives from two sources -1) electronic interaction with other chromophores (eg Phe) and polarisable groups (eg sidechains) in the closely packed interior of the protein, and 2) electronic interaction with neighbouring peptide groups which are arrayed asymmetrically (owing to the L-chiral configuration of natural amino acids). The CD properties of a chromophoric side chain thus reflect both secondary and tertiary structure. Near UV CD is a sensitive indicator of relatively small changes in protein conformation in the vicinity of the aromatic group, as well as an indicator of different chiral environments within a protein. The very different near UV CD spectra indicate distinct chiroptical environments of the Trp residue in the two peptide complexes and are consistent with interaction of Trp4 (in peptide WFF) and Trpl7 (in peptide FFW) with different domains of the protein. This would suggest that both target peptides are binding to calmodulin in the same orientation i.e. with residue 4 interacting with the C-domain of calmodulin and residue 17 interacting with the N-domain, as was found for the homologous 26-residue M13 peptide bound to calmodulin (2). The affinities of the two peptides for four calcium binding site mutants of calmodulin also provide important information. The B2K and B2Q calmodulins have Glu67 (in binding site 2) mutated to Lys and Gin respectively, and B4K and B4Q calmodulins have Glul40 (in binding site 4) mutated to Lys and Gin respectively. Each of these mutations effectively eliminates calcium binding to the altered site (3). As shown in Table 1, the affinity of each of the mutant proteins for either peptide is at least 10-fold lower than that of wildtype calmodulin. The B2K mutant has the highest affinity for both peptides, suggesting that it is the least altered in function. The B4K mutant has the lowest affinity for both peptides - more than 200-fold lower than wildtype calmodulin suggesting that it is the most altered in function. Although the B2Q and B4Q mutants both have about 100-fold lower affinity for the WFF peptide than wildtype CaM, there is a 10-fold difference in their affinities for the FFW peptide. The fluorescence enhancement upon binding of the FFW peptide to the B2Q mutant is also much lower than that for any of the other proteins. These results suggest that the E67Q (B2Q) but not the E67K (B2K) mutation in site 2 of the N-domain has significantly altered the interaction with the sidechain of the residue in position 17 of the peptide. It is interesting to note that two different replacements for a single residue in the protein result in significantly different affinities.
Wendy A. Findlay et al.
406
Table I - Dissociation constants of wildtype and four mutant calmodulins for WFF and FFW peptides and fluorescence enhancement (at X =330 nm) upon complex formation CaM
WFF peptide K,(nM) F/Fo
FFW peptide F/Fo
Ksi(nM)
WT
<0.22 (1)'
2.4
1.6
(1)
3.0
B2K
5.2 (24)
2.7
19
(12)
3.1
320 (200)
2.0
B2Q
21
(95)
2.8
B4Q
21
(95)
2.5
38
(24)
2.9
B4K
48
(218)
2.2
340 (213)
2.4
• Values in brackets are relative to the wildtype K^.
Of all the mutant calmodulins studied, the B4K mutant has the most altered a-helical secondary structure, based on the CD signal at 222nm, (Fig. 2A). Values obtained from current samples of B2K and B2Q mutants show somewhat weaker far UV-CD than previously reported, (4), although the absolute values for a given mutant are critically dependent upon determination of protein concentration. When the WFF peptide is added, the CD (222nm) increases for all the proteins, as shown in Fig. 2B. For WT-protein this increase in intensity is interpreted as deriving mainly from the bound peptide adopting an a-helical conformation (2). 200
210 220 230 wavelength (nm)
210 220 230 wavelength (nm)
Figure 2 - Far UV CD spectra of wildtype and four mutant calmodulins A) alone and B) in (1:1) complex with WFF peptide (Ae is per mole calmodulin).
Calmodulin-Target Sequence Interactions
407
As shown in Fig. 2B, the far UV CD spectra of the mutant calmodulins in complex with the WFF peptide more closely resemble that of the wildtype calmodulin:WFF peptide complex than do the corresponding spectra in the absence of peptide (Fig. 2A). Quantitation of this effect shows that in addition to induction of of-helix in the bound peptide, the mutant calmodulins have recovered at least some of the "native" helical structure. IV. Conclusions The study of the interaction of peptides with proteins by optical spectroscopy is greatly facilitated if the peptide contains tryptophan and the protein does not, as in the case of the calmodulin/peptide systems studied here. Even for proteins which contain one or more tryptophans this approach can still be used provided that the spectral changes associated with binding of the peptide are sufficiently large. The use of synthetic peptides or site specific mutagenesis allows almost any residue to be replaced by tryptophan. The most conservative substitutions on the basis of size and hydrophobicity would be Phe - > Trp or Tyr - > Trp. In general, binding of the peptide to a protein will result in a shift in the tryptophan fluorescence emission maximum from approximately 355 nm (free peptide) to shorter wavelength as the tryptophan enters a more hydrophobic environment, and an overall intensification of the fluorescence emission intensity. The extent of the wavelength shift gives some information about the environment of the tryptophan in the complex. More importantly, the fluorescence enhancement on binding of the peptide may be used to determine the dissociation constant (K^) at the low protein/peptide concentrations required to study high affinity interactions. The principal near UV circular dichroism bands of tryptophan are generally distinct (at longer wavelength) from those of tyrosine and phenylalanine and ^so differ in characteristic band shape. Since near UV CD bands can be either positive or negative, the near UV CD spectrum can provide more information about the properties of aromatic residues than absorption spectroscopy. Free peptides (< 20 residues) generally have negligible near UV CD because of the conformational mobility of the chromaphoric aromatic side chains. However, as shown in this work, the immobilization of an aromatic group of a peptide when bound to a protein means that the near UV CD spectrum may be used to distinguish different chiral environments for aromatic residues such as tryptophan introduced in
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Wendy A. Findlay et al.
different positions in the peptide. Far UV circular dichroism spectra contain information about secondary structure content in proteins and peptides. Small peptides are almost invariably unstructured in aqueous solution, but can adopt regular secondary structure upon binding to a protein. In general, the secondary structure of a protein is unlikely to change dramatically upon binding to a peptide. However, as shown in this work, the binding of a target peptide to a mutated protein with an altered structure (compared with the wildtype) may partially restore the "native" structure of the protein. The use of site specific mutagenesis of a protein in identifying individual residues involved in enzyme function or interaction with ligands is well established. In the case of calmodulin, whose mode of action is dependent on its calcium sensitive interaction with target proteins, a mutant protein may retain the ability to activate its target enzymes (5). This may mask the fact that a structural change in the mutant calmodulin has actually been compensated for by the strength of the interaction with the target sequence. Such a mechanism can be identified and characterized by quantitative examination of the interaction of the mutant protein with peptides related to the calmodulin binding sequence of the target enzyme, as outlined here.
Acknowledgments We thank Dr. K. Beckingham and colleagues (Rice University, Texas, USA) for providing the mutant calmodulins and Peter Fletcher (N.I.M.R.) for synthesis and purification of the peptides. References 1. O'Neil, K.T. and Degrade, W.F. (1990). TJ.B.S. 15, 59. 2. Ikura, M., Clore, G.M., Gronenbora, A.M., Zhu, G., Klee, C.B., and Bax, A. (1992) Science 256, 632. 3. Maune, J.F., Klee, C.B., and Beckingham, K. (1992)7. Biol Chem. 161, 5286. 4. Maune, J.F., Beckingham, K., Martin, S.R., and Bayley P.M. (1992)Biochemistry 31, 7779. 5. Gao, Z.H., Krebs, J., VanBerkum, M.F.A., Tang, W.-J., Maune, J.F., M e a n s , A.R., Stull, J.T., and Beckingham, K. (1993)7. Biol Chem. 268, 20096. 6. Bevington, P.R. (1969) in "Data Reduction and Error Analysis for the Physical Sciences", McGraw-Hill, New York, USA. 7. Strickland, E.H. (1974) Crit. Rev. Biochem. 2, 113.
I. Introduction Protein-protein interactions are important in the function and regulation of many biological pathways. Associations between proteins are often characterized by strong and extremely specific noncovalent interactions between complementary surfaces. One way of looking at the details of these interactions is to use peptides corresponding to the "interaction region" of one of the proteins to determine the minimum sequence needed for the interaction as well as the effect of changing individual residues. We report here on the use of this approach to study the interaction of calmodulin with a target sequence from skeletal muscle myosin light chain kinase (sk-MLCK). The strategy is to use a number of sequence variants of the peptide and site directed mutants of calmodulin. Calmodulin is a small ubiquitous calcium binding protein which regulates a variety of enzymes in several different metabolic pathways. Calmodulin interacts with many of its target proteins with very high affinity (K^ — nM), usually in a calcium specific manner. It also binds target sequence peptides derived from the calmodulin binding regions of many of these proteins with affinities close to those for the intact enzymes. Many target sequences are predicted to form basic amphipathic helices and this has been proposed as a common structural motif for calmodulin binding (1). In the solution structure of a complex of calmodulin with a 26-residue target peptide derived from the sequence of sk-MLCK, the two domains of TECHNIQUES IN PROTEIN CHEMISTRY VI Copyright © 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
401
402
Wendy A. Findlay et al.
calmodulin surround the Ml3 peptide, which has adopted an oj-helical conformation (2). The peptide lies in a hydrophobic channel and the sidechains of Trp4 and Phel7 of the peptide appear to anchor the peptide to the two domains by fitting into the hydrophobic "pockets". Maune et al. (3) have produced a set of single site mutants of Drosophila melanogaster calmodulin, each of which has the conserved glutamic acid residue in the 12th position of one of the four calcium binding loops mutated to either glutamine or lysine. Mutations to sites 2 or 4 effectively eliminate calcium binding to the mutated site and cause structural changes in the protein (3, 4) as well as decreasing the ability of calmodulin to activate target enzymes (5). The availability of these mutant calmodulins and the choice of synthetic peptides derived from the sk-MLCK target sequence allows the manipulation of both components involved in the interaction. The 18residue sk-MLCK target sequence has 3 aromatic residues: Trp4, Phe8, and Phel7. In this work, we use peptides with tryptophan in either position 4 (WFF peptide) or position 17 (FFW peptide). Since the calmodulin itself has no tryptophan residues, we can use optical spectroscopy to monitor the interaction of a tryptophan in a specific position in the target sequence with an individual domain of calmodulin. We have studied binding of the two peptides to wildtype calmodulin and to the site 2 and site 4 mutants (B2Q, B2K, B4Q, and B4K) to see how mutations which effectively eliminate calcium binding to a particular site affect the interaction of the protein with the two target sequence analogues.
n . Materials and Methods Proteins and peptides - Drosophila melanogaster calmodulin and the various mutants expressed in E. coli were purified essentially as previously described (3). Peptides were synthesized on an Applied Biosystems 430A peptide synthesizer and purified by reverse phase HPLC on a CIS column (WFF peptide) or a C8 column (FFW peptide) and were provided with free carboxy and amino termini. All concentrations were determined spectrophotometrically using a calculated extinction coefficient of 5560 M"^ cm"^ at 280 nm for the peptides (2 Phe and 1 Trp) and published extinction coefficients for wildtype and the four mutant calmodulins (3).
Calmodulin-Target Sequence Interactions
403
Fluorescence and affinity measurements - Peptide in 25 mM Tris, 100 mM KCl and 1 mM CaCl2 at pH 7.5 and 30 C was titrated with a stock solution of calmodulin in UV transmitting plastic cuvettes since the peptides appear to bind to glass. Fluorescence titration spectra were recorded using a SPEX FluoroMax fluorescence spectrometer with excitation at 280 nm and emission scanned from 310 to 390 nm. The value of fluorescence intensity at 330nm was plotted as a function of calmodulin concentration and fitted using standard non-linear least squares methods (6) to obtain optimal values of the dissociation constant (K^) and the maximum fluorescence enhancement (F/FQ). The detection limit under our experimental conditions was 50 nM peptide and all quoted K,, values are the average of at least 3 independent determinations. Circular dichroism spectra - Spectra were recorded on a Jasco J-600 spectropolarimeter at room temperature. Far UV CD spectra (190 to 260 nm) of 7.5 fiM peptide:calmodulin complex in 25 mM Tris, 100 mM KCl and 1 mM CaCl2 were measured in a 0.1 cm path length cuvette. Near UV CD spectra (250 to 340 nm) of 20 /xM peptide:calmodulin complex in the same buffer were measured in a 1 cm path length cuvette.
n i . Results We have used two synthetic 18-residue peptides related to the target sequence of sk-MLCK to study their interaction with calmodulin in the presence of calcium. The WFF peptide (KKRWKKNFIAVSAANRFK) corresponds to residues 577 to 594 of rabbit sk-MLCK. In the FFW peptide (KKRFKKNFIAVSAANRWK) the W4 and F17 residues have been interchanged. Upon binding to the protein the tryptophan fluorescence emission maximum for each peptide is shifted from 356 nm to 334 nm as shown in Fig. lA, with an enhancement in fluorescence intensity at 330 nm of about 2.4-fold for the WFF peptide and 3-fold for the FFW peptide (Table 1). These results indicate that the Trp residue is in a hydrophobic environment when either peptide binds to the protein. By monitoring fluorescence intensity at 330 nm while titrating either peptide with calmodulin, we determined the affinities of calmodulin for the FFW peptide (Kd= 1.6 nM) and the WFF peptide (K^<0.2 nM). The affinity of calmodulin for the native sequence (WFF peptide) is at least an order of magnitude higher than that for the modified sequence (FFW peptide).
404
Wendy A. Findlay et al.
We used near UV CD spectroscopy to obtain information about the steric environment of the Trp sidechain in each of the protein:peptide complexes. Figure IB shows the near-UV CD spectra of wild-type Drosophila calmodulin in the presence of calcium alone and in (1:1) complexes with the WFF and FFW peptides. Free calmodulin shows prominent bands at 262 and 268 nm, which derive from the nine Phe residues. The signal at longer wavelengths (X > 275 nm) derives from the single Tyr located at position 138 in the C-terminal domain. The free peptides show negligible circular dichroism in this wavelength range. The spectra of the complexes of calmodulin with the WFF or FFW peptide show clear evidence of a major contribution from the Trp residue in the peptide. Tryptophan model compounds (7) generally show two sharp bands (from I^ transitions), one at 289 - 294 nm and the second some seven nanometres to shorter wavelength, which generally has the same sign. Bands corresponding to the L. transitions usually occur at shorter wavelengths (265-275 nm) and show little fine structure. The Ae values for these bands are expected to lie in the range ± 3 M"^ cm"^ (7). The changes in the near UV CD spectrum upon binding of peptide conform to the general pattern described for Trp CD (indicating that there is little contribution from the two Phe residues in the peptides), but the two spectra differ significantly in both magnitude and sign. The large negative intensity of the Trp of the bound FFW peptide clearly indicates that the indole chromophore is strongly immobilized in an asymmetric environment. Based on the solution structure of the CaM:M13 CaM:WFF
280
300
320
340
360
wavelength (nm)
380
400
250
260
270
280
290
300 310
320
wavelength (nm)
Figure 1 - A) Fluorescence spectra of WFF and FFW peptides, free and bound to wildtype calmodulin, [peptide] = 200 nM, [CaM] = 200 nM in 25 mM Tris (pH 7.5), 100 mM KCl, and 1 mM CaClj. B) Near UV CD spectra of 20 /xM wildtype calmodulin alone and in (1:1) complex with WFF and FFW peptide (Ae is per mole calmodulin).
Calmodulin-Target Sequence Interactions
405
peptide complex (2) the Trp sidechain of the bound WFF peptide is also expected to be strongly immobilized. The weaker CD signal of the WFF Trp is diagnostic of lower asymmetry but not necessarily greater mobility. The asymmetry derives from two sources -1) electronic interaction with other chromophores (eg Phe) and polarisable groups (eg sidechains) in the closely packed interior of the protein, and 2) electronic interaction with neighbouring peptide groups which are arrayed asymmetrically (owing to the L-chiral configuration of natural amino acids). The CD properties of a chromophoric side chain thus reflect both secondary and tertiary structure. Near UV CD is a sensitive indicator of relatively small changes in protein conformation in the vicinity of the aromatic group, as well as an indicator of different chiral environments within a protein. The very different near UV CD spectra indicate distinct chiroptical environments of the Trp residue in the two peptide complexes and are consistent with interaction of Trp4 (in peptide WFF) and Trpl7 (in peptide FFW) with different domains of the protein. This would suggest that both target peptides are binding to calmodulin in the same orientation i.e. with residue 4 interacting with the C-domain of calmodulin and residue 17 interacting with the N-domain, as was found for the homologous 26-residue M13 peptide bound to calmodulin (2). The affinities of the two peptides for four calcium binding site mutants of calmodulin also provide important information. The B2K and B2Q calmodulins have Glu67 (in binding site 2) mutated to Lys and Gin respectively, and B4K and B4Q calmodulins have Glul40 (in binding site 4) mutated to Lys and Gin respectively. Each of these mutations effectively eliminates calcium binding to the altered site (3). As shown in Table 1, the affinity of each of the mutant proteins for either peptide is at least 10-fold lower than that of wildtype calmodulin. The B2K mutant has the highest affinity for both peptides, suggesting that it is the least altered in function. The B4K mutant has the lowest affinity for both peptides - more than 200-fold lower than wildtype calmodulin suggesting that it is the most altered in function. Although the B2Q and B4Q mutants both have about 100-fold lower affinity for the WFF peptide than wildtype CaM, there is a 10-fold difference in their affinities for the FFW peptide. The fluorescence enhancement upon binding of the FFW peptide to the B2Q mutant is also much lower than that for any of the other proteins. These results suggest that the E67Q (B2Q) but not the E67K (B2K) mutation in site 2 of the N-domain has significantly altered the interaction with the sidechain of the residue in position 17 of the peptide. It is interesting to note that two different replacements for a single residue in the protein result in significantly different affinities.
Wendy A. Findlay et al.
406
Table I - Dissociation constants of wildtype and four mutant calmodulins for WFF and FFW peptides and fluorescence enhancement (at X =330 nm) upon complex formation CaM
WFF peptide K,(nM) F/Fo
FFW peptide F/Fo
Ksi(nM)
WT
<0.22 (1)'
2.4
1.6
(1)
3.0
B2K
5.2 (24)
2.7
19
(12)
3.1
320 (200)
2.0
B2Q
21
(95)
2.8
B4Q
21
(95)
2.5
38
(24)
2.9
B4K
48
(218)
2.2
340 (213)
2.4
• Values in brackets are relative to the wildtype K^.
Of all the mutant calmodulins studied, the B4K mutant has the most altered a-helical secondary structure, based on the CD signal at 222nm, (Fig. 2A). Values obtained from current samples of B2K and B2Q mutants show somewhat weaker far UV-CD than previously reported, (4), although the absolute values for a given mutant are critically dependent upon determination of protein concentration. When the WFF peptide is added, the CD (222nm) increases for all the proteins, as shown in Fig. 2B. For WT-protein this increase in intensity is interpreted as deriving mainly from the bound peptide adopting an a-helical conformation (2). 200
210 220 230 wavelength (nm)
210 220 230 wavelength (nm)
Figure 2 - Far UV CD spectra of wildtype and four mutant calmodulins A) alone and B) in (1:1) complex with WFF peptide (Ae is per mole calmodulin).
Calmodulin-Target Sequence Interactions
407
As shown in Fig. 2B, the far UV CD spectra of the mutant calmodulins in complex with the WFF peptide more closely resemble that of the wildtype calmodulin:WFF peptide complex than do the corresponding spectra in the absence of peptide (Fig. 2A). Quantitation of this effect shows that in addition to induction of of-helix in the bound peptide, the mutant calmodulins have recovered at least some of the "native" helical structure. IV. Conclusions The study of the interaction of peptides with proteins by optical spectroscopy is greatly facilitated if the peptide contains tryptophan and the protein does not, as in the case of the calmodulin/peptide systems studied here. Even for proteins which contain one or more tryptophans this approach can still be used provided that the spectral changes associated with binding of the peptide are sufficiently large. The use of synthetic peptides or site specific mutagenesis allows almost any residue to be replaced by tryptophan. The most conservative substitutions on the basis of size and hydrophobicity would be Phe - > Trp or Tyr - > Trp. In general, binding of the peptide to a protein will result in a shift in the tryptophan fluorescence emission maximum from approximately 355 nm (free peptide) to shorter wavelength as the tryptophan enters a more hydrophobic environment, and an overall intensification of the fluorescence emission intensity. The extent of the wavelength shift gives some information about the environment of the tryptophan in the complex. More importantly, the fluorescence enhancement on binding of the peptide may be used to determine the dissociation constant (K^) at the low protein/peptide concentrations required to study high affinity interactions. The principal near UV circular dichroism bands of tryptophan are generally distinct (at longer wavelength) from those of tyrosine and phenylalanine and ^so differ in characteristic band shape. Since near UV CD bands can be either positive or negative, the near UV CD spectrum can provide more information about the properties of aromatic residues than absorption spectroscopy. Free peptides (< 20 residues) generally have negligible near UV CD because of the conformational mobility of the chromaphoric aromatic side chains. However, as shown in this work, the immobilization of an aromatic group of a peptide when bound to a protein means that the near UV CD spectrum may be used to distinguish different chiral environments for aromatic residues such as tryptophan introduced in
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Wendy A. Findlay et al.
different positions in the peptide. Far UV circular dichroism spectra contain information about secondary structure content in proteins and peptides. Small peptides are almost invariably unstructured in aqueous solution, but can adopt regular secondary structure upon binding to a protein. In general, the secondary structure of a protein is unlikely to change dramatically upon binding to a peptide. However, as shown in this work, the binding of a target peptide to a mutated protein with an altered structure (compared with the wildtype) may partially restore the "native" structure of the protein. The use of site specific mutagenesis of a protein in identifying individual residues involved in enzyme function or interaction with ligands is well established. In the case of calmodulin, whose mode of action is dependent on its calcium sensitive interaction with target proteins, a mutant protein may retain the ability to activate its target enzymes (5). This may mask the fact that a structural change in the mutant calmodulin has actually been compensated for by the strength of the interaction with the target sequence. Such a mechanism can be identified and characterized by quantitative examination of the interaction of the mutant protein with peptides related to the calmodulin binding sequence of the target enzyme, as outlined here.
Acknowledgments We thank Dr. K. Beckingham and colleagues (Rice University, Texas, USA) for providing the mutant calmodulins and Peter Fletcher (N.I.M.R.) for synthesis and purification of the peptides. References 1. O'Neil, K.T. and Degrade, W.F. (1990). TJ.B.S. 15, 59. 2. Ikura, M., Clore, G.M., Gronenbora, A.M., Zhu, G., Klee, C.B., and Bax, A. (1992) Science 256, 632. 3. Maune, J.F., Klee, C.B., and Beckingham, K. (1992)7. Biol Chem. 161, 5286. 4. Maune, J.F., Beckingham, K., Martin, S.R., and Bayley P.M. (1992)Biochemistry 31, 7779. 5. Gao, Z.H., Krebs, J., VanBerkum, M.F.A., Tang, W.-J., Maune, J.F., M e a n s , A.R., Stull, J.T., and Beckingham, K. (1993)7. Biol Chem. 268, 20096. 6. Bevington, P.R. (1969) in "Data Reduction and Error Analysis for the Physical Sciences", McGraw-Hill, New York, USA. 7. Strickland, E.H. (1974) Crit. Rev. Biochem. 2, 113.