(Received for publication, October 17, 1996, and in revised form, December 19, 1996)
From the Department of Pharmacology, Duke University
Medical Center, Durham, North Carolina 27710 and the
§ Department of Biochemistry, Howard Hughes Medical
Institute, Baylor College of Medicine, Houston, Texas 77030
To test the relevance of the calmodulin-peptide
crystal structures to their respective calmodulin-enzyme interactions,
amino acid side chains in calmodulin were altered at positions that interact with the calmodulin-binding peptide of smooth muscle myosin
light chain kinase but not with the calmodulin kinase II peptide.
Since shortening the side chains of Trp-800, Arg-812, and Leu-813 in
smooth muscle myosin light chain kinase abrogated calmodulin-dependent activation (Bagchi, I. C., Huang, Q.,
and Means, A. R. (1992) J. Biol. Chem. 267, 3024-3029), substitutions were introduced at positions in calmodulin
which contact residues corresponding to Arg-812 and Leu-813 in the
smooth muscle myosin light chain kinase peptide. Assays of smooth
muscle myosin light chain kinase with the calmodulin mutants
M51A,V55A, L32A,M51A,V55A, and L32A,M51A,V55A,F68L, M71A
exhibited 60%, 25%, and less than 1% of maximal activity
respectively, whereas the mutants fully activated calmodulin kinase
II
. Alanine substitutions at positions on the smooth muscle myosin
light chain kinase peptide, corresponding to Trp-800 and Arg-812 in the
enzyme, produced an 8-fold increase in the enzyme inhibition
constant in contrast with the abolition of calmodulin binding by
similar mutations in the parent enzyme.
Calmodulin (CaM)1 is an intracellular
protein that binds to a wide variety of enzymes in a
Ca2+-dependent manner and serves as a
regulatory subunit. Three examples of such enzymes are the protein
kinases smooth muscle myosin light chain kinase (smMLCK),
CaM-dependent protein kinase II (CaMKII
), and
skeletal muscle myosin light chain kinase (skMLCK). Since peptides
derived from these enzymes can bind Ca2+/CaM with high
affinity (Kd = nM), they have been
treated as representative models of their respective CaM-enzyme
interactions. The three-dimensional structures of the
Ca2+/CaM-peptide complexes derived from these three protein
kinases have been solved by x-ray crystallography or NMR spectroscopy (1-3). Although the amino acid sequence of each peptide is distinct, the three structures show similar themes. The peptides adopt a helical
structure and orient antiparallel to the NH2- and
COOH-terminal domains of CaM. To accommodate the different primary
structures, the linker connecting the two domains of CaM serves as a
variable expansion joint thus facilitating numerous van der Waals
contacts between the peptides and hydrophobic pockets located in the
two domains of Ca2+/CaM. On closer inspection, however, the
Ca2+/CaM-peptide complexes exhibit differences in the
specific details of their interactions. A comparison of the
CaMKII
peptide (CaMKII
p)·CaM complex with the smMLCK peptide
(smMLCKp)·CaM complex reveals that the NH2-terminal
domain of CaM interacts differently with the COOH-terminal ends of
either the CaMKII
p or the smMLCKp (2). The specific contacts between
the CaM domains and the peptides are important for the formation of
these structures since the same contacts are maintained between the
CaMKII
p and CaM mutants containing multiple residue deletions and
insertions in the linker between the CaM domains (4).
The relevance of the CaM-peptide structural models to the interaction
between CaM and the intact enzymes remains largely untested. Previous
mutagenesis studies on CaM and CaM-binding enzymes did not address this
point since most failed to target the binding interface between the two
proteins. One exception was an alanine scan of the CaM binding domain
of smMLCK. These studies demonstrated that the side chains of Trp-800,
Arg-812, and Leu-813, which interact with multiple side chains of CaM
in the Ca2+/CaM·smMLCKp structure, are critical for CaM
binding and activation of the enzyme (5). Collectively, the available
data presented an opportunity to test whether the different CaM-peptide
complexes provide information on how CaM interacts with and activates
the target enzymes. To address this question two complementary
approaches were taken. First, in an attempt to perturb the CaM-smMLCK
interaction without interfering with the CaM·CaMKII complex
we studied the effects of truncating amino acid side chains in the
NH2-terminal domain of CaM which differentially interact
with the smMLCKp and the CaMKII
p. Consequently Ala residues were
sequentially substituted for CaM residues Leu-32, Met-51, Val-55, and
Met-71. These side chains interact with residues in the smMLCKp,
corresponding to Arg-812 and Leu-813 in the enzyme smMLCK but have no
contacts with the CaMKII
p. Both enzymes were then tested for
activation by the resulting double, triple, or multiple substitution
mutants of CaM. Second, we tested whether simultaneous Ala
substitutions of the Trp-5 and Arg-17 residues in the smMLCKp would
eliminate CaM binding as was demonstrated when similar changes were
made at the corresponding Trp-800 and Arg-812 in the context of the enzyme (5).
Mutants of CaM were
generated by the combination of two protocols. First the double mutant
of CaM, M51A,V55A, was introduced into the pCaM23N plasmid containing
the chicken CaM cDNA (6) by unique restriction site elimination
mutagenesis (7). This procedure simultaneously eliminated the unique
AccI restriction site. The protein coding region was then
subcloned into the pCaMpl vector (8) via the unique 5 NcoI
and 3
XbaI restriction sites to facilitate bacterial
expression. The triple Ala mutant of CaM, L32A,M51A,V55A, was created
by the method of site-directed polymerase chain reaction mutagenesis
(9) using the pCaM23N construct containing the M51A,V55A mutant as
template. The resulting NcoI-XbaI fragment was
then ligated into the pCaMpl expression vector. The L32A,M51A,V55A,F68L,M71A multiple mutant of CaM was also created by
polymerase chain reaction mutagenesis, but instead using the plasmid
from the L32A,M51A,V55A mutant as DNA template. The identities of the
mutant cDNA constructs were verified by double-stranded DNA
sequencing of the entire protein coding region of CaM.
CaM mutant proteins were expressed in the heat-inducible bacterial strain N5151 and purified by conventional column chromatography as described previously (10). Briefly, bacteria were grown in 1 liter of LB broth at 30 °C to A595 = 1 and induced to express protein by heat induction at 42 °C for 1.5 h. The bacterial pellet was then collected after centrifugation, suspended in a solution of 2.4 M sucrose, and subjected to overnight lysis in a lysozyme solution. CaM was purified from the resulting lysis supernatant by phenyl-Sepharose and gel filtration chromatography. The purified proteins subsequently were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Concentrations of CaM were determined by UV absorbance (11) and by the method of Bradford (12).
CaM-binding Peptides of smMLCKThe peptides containing the sequence of the CaM binding domain of smMLCK residues 796-813 (smMLCKp) ARRKWQKTGHAVRAIGRL and the two alanine substitutions (W5A,R17A) ARRKAQKTGHAVRAIGAL were made by automated solid phase synthesis and purified by reverse phase high performance liquid chromatography. The compositions of the peptides and their respective concentrations were verified by amino acid analysis.
Assay of CaM-dependent Activation of MLC Kinase and Autophosphorylation of CaMKIISmMLCK was purified from a baculovirus expression system and assayed for light chain phosphorylation in the presence of increasing amounts of CaM as described previously (10). Assays were initiated by the addition of enzyme (2 nM final concentration) in a total volume of 50 µl and performed for 10 min at 30 °C in 50 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM CaCl2, 1 mM dithiothreitol, 0.1% Tween 80, 0.5 mg/ml bovine serum albumin, 0.1 mM ATP (0.2 µCi), and 50 µM MLCs (17). Aliquots of 40 µl were loaded onto a Whatman 3MM filter and washed with four or five changes in a solution of 10% trichloroacetic acid and 2% sodium pyrophosphate. Assays were also conducted under similar conditions except using 200 µM of the MLC peptide MLC 11-23 as substrate. In this case aliquots were loaded onto Whatman P81 filters and washed in 75 mM phosphoric acid. Filters were counted on a Beckman LS 6000 scintillation counter. Kinetic constants for CaM and the mutant CaM were derived from the equation
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(Eq. 1) |
The inhibition constant for the CaM multiple mutant L32A,M51A, V55A,F68L,M71A with smMLCK was derived from a modified version of the assay with MLC as substrate. A competition assay was performed with either 1, 2, 4 or 8 nM wild type CaM and increasing concentrations of the CaM mutant protein. Inhibition constants were derived from double-reciprocal plots and linear regression analysis. The enzyme inhibition constants for the smMLCK peptides were obtained by adding increasing concentrations (20-1000 nM) of either peptide to the smMLCK assay containing 200 nM CaM and 100 nM enzyme. Inhibition constants were derived by fitting the results from the competition assay with the CURVE-FIT program to an equation describing an activator depletion model.
Ca2+/CaM-dependent autophosphorylation of
baculovirus-expressed CaMKII was measured at 1 mM
CaCl2 as described (10). Kinetic constants
(KCaM and percent maximum activity) were derived
in a manner similar to that for smMLCK.
The aim of this study was to investigate the functional
implications of the CaM-peptide structures by correlating incremental structural changes at the protein-peptide interface with changes in CaM
binding or enzyme activation. Therefore perturbations were introduced
at the CaM·smMLCKp binding site by either by shortening side chains
of CaM which exhibited differences in contacts between the smMLCKp and
the CaMKIIp or by shortening side chains on the smMLCKp which were
required for binding and activation of the parent smMLCK enzyme.
In the first case mutations were introduced into the CaM NH2-terminal domain by combining the techniques of unique site elimination and polymerase chain reaction mutagenesis. The resulting CaM mutant constructs contained either two amino acid side chain changes, the double mutant M51A,V55A; three side chain substitutions, the triple mutant L32A,M51A, V55A; or five side chain substitutions, the multiple mutant L32A,M51A,V55A,F68L,M71A. The different CaMs were inducibly expressed in bacteria and purified using chromatographic procedures identical to those for the wild type CaM. This resulted in similar yields for all proteins of approximately 10-20 mg of protein/liter of liquid bacterial culture.
The mutant proteins were then compared with authentic CaM in their
respective abilities to support autophosphorylation of the enzyme
CaMKII. The resulting activation profiles at increasing concentrations of CaM are presented in Fig.
1A. Similar to authentic CaM, all mutant CaMs
were capable of maximal (100%) activation of CaMKII
autophosphorylation. In the specific case of the double mutant
M51A,V55A, the activation constant (KCaM = 50 nM) was almost identical to that of CaM (45 nM). In contrast, the triple and multiple mutants of CaM
exhibit larger increases in KCaM, which are
approximately 3-8-fold greater than the normal protein, at 120 and 350 nM, respectively.
The results from testing the ability of the CaM mutants to activate
smMLCK phosphorylation of MLCs are plotted in Fig. 1B. In
contrast to CaMKII, none of the CaM mutant protein was capable of
maximally activating smMLCK. The double mutant M51A,V55A achieved 60%
of the maximal activity of the wild type, whereas the triple mutant
L32A,M51A,V55A resulted in only 25% of maximal activity. The multiple
mutant L32A,M51A,V55A,F68L,M71A showed the largest change with less
than 1% of maximal activity. Interestingly, the KCaM values for the double and triple CaM
mutants were not significantly different from CaM (1.5 nM)
as they increased less than 3-fold to 4.0 nM for the double
mutant and 3.0 nM for the triple mutant. Since the multiple
mutant L32A,M51A,V55A,F68L, M71A showed scant ability to activate
smMLCK, an inhibition constant of 4.5 nM was determined
from competition assays with authentic CaM. In addition, the use of the
13-residue MLC peptide MLC 11-23 as substrate in the assays gave
essentially the same results as with the full-length 20-kDa light
chains.
A comparison of the parameters for CaM and mutant CaM proteins is
presented in Table I, listing activation constants and maximal activity for autophosphorylation of CaMKII and for
phosphorylation of MLC or MLC peptide 11-23 by smMLCK. Sequential
mutations of the side chains Leu-32, Met-51, Val-55, and Met-71 of CaM
to Ala and Phe-68 to Leu in the NH2-terminal domain of CaM
had little effect on the activation of CaMKII
since the most
deleterious CaM mutant, L32A,M51A,V55A,F68L,M71A, produced less than an
8-fold increase in KCaM for CaMKII
while
retaining 100% maximal activity. These results are consistent with the
CaM·CaMKII
p structural model since the truncated amino acid side
chains of CaM do not interact with the CaMKII
p. They also correlate
closely with results from earlier studies on the
NH2-terminal domain of CaM where chemical modification of
Lys-75 with the phenothiazine affinity reagent norchlorpromazine
isothiocyanate had little effect on the activation of CaMKII (13). In
addition a chimeric protein containing the NH2-terminal
domain E-F hand I of cardiac troponin C joined in-frame with the
remaining portion of CaM (TaM-BM1) had at most a 5-fold increase in the
activation constant for CaMKII (14).
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In contrast with the results from CaMKII, the same CaM mutant
proteins produced large reductions (>40-99%) in the maximal activity
of smMLCK with little change in the activation or inhibitory constants
(Table I). The mutated residues on CaM interact with a residue on the
smMLCKp, corresponding to Leu-813 on the intact enzyme, which has been
proposed to play an important role in the recognition of CaM (1). The
small effect of these mutations on the affinity of smMLCK for CaM, as
reflected in the similar activation constants (Table I), suggests that
the van der Waals packing with smMLCK Leu-813 is important for
activating the enzyme and less critical in the formation of a high
affinity CaM·smMLCK complex. This result also agrees with those from
the norchlorpromazine isothiocyanate derivative of the CaM
NH2-terminal domain which was unable to activate smMLCK but
still bound to the enzyme with high affinity (15). Finally, the results
from a complementary study on the kinase domain of smMLCK by alanine
scanning mutagenesis provide support for this point since the L813A
mutant lost more than 85% of maximal activity while retaining some
ability to bind CaM as assessed by a gel overlay assay (5).
It is remarkable to observe the extent to which the molecular details derived from these and other recent mutagenesis studies on the Ca2+/CaM·smMLCK interaction closely follow an earlier two-step model proposing a mechanism of CaM function based on the gross thermodynamics of the Ca2+/CaM·skMLCK interaction (17). The first event in this model involves the hydrophobic interaction between undetermined groups of the enzyme and Ca2+/CaM which function to immobilize partially both proteins while stabilizing the formation of a high affinity complex. The site of the initial, high affinity interaction between smMLCK and Ca2+/CaM has been localized to the COOH-terminal hydrophobic pocket of Ca2+/CaM by scanning mutagenesis of the nine widespread Mets of CaM (10). These experiments demonstrated that a solvent accessible hydrophobic surface in the COOH-terminal domain of Ca2+/CaM, which includes Met-109 and especially Met-124, is critical for high affinity binding with smMLCK. Importantly, this region of Ca2+/CaM has hydrophobic interactions with nonpolar residues in skMLCKp (3) and smMLCKp (1), including the equivalent of Trp-580 in skMLCK and the homologous Trp-800 in smMLCK, which is required for binding Ca2+/CaM (5).
The second step in the proposed model involves the operation of short range forces including van der Waals, hydrogen bonding, and ionic interactions that lead to enzyme activation. The results presented herein and elsewhere (10) provide compelling evidence that the disruption of van der Waals interactions between nonpolar residues of smMLCK and those in the NH2-terminal domain of CaM have dramatic effects on the maximal activity of the enzyme while scarcely affecting the affinity of the Ca2+/CaM·smMLCK complex. Other studies also suggest that van der Waals interactions between smMLCK and nonpolar residues in the COOH-terminal domain of CaM such as Met-124 and Leu-112 play a similarly important role in smMLCK activation as well (10, 18, 19). Evidence for the role of hydrogen bonding and ionic interactions comes primarily from CaM-troponin C chimeric proteins. These studies reveal that mutation of polar or charged residues located on an external "latch" surface of CaM, distinct from the CaM·smMLCKp interface, will impair activation of smMLCK but still maintain a high affinity complex (14, 16, 18, 19).
Mutagenesis studies on an enzymatically active fragment of smMLCK had shown that Trp-800 and Arg-812 were individually necessary for Ca2+/CaM-dependent binding and enzyme activation as the substitution of either residue markedly decreased both parameters (5). Since both residues interact with CaM in the Ca2+/CaM·smMLCKp complex, we prepared a peptide in which Ala substitutions were introduced simultaneously at positions corresponding to Trp-800 and Arg-812. The resulting double Ala peptide was compared with the original peptide for their respective abilities to bind to Ca2+/CaM and thus inhibit the activation of smMLCK. The two amino acid changes did not abolish the ability of the smMLCKp to bind CaM, but rather resulted in only an 8-fold diminution in the enzyme inhibition constant (Ki) from 1.3 nM to 10.4 nM (Table II).
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This result is consistent with those from other studies which have determined Ca2+/CaM to be capable of high affinity binding to peptides with diverse amino acid sequences (20). A major feature of peptides that bind to Ca2+/CaM is the capability to form an amphipathic helix following the binding of Ca2+/CaM. Using this premise it was possible to design a high affinity CaM-binding peptide de novo consisting only of Leu and Lys residues (LK peptide) (21). The substitution of Trp for Leu at the position of the LK peptide corresponding to Trp-5 in the smMLCKp resulted in a 10-fold increase in affinity for Ca2+/CaM (22). This result is comparable to the effect of the double Ala-substituted smMLCKp (Table II). Other studies on CaM-peptide binding demonstrate even greater plasticity on the part of the peptides. The smMLCKp composed solely of D-amino acids binds just as well to Ca2+/CaM as the peptide composed of normal L-amino acids (23). Also, a study of the skMLCKp shows that no individual Ala substitutions decreased but in all cases instead increased the affinities of the peptide for Ca2+/CaM (24).
The small effects of the double Ala substitutions on the CaM binding properties of the smMLCKp, however, do not agree with the abolition of CaM binding due to individual substitutions at the equivalent positions of Trp-800 and Lys-813 in the smMLCK enzyme. This discrepancy is an indicator of significant differences in the binding of Ca2+/CaM by nonnative peptide sequences compared with the same sequence in the context of an intact enzyme. Insight into the differences in the molecular recognition process between a free CaM-binding peptide and the same peptide segment in the environment of a folded enzyme is provided by the recently determined x-ray structure of Ca2+/CaM-dependent protein kinase I (CaMKI) (25). In this structure, the CaM binding domain of CaMKI is held in place on the surface of the kinase and makes numerous contacts with neighboring groups in both major domains of the enzyme. In one exceptional case, Trp-303 (the structural homolog to Trp-800 in smMLCK) is exposed to solvent and is thus thought to play an important role in the binding of Ca2+/CaM. In contrast to the enzyme, the free CaM-binding peptide, in the absence of constraints imposed by the remainder of the protein, would likely present a markedly different target for recognition and binding by Ca2+/CaM. However, since the peptide can form an amphipathic helix it could still bind to Ca2+/CaM with similar or even higher affinities than the parent enzyme. An intriguing question pertinent to the activation process is whether the CaM binding domain of CaMKI is transformed to a helical segment following the binding of Ca2+/CaM.
We thank Dr. Apolinary Sobieszek for assistance with the inhibition kinetic analyses.