Protein Science (1996), 5:468-477. Cambridge University Press. Printed in the USA.
Copyright
0 1996 The Protein Society
Interlobe communication in multiple calcium-binding
site mutants of Drosophila calmodulin
.
.-
..-
."
POUSHALI MUKHERJEA,' JOHN E MAUNE,
AND
~
~~
KATHY BECKINGHAM
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005-1892
(RECEIVED November
20, 1995; ACCEPTED December 21, 1995)
Abstract
We have generated mutants ofDrosophila calmodulin in which pairs of calcium-binding sites are mutated so as
to prevent calcium binding. In all sites, the mutation involves replacement of the -Z position glutamate residue
with glutamine. Mutants inactivated in both N-terminal sites (B12Q) or both C-terminal sites (B34Q), and two
mutants with one N- and one C-terminal site inactivated (B13Q and B24Q) were generated. The quadruple mutant with all four sites mutated was also studied. UV-difference spectroscopy and near-UV CDwere used to examine the influence of these mutations upon thesingle tyrosine (Tyr-138) of the protein. These studies uncovered
four situations in which Tyr-138 in the C-terminal lobe responds to a change in the calcium-binding properties
of the N-terminal lobe. Further, they suggest that N-terminal calcium-binding events contribute strongly to the
aberrant behavior of Tyr-138 seen in mutants with a single functional C-terminal calcium-binding site. The data
also indicate thatloss of calcium bindingat site 1 adjusts the aberrant conformation
of Tyr-138 producedby mutation of site 3 toward the wild-type structure. However, activation studies for skeletal
muscle myosin light chain
kinase (SK-MLCK) established that all of the multiple binding site mutants are pooractivators of SK-MLCK. Thus,
globally, the calcium-induced conformationof B13Q is not closer to wild type than thatof either the site 1 or the
site 3 mutant. The positioning of Tyr-138 within the crystal structure of calmodulin suggests that effects of the
N-terminal lobe on this residue may be mediated via changes to the central linker region of the protein.
Keywords: calcium-binding proteins; circular dichroism; skeletal muscle myosin light chain kinase; UV-difference
spectroscopy
The calcium-binding protein calmodulin is widely distributed in
eukaryotes and plays a central role in mediating intracellularcalcium signaling. The structure of the calcium-saturated (holo)
form of the proteinis relatively well understood. Crystal structures for the holo forms of mammalian andDrosophila calmodulins have been determined (Babu et al., 1988; Taylor et al.,
1991). These two calmodulins differby only three conservative
amino acid differences (Smith et al., 1987) and the two structures arevery similar. Both are dumbbell-shapedmolecules with
two globular terminal lobes, each containing
a pair of the
calcium-binding sites. The centralregion separating the termi-
"
~
~
~
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~~
Reprint requests to: K. Beckingham, Department Biochemistry and
Cell Biology, MS140, Rice University, 6100 Main Street, HoustonTexas,
77005-1892; e-mail: kate@bioc.rice.edu.
Present address: Department of Neuroscience and Cell Biology,
UMDNJ-RW Johnson Medical School, Piscataway, New Jersey 088545635.
Abbreviations: EGTA, [ethylenebis(oxyethylenenitrilo)]tetra-acetic
acid; Hepes, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid;
IPTG, isopropyl-thio-P-D galactoside; MOPS, (3-[N-morpholino]propane-sulfonic acid; PEG, polyethylene glycol; SAXS, small angle X-ray
scattering; SK-MLCK, skeletal muscle myosin light chain kinase.
'
468
nal lobes is an a-helix of about eight turns. Small angle X-ray
scattering analysis (Heidorn & Trewhella, 1988) and NMR (Barbat0 et al., 1992) studies indicate thatthis overall conformation
is maintained in solution, but also indicate that part of thecentral region is unstructured rather that helical.
Less is known about the conformation of the calcium-free
(apo) form of calmodulin and hence of the conformational
changes induced by calcium binding. SAXS analysis has indicated that the terminal lobes of the protein are maintained in
the absence of calcium, but are closer together by about 5 A
(Seaton et al., 1985; Heidorn & Trewhella, 1988). Nevertheless,
biophysical and biochemical studies with both the intact protein
and the separatedN- and C-terminalhalves have suggestedthat
the calcium-induced conformational changes in the two terminal
lobes are independent of one another (Forsen
et al., 1986; Martin & Bayley, 1986). Similarly, the model for apo-calmodulin
proposed by Strynadka and James(1988) predicted that calciuminduced events in each of the terminal lobes would occur independently. The recent NMR determinations of thesolution
structure for apo-calmodulin (Kuboniwa et al., 1995; Zhang
et al., 1995) have largely confirmed the Strynadka and James
model and reinforce the view that calcium-induced events in the
two halves of the molecule are independent phenomena.
469
Multiple CaZi binding site mutants of calmodulin
We have used site-directed mutagenesis ofDrosophila calmodulin to investigate the contributionof the individual “E-F hands”
(Kretsinger & Nockolds, 1973) to overall calcium binding and
calcium-induced conformational changes in the protein. Initially, two series of mutants were generated and studied (Maune
et al., 1992b). In each mutant, the conserved glutamic acid
residue at the -Z coordination position of the calcium-binding sites
(Kretsinger & Nockolds, 1973) was mutated in one site. In the
four proteins of the Q series, the -Z residue was mutated to glutamine. Direct calcium-binding studies demonstrated that, in
each site, theQ mutation effectively prevents calcium binding,
such that under the quasi-physiological conditions used for binding studies, calcium binding at the mutated site was undetectable (Maune et al., 1992b).
Surprisingly, calcium binding and conformational studies of
these single binding site mutants (Martin et al., 1992; Maune
et al., 1992a,1992b) providedsome evidence foreffects of
N-terminal mutations upon calcium-induced conformational
changes to the single tyrosine of the protein (Tyr-138) located
in the C-terminal lobe. Someof the most striking effects on this
residue were detected when one of the C-terminal sites was mutated. However, given the presence of at least one functional
calcium-binding site in the C-terminusof these mutants, it was
impossible to determine whetherthese effects originated from
N-terminal binding or aberrant C-terminal binding. We have investigated these phenomena further through theuse of calmodulin mutants inwhich twoofthecalcium-binding
sites are
disabled. TheE to Q mutations of the -Zcoordinate described
above, with their known effects on calcium binding, were used
to create these mutants. The mutantwith both N-terminal binding sites mutated and the mutantwith both C-terminal sites mutated were generated to allow study of calmodulins in which
calcium binding was primarily to only one terminal
lobe of the
protein. Using our previous nomenclature (Maune et al. 1992b),
these two calmodulins have been termed B12Q and B34Q, indicating that the E to Q mutation is present in sites 1 and 2, or
sites 3 and 4, respectively (see Table 1). In addition, two proteins in which one N-terminal and one C-terminalsite contain
Table 1. Multiple calcium-binding site
mutants of calmodulin”
~~~~
~
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~
Mutant name
.~
~~
”
~
Residues mutated
Sites mutated
~
Bl2Q
E31 -+ Q
E67 + Q
Sites 1 , 2
B13Q
E31 + Q
E104 + Q
Sites I , 3
B24Q
E67 + Q
E140 + Q
Sites
4 2,
B34Q
E104 + Q
E140 -+ Q
Sites 3, 4
B 1234Q
E31 +
E67 -+ Q
E104 + Q
El40 + Q
Sites 1 , 2 , 3 , 4
~”
Results
Protein purification
The mutant calmodulins studied here are detailed in Table 1.
Each has two or more
calcium-binding sites containing the E to Q
mutation at the -Zcalcium coordination position. In previous
studies (Maune et al., 1992b), it was found that mutant calmodulins carrying this E to Q mutation in a single calcium-binding
site could be purified by phenyl-Sepharose affinity chromatography in the presence of high salt. This purification procedure
relies upon the induction of hydrophobic surfaces on calmodulin in response to calcium binding. However, no conditions
could be found under which calmodulins with more than one
mutant binding site would bind to phenyl-Sepharose. A more
conventional procedure, based on that of Newtonet al. (1988)
and involving ammonium sulfate fractionation, DEAE-Sephacel
chromatography, and HPLC anionexchange chromatography,
was therefore developed for purification ofthese proteins (see
the Materials and methods). Drosophila calmodulin contains no
tryptophan, nine phenylalanines, anda single tyrosine residue.
As a result, the ratio of the tyrosine peak at
279 nm and the
shortest wavelength absorbance peak for phenylalanine at 253 nm
is a extremely sensitive measure of protein purity. For pure wildtype calmodulinin the presence of calcium, the279/253-nm ratio is less than 1.00.All mutant calmodulins were therefore
purified until spectra revealed 279/253-nm ratios lower than
1 .OO.SDS-PAGE was used to confirm thepurity of the proteins.
Extinction coefficients for the five purified proteins were determined in the presence and absence ofcalcium and are shownin
Table 2.
Conformational change during calcium titration
.”
In all mutants, the residues mutated are the conserved glutamates
found at the - Z position of the calmodulin Ca2+-binding sites.
a
the Q mutation have been examined. Of the four possible mutants of this class, thosewith (1) site 1 and site 3 mutated, and
(2) site 2 and site 4 mutated were chosen for study. Previous
work (Martin et al., 1992; Maune et al., 1992a, 1992b; Starovasnik et al., 1992; Gao et al., 1993; Mukherjea & Beckingham,
1993) indicates these two mutants, B13Q and B24Q respectively,
represent the combinations of binding site mutations with (1)
the mildest effects on calcium binding and conformational
change (theB13Q mutant) and(2) the strongesteffects on these
properties (the B24Q mutant). The mutant calmodulinwith all
four calcium-binding sites mutated (B1234Q) was also generated
and studied.
UV-difference spectroscopy and near-UV CD (Mauneet al.,
1992a, 1992b) havebeenused to monitor conformational
changes to Tyr-138. In addition, theability of these mutants to
activate one of the better characterized target enzymes of calmodulin, skeletal muscle myosin light chain kinase, has been examined. The findings provide strong evidence that events in the
N-terminal lobe of calmodulin are detected in the C-terminal
lobe, as monitored by Tyr-138. Mechanisms whereby such interlobe communication could occur are consideredin the light
of current knowledge on the positioning of Tyr-138 and the
calcium-induced changes to this residue.
The single tyrosine (Tyr-138) of Drosophila calmodulin responds
to calcium binding to the protein and thus can
be used to monitor conformational changes to its environment in the fourth
79.3
470
, , , i A v l IB
Table 2. Molar extinction coefficients for wild-type
and mutant calmodulins at the tyrosine
absorbance maxima
~~~
~~~~~
~
~~
~~~~~
~~~~~~~~~
~~
~
X.,
~
~
~~
~~~~
~
~~
~~~
~~
1.578
1.722
279.9
1,688
1,720
1,799
1,799
~
~~
~
~
~~~
1,874
278.9
1,860
~~
~
~
~~~~~~
-200
-300
278.9
-400
1,848
278.4
277.7
1,900 278.6
278.6
E-"
~~~~
~~~
+EGTA
+Ca2+
.~
WT
B12Q 278.3
B13Q 1,878
B24Q
279.6 B34Q 1,946
B 1234Q
~~
~
839
(k0.3 nm)
~
+EGTA
Calmodulin
+Ca2+
~
~~~
Extinction
coefficient
100
0
P. Mukherjea et al.
278.9
277.3
-500
."
100
u
~~
~~~~~~~
~~~
~~~~~
d
-300
calcium-binding domain of the protein. We have used UV-difference spectroscopy and near-UV CD to monitor changes to
-400
Tyr-138 during titrations athigh protein concentration (100 pM)
-500
with progressive addition of equivalents of calcium. Under these
conditions, the wild-type protein shows stoichiometric calcium
binding, with the higher-affinity sites being occupied first and
all changes to Tyr-138 are completed after addition of four
equivalents of calcium. For the mutant proteins, however, two
further factors influence the titration behavior. Although the Q
t
mutation used here effectively eliminates calcium binding at the
mutated site under the conditions studied originally (Maune
et al., 1992b), NMR analysis has shown that, under high protein concentrations, binding at some mutatedsites is detectable
late in calcium titrations (Starovasnik et al., 1992). In addition,
10
the presence of the Q mutationin some binding sites was shown
to lower calcium affinity at someof the residual functional sites
Equivalents Ca2+
on the protein (Mauneet al., 1992b). Thus, as aresult of these
Fig. 1. Changes to the UV-difference spectra peaks of wild-type and
two phenomena, spectral changes to some of the mutant promutant calmodulins during titration with calcium. Changes
in A€ assoteins continue at levels of calcium higher than those nominally
ciated with the two tyrosine peaks at279 nm (0)
and 286 nm).( of the
required to fill all remaining functional calcium-binding sites on
UV-difference spectra are shownas a function of equivalents of calcium
the protein. These factors are considered
in discussing the conadded per calmodulin. Titrations for wild-type protein, B3Q, and B4Q
have been published previously (Maune et al., 1992b). Where error bars
formational changes seen during calcium titration of the mutant
are not visible, they were smaller than the symbols
used for the data
proteins.
1
1
J
L
points.
UV-difference spectroscopy
In previous work, UV-difference spectra produced forwild-type
Drosophila calmodulin during calcium titrationwere shown to
contain two negative peaks corresponding to changes
in absorbance at the peak and shoulder for tyrosine absorbance (279 and
286 nm) (Mauneet al., 1992b). A negative difference spectrum
in the region 279-287 nm is usually interpreted as increasedexposure of tyrosine residues to the solvent (Donovan, 1973).
However, the ratioof the 279- and 286-nm peaks for calmodulin is unusual in that, unlike other proteins, the 279-nm peak
gives stronger differential absorbance than the 286-nm shoulder. During calcium titration, the major changes to these two
peaks occur during the addition of thefirst two equivalents of
calcium (see Fig. IA), indicating that they are induced by binding to the high-affinity C-terminal sites. Findings for calmodulins containing mutations in single calcium-binding sites
reinforced this interpretation (Maune et al., 1992b). Thus, for
mutants to site 1, the unusual ratioof the 279/286-nm UV-difference spectrum peaks is maintained and theresponse of these
peaks is quantitatively similar to that of the wild type in the initial phases of the calcium titration.
The single B2Q mutant was not examined previously and was
investigated herein order to aid with interpretation of multiple
mutants containing a mutation to site 2. As can be seen from
of Maune et al.
Figure ID and a comparison to Figure 6B
(1992b), the response of B2Q is very similar to that of BlQ.
Changes very like those for thewild-type protein are seen over
the addition of the first two equivalents of calcium and theonly
detectable difference is that the small incremental increases in
both the 279-nm and 286-nm peak intensities seen for the wildtype protein upon additionof the third and fourth aliquotsof
calcium are not detected for B2Q.
Given that mutation of either site 1 or site 2 has essentially
no effect on the early response of Tyr-138,the doubleB12Q mutant might be expected to behave similarly. As shown in Figure IC, however, this is not the case. Unexpectedly, the entire
calcium-induced change in the environment
of Tyr-138 is com-
Muitipie Ca2+ binding site mutants of calmodulin
47 1
tant (compareFig. 1A and E). Although the relative intensities
pleted upon the addition of one equivalent of calcium as opof the 279- and 286-nm absorbance values are not completely
posed to two. Data for up 10
to additions of calcium are shown
restored to the wild-type situation, in other aspects the B13Q
in Figure IC, but addition of up to 16 equivalents produced no
further changes (data not shown). The overall responses of themutant UV-difference titration curve is more like that of the
279- and 286-nm peaks and the279/286-nm peak ratio are also wild-type protein than that of any other mutant studied. These
somewhat reduced in the B12Q mutant as compared to BlQ and findings thus suggest that calcium binding atsite 1 contributes
strongly to the aberrant behavior of Tyr-138 in B3Q and that
B2Q (compare Fig. ID and G).
loss of binding at thissite has a “corrective” effect on theenviMaune et al. (1992b) showed that mutation of either site in
the C-terminus producesa response that dramatically alters the ronment of this residue.
A comparison of theB4Q mutant with the B24Q mutant adUV-differencechangesseenduringcalciumtitration.The
dresses a similar question concerning the contribution of bind279/286-nm peak ratio, the intensity of the peaks, and behaving at site 2 to the unusual UV-differences changesseen when
ior of these peaks during titration areall very different from the
site 4 is mutated. As shown from a comparison of Figure IC
wild-type protein. These titrations for B3Q and B4Q have been
and F, loss of calcium binding at site 2 stronglymodifies the beinvestigated
repeated to provide controls for the double mutants
havior of Tyr-138, butin this case, most of the response of the
in this study and are shownin Figure IB and C.As can be seen,
tyrosine is lost and minimal changes in the 279/286-nm peaks
B3Q shows a biphasic response, whereas the response of B4Q
are seen during the calcium titration. Thus, calcium binding at
is essentially monophasic. Therelative affinities of the three nonsite 2 appears tobe a significant component of the aberrant Tyrmutated calcium-binding sites of B3Q and B4Q are closer than
138 response of mutant B4Q. From a comparison of FigureI F
those of the wild-type protein (Maune et al., 1992b), and NMR
with Figures 1H and 1 J, it can be seen that the response of the
experiments indicate that they fill more or less simultaneously
tyrosine in B24Q mutant is as poor as that in the B34Q and
(Starovasnik et al., 1992). Thus, it was impossible to determine
B1234Q mutants, suggesting that mutation of sites 2 and 4 has
which calcium-binding events produced the dramatically altered
at least as deleterious aneffect on the overallresponsiveness of
behavior of Tyr-138 seen in these two mutants. Thecalcium afTyr-138 as does mutation of sites 3 and 4.
finities of the residual sites were consistent, however, with the
interpretation offered by Maune et al. (1992b) that the initial
phase of the B3Q titration represents changes due tothe unusual
Near- U V CD
situation of calcium binding atsite 4 in the absence of binding
The near-UV C D signal at 280 nm (AE280) provides different inat site 3, and that the followingrecovery phase represented ocformation on the conformation of the single tyrosine in Drocupancy of the mutated site 3 itself. For B4Q, the changes were
consistent with the entire titration curve representing the occu- sophila calmodulin, probably reflecting the rotational freedom
of its side-chain moiety. In a previous study (Maune et al.,
pancy of the partnersite, site 3. However, for both mutants, the
1992a), Atzso values for the wild-type protein and mutants carpossibility that some of the dramatic changes seen result from
rying the single Q mutants were determined in the absence of
binding at the N-terminal sites remained open.
calcium (apo At28o) and in a IO-fold molar excess of calcium
Examination of the UV-difference changesin mutant B34Q,
(holo AeZso). In the absence of calcium, the wild-type protein
in which both C-terminal sites are mutated, addresses the quesshows a strong positive AezSo (3.2) that changes to an even
tion of whether calcium binding at the residual C-terminalsite
higher negative ALt280 value upon calcium saturation (-5.00).
is indeed required for any aspect of theresponses seen in B3Q
and B4Q. As shown in Figure IH, the changes during addition This indicates a conformational change for the tyrosine side
chain from one restricted conformation to a second restrained
of the first four equivalents of calcium to this mutant are very
position upon calcium binding. Examination of At280 during
limited and do not show the unusual
279/286-nm ratio characwere largely
teristics seen for the B3Q and B4Q responses. A comparison to calcium titration demonstrated that these changes
completed after additionof two equivalents of cakium, suggestB34Q early in the titration
Figure 1J shows that the behavior of
of conformation examined by UV-differing, as for the aspect
is not significantly different from thatof the quadruple mutant
ence spectroscopy, that binding to the C-terminalsites largely
in which all four calcium-binding sites are mutated. Thus,in the
governs this change in the environment of Tyr-138.
absence of any functional calcium-bindingsites in the C-termAs for UV-difference spectroscopy, the calcium-induced
inus, calcium binding to the N-terminus alonedoes not produce
changes in the environment of Tyr-138 similar to thoseseen for
changes to Atzso seen for N-terminal site mutants largely reB3Q and B4Q. Calcium binding at theresidual C-terminal site
inforced this idea. Thus, the overall change in A L for ~mu-~ ~ ~
is therefore at least a component of the B3Q and B4Q responses.
tants BlQ and B2Q is similar to that seen for the wild-type
The B13Q and B24Q mutants each contain asingle N-terminal
protein, although the A6280 value for holo B2Q is somewhat
and a single C-terminal calcium-binding site mutation. These
greater than that of the wild-type protein (Table 3; Fig. 2). Given
mutants thusallow a different question to be posed in relation
that mutant B12Q behaved differently with respect to UV-difto the unusual UV-difference titration curves
seen for Tyr-138
ference titrations than either mutant BlQ or B2Q alone (see
in B3Q and B4Q. Is there any effect of calcium binding at the
above), it was of interest to determine whether it would demN-terminal sites on the behavior of Tyr-138 when one of the
onstrate differences with respect to A6280. As shown in Figure2,
C-terminal sites is functional? For the mutant pair B13Q and
the
values fortheapoandholoforms
of B12Q arenot
B3Q, the UV-difference changes during calcium titration are
strikingly different from wild type or the individual BlQ (Fig. 2;
shown in Figure 1B and E. As can be seen, the UV-difference
Maune et al., 1992a) and B2Q mutations. However, a significalcium titrations are strikingly different. Surprisingly, the
becant difference is that changes to ALtz80 do not saturate until behavior of the 279/286-nm peaks in theB13Q double mutantis
tween two and four equivalents
of calcium are added, whereas
more similar to thewild-type titration than that of the B3Q mufor BlQ andB2Q, changes are completed after additionof two
412
P . Mukherjea et al.
Table 3. Near-UV CD properties
of Drosophila
calmodulin calcium-binding site mutants
Calmodulin
~~
.".~
At280
Ac2s0
(Ca2+-free)
( + l o equivalents Ca2+)
3.17
2.88
2.36
2.03
1.86
0.83
I .52
0.095
-5.02
-6.50
-3.1 1
-4.44
-4.54
- 1.34
1.17
-0.80
~~
Wild-type
B2Q
B3Q
8124
B13Q
B24Q
B34Q
B 1234Q
~
equivalents of calcium.This suggests that when both N-terminal
or both sites in the
sites are mutated, the affinity of one
C-terminus is decreased significantly.
Mutation of either C-terminal site was shown previously to
affect the &280 signal severely, again indicating a dominant
role for C-terminal binding in the Tyr-138 A t 2 8 0 signal. B3Q
nevertheless gave a change to a final negative k 2 8 0 value of
-1.35, whereas for B4Q the decrease in&280 was so low as to
retain a final positive value (+ 1.05). In repeating studies of the
single mutants as controls for the experimentsdescribed here,
we found our values to be in good agreement with those of
Maune et al. (1992a), although our holo h 2 8 0 value for mutant B3Q is somewhat more negative than previously reported
(Table 3).
The role of the C-terminal sites in generating the A t 2 8 0
change to Tyr-138 is most clearly demonstrated by the double
C-terminalsitemutant B34Q. Calcium-inducedchanges to
A t 2 8 0 are almostnonexistent (Fig. 2), and, in addition, the A t 1 8 0
41
WT
824
B3Q
B4Q
B12Q
B34Q
E l 38 02 4 4
B1234Q
h
e
value for the apo form of this protein
is significantly reduced.
Thus, mutation of the two C-terminal calcium-binding
sites
alters the initial environment of Tyr-138 and prevents almost entirely theconformationalchangedetected
by
in the wildtype protein.
Interestingly, for the aspect of conformational change measured by A t 2 8 0 , the response of the quadruple mutant B1234Q
is noticeably different from that of the B34Q mutant (see Fig. 2).
Strikingly, the apo A t 2 8 0 value is almost zero, whereas for the
B34Q mutant, as noted above, relatively
a
strong positive value
is maintained. A further differencebetween B1234Q and B34Q
is that a greater change toward a more negative A t 2 8 0 is seen
upon addition of calcium toB1234Q with a total changeof 0.9
as opposed to 0.35 for the B34Q mutant (Table 3). These differences indicate a contribution from calcium binding in the
N-terminal domain to the positioning of Tyr-138 and demonstrate that two aspectsof the conformationof Tyr-138 are altered when this binding is lost. These are (1) loss of the restricted
rotational conformation, seen in the apo formof the B34Q mutant and (2) gain of some ability to move into a conformation
showing greater rotational restriction upon calcium binding.
Examination of the At28() changes for the B13Q and B24Q
double mutants againallows the effects of mutating oneof the
N-terminal sites upon the altered responses seen for mutants
with one functional C-terminal site to be assessed. The apo
Atzso for B13Q is similar to that of B3Q (Fig. 2), but surprisingly the overall change in At2*,, upon calcium binding is increased significantlyrelative to B3Q. This value is closer to the
wild-type value (see Table 3; Fig. 2) and is close to that for the
BlQ mutation alone (see Fig. 3 of Maune et al., 1992a). Thus,
as found in the UV-difference experiments described above, mutation of site 1 and site 3 appears to adjust the conformational
change experienced by Tyr-138 toward that seen for the wildtype protein.
Comparison of the apo and holoA t 2 8 0 values for B24Q with
those for B4Q alone reveals that mutation of site 2 also modifies the response ofits homologous mutation in the C-terminal
domain. The apovalue is decreased strongly relative to the B4Q
value, to a At28o that is the second lowest recorded among this
set of mutants (Table 3; Fig. 2). However, the overall change
in Atzxo upon calcium saturation is approximately the same
as that seen for the B4Q mutation, that is, a change of about
-2 At28o units (Table 3).
SK-MLCK activation
Fig. 2. Calcium-induced changes to the near-UV C D signal (Atzso) for
wild-type and calcium-binding site mutant calmodulins.
Values for
AtZIO in the absenceof calcium and in the presenceof 10 equivalents of
calcium are shown for all mutants. For the double and quadruple
2 and 4 equivalents of calcium
calcium-binding site mutants, values at
are also shown. For clarity, error bars for these values are not shown,
but they were comparable to, or smaller than, those shown for the 10
equivalents of calcium values. Data for the wild-type protein and the
single mutants BlQ, B3Q, and B4Q have been published previously
(Maune et al., 1992a). Measurements for most of these proteins were
repeated as partof this study, and proved to be very similar to the
viously reported values (see text).
In a previous study (Gaoet al., 1993), the ability of the single
site Q mutations to activate SK-MLCK
was examined. For this
enzyme, B l Q proved to be poorest activator, followed by B2Q,
then B3Q, with B4Q showing the best activation capacity. Given
the findings described above,it was of interest to examine the
capacity of these multiple calcium-bindingsite mutants toactivate SK-MLCK. The activation curves are shown in Figure 3 and
the derived kinetic parameters listed in Table 4. Data for wildtype calmodulin and the BlQ and B3Q mutants, generated as
controls for these experiments, are shown. Although the K,,.,
values derived for our data are somewhat
higher than those of
Gao et al., qualitatively the activationabilities of BlQ andB3Q
pre-relative to the wild-type protein are as determined previously
(compare Table 4 with Table 1 of Gao et al., 1993).
473
Multiple Ca2+ binding site mutants of calmodulin
120
I-
/
60 -
I
-
40
G
.-;;i
.->
0
20
-
Y
-9
0:
2
120
cd
.-E
Y
5
s
-IO
-11
-8
-9
-6
-7
B
. o
w
1
o
B
3
Q
100
a
-
80 -
XBlQ
p11 B13Q
60 -
lar range- approximately 1,000-fold higher than that of the
wild-type protein (see Table 4). A K,,., could not be determined
for the B34Q and B1234Q mutants because, even at micromolar concentrations, they showed negligible activity. The findings
of Gao et al. (1993) demonstrated that mutation of either
N-terminal calcium-binding site was more detrimental to activation of SK-MLCK than mutation of either of the C-terminal
sites. Thus, the finding that the B34Q mutation is a much worse
activator of the enzyme than the
B12Q mutant would not have
been predicted from those studies.
The UV-difference spectra and near-UV CD analyses described above indicate that the conformationof Tyr-138 in mutant B13Q mutant is closer to thatof the wild-type protein than
that of the B3Q mutation alone, suggesting that loss of calcium
binding at site 1 "corrects" somewhat the conformational problems produced by the B3Q mutation in the region of this residue. Thus, it was of interest to compare theability of the B13Q
mutant to activate SK-MLCKwith those of the BlQ andB3Q
mutants individually. As can be seen from Figure 3B and Table 4, the B13Q is a much worse activator of SK-MLCK than
either B3Q or B l Q alone. Thus, atleast in terms of this enzyme,
the two mutations appear to act additively to debilitate the
enzyme.
40 -
20
Discussion
-
Evidence for interdomain interactions
0,
-1 1
,
.
,
l
l
-9
-10
,
,
l
.
l
,
,
-8
,
.I
,
.
.
I
t
-6
-7
log [Calmodulin] M
Fig. 3. Activation of SK-MLCK by multiple calcium-binding site mutants of calmodulin. Activation is normalized as percentage maximal
activation for the wild-type protein.
All of the double calcium-bindingsite mutants proved to be
poor activators of SK-MLCK (Fig. 3), considerably worse than
any of the single-site mutants examined previously. TheB12Q
mutant proved to be thebest activator of the group (Fig. 3A),
but even for this mutant, theKO,,was shifted to the micromo-
Table 4. Kinetic parameters for activation of SK-MLCK
by calcium-binding site mutantsa
Calmodulin
Wild-type
BlQ
93Q
B13Q
B 12Q
B34Q
B1234Q
Koc,
(nM)
1.66
43. I
33.4
2,236.8
1,311.7
N D ~
N D ~
070
activation
Maximal
100
48
89
48
ND
8'
8'
a KO,,is the concentration of calmodulin required for half-maximal
activation of the enzyme. VO Maximal activation is the Vmaxof a given
mutant expressed as a percentage of V,,,, for the wild-type protein.
ND, not determined.
Activity at 1,000 nM mutant protein.
Previous studies with single calcium-binding site mutants suggested that calcium-binding events in the lobe of calmodulincontaining Tyr-138 (that is, the C-terminal lobe)play a major role
in determining conformation in the environment of Tyr-138
(Maune et al., 1992a, 1992b). The present study reinforces that
interpretation. Thus, the near-UV CD responseof Tyr-138 in
B34Q is the smallest among all the mutants studied and similarly the UV-difference titration changes are minimal. However,
from both theUV-difference and near-UV CD data, four comparisons provide striking evidence for effects of events in the
N-terminus upon the environmentof Tyr-138. These comparisons are:(1) the effects of the double N-terminal mutantB12Q
upon Tyr-138 compared to those of the single-site N-terminal
mutants; (2) the effects of the quadruple mutantB1234Q compared to the those of the C-terminal double mutantB34Q; (3)
the effects of the B13Q mutant compared to those of the B3Q
mutant; and (4) the effects of theB24Q mutation compared to
those of the B4Q mutation.
We consider each of these comparisons below. In offering interpretations of the effects seen, two assumptions concerning the
behavior of the mutants proteins have been made. The first is
that the mutated sites, if occupied at all during titrations, will
fill after any remaining nonmutated functionalsites on the protein. This assumption is founded on the known effects of the
E to Q mutation in each of the binding sites as determined in
previous studies (Maune et al., 1992a, 1992b; Starovasnik et al.,
1992). The second assumption is that the conformation of the
apo form of the mutant proteins is very similar to that of the
wild-type protein, but thatloss of calcium binding at a particular site results in loss of those components of the calciuminduced conformational changes normally associated
with calcium binding at those sites. This assumption is supported by farUV CD studies of these mutant proteins (Mukherjea, 1995). For
414
P. Mukherjea et ai.
suggests that, after mutation of all four calcium-binding sites,
all four double mutants studied here, the
UV C D signal at
Tyr-138 is essentially unrestrained in this protein. In contrast,
222 nm for the apo form is very similar to wild type, but the
calcium-induced change is diminished. For the quadruple muin the apo formof B34Q mutant, the residue gives a significant
tant, there is a very limited calcium-induced change and the apo AtrRovalue. This suggests a role for the wild-type N-terminus
in providing structure in the environment of Tyr-138. As noted
222-nm signal is approximately 80% of the wild-type value.
above, the far-UV CD
signal for the apo form of
B1234Q is diThus, for this mutant, far-UV CD indicates changes to the apo
structure that are relevant to our observations (see below).
minished relative to the wild-type apo signal.
B12Q versus BlQ, B2Q, and wild-type calmodulin
The UV-difference data revealed an unexpected difference between mutant B12Q and the single-site mutants BlQ andB2Q
and thewild-type protein; the aspect of conformational change
at Tyr-138 monitoredby this parameter is completed upon addition of one equivalent of calcium instead of two.This surprising finding requires consideration of why the UV-difference
spectral changes to Tyr-138 for thewild-type protein require addition of two equivalentsof calcium. One obviouspossibility is
that calcium binding to both C-terminal sites affects Tyr-138.
Our findings for mutantsB3Q, B4Q, and B34Q clearly support
this argument. If only one of the C-terminal sites could affect
Tyr-138, the titration curve for B34Q would resemble that of
either B3Q or B4Q, as opposed the observed result, which is that
the Tyr-138 response is essentially eliminated. However, even
if calcium binding to a single C-terminal site were responsible
for the Tyr-138 UV-difference changes, they would still occur
over addition of two equivalents of calcium, because cooperativity in the C-terminus (Linse et al., 1991) produces overlapping occupancy of the two sites on individual molecules, and the
critical site would not be occupied on all calmodulin molecules
of the two N-terminalsites
until this point. Thus, for mutation
to produce a change in Tyr-138 that requires addition of only
one equivalent of calciumimplies, at the very least, that cooperativity in the C-terminus is altered and occupancy of one
C-terminal site lags behind its partner. The near-UV CD data
for B12Q provide some support for this interpretation. Asdiscussed in the Results, the change to A t 2 ~ oto B12Q is not complete upon addition of one equivalent of calcium, but rather
takes addition ofbetween two and four equivalentsof calcium
to saturate. This finding indicates that the calcium affinity of
at least one C-terminal site has been reduced.
Whatever the explanation, someinterdomain interaction must
be evoked to explain the effects
of the B12Q mutant upon Tyr138. However, it is important to recognize that the data forthis
interaction (and for all the other interdomain interactionsdiscussed here) can be interpreted as evidence for interdomain communication before or after mutational changes. Thus, the
wild-type behavior of Tyr-138 may reflect an interaction of
the N- and C-terminal domains thatis lost upon mutation of the
N-terminal sites or, alternatively, the interaction is newly induced as a result of mutating these sites.
B34Q versus B1234Q
For the comparisonof B1234Q with B34Q, the near-UV CD
data provide evidence for a difference in the behavior of Tyr138 and, thus, for a role for N-terminal
events upon conformation in the vicinity of this residue. Although for B1234Q the
magnitude of the near-UV C D change to Tyr-138 is somewhat
greater than thatseen for B34Q, the moresignificant difference
between them is seen in the AeZso values for the apo forms of
the two proteins. The A€,,, value of close to zero for B1234Q
B13Q versus B3Q
In mutant B3Q, the behavior of Tyr-138 is markedly affected
(Maune et al., 1992a,1992b). The studies presented here show
that mutating site 1 in the N-terminus strongly modifies theresponses of Tyr-138 associated with the B3Q mutation, producing near-UV CD and UV-difference changes that are more
similar to the wild-type behavior. The striking UV-difference
spectral changes seen in B3Q were interpreted previously as originating entirely from anomalous events in the C-terminus. However, our discovery here that loss of binding at site l dramatically
modifies those changes leads to a new interpretation of the B3Q
UV-difference spectra. It suggests that the aberrant behaviorof
the B3Q mutant during additionof the first two aliquotsof calcium is produced by generation of one or morecalmodulin conformers not normally seen in the wild-type titration in which the
remaining C-terminal site (site 4) and oneN-terminal site are occupied. Given that the B13Q mutation, a protein in which calcium binding is primarily to sites 2 and 4,gives UV-difference
titration changes for Tyr-138 similar to the wild-type protein,
it would be predicted that the unusual species with sites 1 and
4 occupiedis the main source of the aberrant behavior for Tyr138 seen during titrationof B3Q rather than the species with sites
2 and 4 occupied.
B24Q versus B4Q
A similar logic can be evoked to explain the differences between the B4Q mutant and the B24Q mutant. That is, the aberrant behavior of Tyr-138 in B4Q can be viewed as arising from
unusual conformers in which site 3 and one of the N-terminal
sites is occupiedratherthanfromeventsrestrictedtothe
C-terminus. The finding that the UV-difference response of Tyr138 is essentially lost in B24Q suggests that a species with calcium bound atsite 2 plays a prominent rolein determining the
spectral properties of the B4Q mutant.
A comparison of the UV-difference spectral properties of mutant B13Q (in which sites 2 and 4 are the
functional binding sites)
with mutant B24Q (in which sites 1 and 3 are the active sites)
permits an evaluationof the efficacy of these two pairs ofsites
in affecting conformation at Tyr-138. In B13Q,Tyr-138responds promptly, giving a calcium titration similar to the wildtype protein. In B24Q, the response ofTyr-138 is minimal. These
findings reinforce previous indications (see the Introduction)
that calcium binding at sites 2 and 4influences calcium-induced
conformational change more strongly than calcium-binding at
sites 1 and 3.
Activation of SK-MLCK by the multiple mutants
Although the UV-difference and near-UV CD data described
above suggest that the local environment of Tyr 138 is closer to
wild type in mutant B13Q than in the single mutant B3Q, the
activation data for SM-MLCK indicate that the overall
cal-
475
Multiple Ca2+ binding site mutants of calmodulin
cium-bound conformationof B13Q is less like thatof the wildtype protein than that of either mutation alone. The previous
studies of enzyme activationby individual calcium-binding site
mutants of calmodulin (Gao et al.,1993) predict that theeffects
of preventing calcium binding at twosites on the protein would
be cumulative. Thus,given that loss of calcium binding and the
associated calcium-induced conformational change at any single binding site of calmodulin significantly decrease the ability
of the protein to activate
several target enzymes, it is reasonable
to expect that preventing two components of the calciuminduced change would be more deleterious than preventing one.
Although all of the multiple mutants examined are poor activators of SK-MLCK, the double mutantwith both N-terminal
binding sites inactivated emerged as thebest activator of the set.
The double C-terminal mutant and the quadruplesite mutant
showed the lowest activity. These rankings would not have been
predicted from the previous studies
of SK-MLCK activation by
the single calcium-binding site mutants (Gao et al., 1993), because mutants BlQ and B2Q both proved to be worse activators than B3Q or B4Q.However,studies with therelated
enzyme, smooth muscle MLCK (VanBerkum& Means, 1991),
suggest that interaction of the SK-MLCK target binding region
with the C-terminal lobeof calmodulin is the initiating event in
loss of calciumenzyme activation, and thus that complete
induced conformational changein this half of calmodulin might
be expected to have
a more deleterious effect on enzyme
activation.
organized system of hydrophobic residues, but, unexpectedly,
the hydroxyl group of Tyr-138 forms a hydrogen bond with glutamate 82- one of three adjacent glutamates
in the continuous
central helix of thecrystallized form of the protein (Babu
et al.,
1988). In solution, however, NMR studies indicate that the
secondary structure of the central linker
region is in dynamic flux
and is influenced by calcium binding. Thus, in the apo form,
residues 76-81 appear to adopta helical conformation approximately one thirdof the time (Kuboniwaet al., 1995), whereas
there is no evidence for any helical structure in this region of
holo-calmodulin (Barbato et al., 1992; Kuboniwa et al.,1995).
It seems possible therefore that, in solution, the hydrogen bond
between Tyr-138 and Glu-82 is a dynamic featureof the protein,
that could be influenced
by calcium-bindingevents in the
N-terminal lobe of the protein.
in the
Thisideathatinformationconcerningevents
N-terminus might be transmitted to Tyr-138 via the central linker
region is given some validity by recent findings for the crystal
structure of calmodulin with one molecule of trifluoperazine
bound to the C-terminal lobe (Cook
et al., 1994). Surprisingly,
this binding event is sufficient to contort the central linkerregion and N-terminal lobe into the bent conformation seen for
calmodulin with a target peptide bound to both the
N- and
C-terminal halves of the protein. Thus, anevent primarily in one
lobe of calmodulin can produce conformational changesin the
central linker region of the protein.
Materials and methods
Analysis in terms of the known properties of Tyr-138
In order to understand how changes
in the calcium-binding
properties of the N-terminusof calmodulin may affect the Tyr138 in the C-terminus, it will be necessary to understand in detail the calcium-induced structural changes to this residue. NMR
studies of both wild type (Seamon, 1980) and single calciumbinding site mutants (Starovasnik, 1992) indicate that Tyr-138
can respond to atleast three different processes during calcium
titration. Time-resolved fluorescence anisotropy (Toroket al.,
1992) has indicated that side-chain movement of Tyr-138 becomes more restricted upon calcium binding. Recently, incorporation of Tyr-138 into the exiting a-helixof site 4 has been
shown to be a calcium-induced event (Kuboniwa et al., 1995)
One possible mechanism whereby N-terminal calcium binding might affect Tyr-138 is by direct interaction between the two
terminal lobes of the protein. However, although the twolobes
can approach moreclosely in solution than indicated by the crystal structure (Seaton et al., 1985; Heidorn & Trewhella, 1988),
no NOE interactions are detectedbetween side chains located
in opposing terminal lobes of calmodulinin either the apo- or
the holo- formof the protein (Ikuraet al.,1991). Therefore, on
the time scale detected by NMR, noresidues of the N-terminus
are within 5 A of any C-terminal residues, suggesting that direct interaction between the two terminal lobes is unlikely.
The alternative route for N-terminal
events to influence Tyr138 would be through the central linkerregion of the protein.
Available data forTyr-138 support this interpretation. The hydroxyl group of Tyr-138 has an unusually high pK that is unaffected by calcium binding (Richman & Klee, 1978, 1979). This
suggests that Tyr-138 is buried within the hydrophobic core of
calmodulin and remains so upon calcium saturation. In the crystal structure of holo-calmodulin, Tyr-138 is clearly part of an
Molecular cloning
Generation of calmodulin cDNA constructs forwild-type Drosophila calmodulin and thesingle-site mutants BlQ, B2Q, B3Q,
and B4Q has been described previously (Maune et al., 1992b).
The double mutantsused here were prepared by combining restriction fragments derived from the appropriatesingle-site mutant constructs. Thus, (1) the double mutantsB12Q and B13Q
were generated by replacing the Pst I-Sal I fragment of BlQ
with the equivalent fragments of B2Q and B3Q, respectively;
and (2) the double mutantsB24Q and B34Q were generated by
replacing the Nco I-EcoR V fragment of B4Q with the equivalent fragments of B2Q and B3Q, respectively. The quadruple
mutant was generated by ligation of the BamHI-Fok I fragment
of theB12Q mutant andthe Fok I-EcoR I fragment of the B34Q
mutant into the BamH I - EcoR I sites of vector pEMBL8’.
All multiple mutant constructs were sequenced throughout the
entire calmodulin coding region prior to transfer to anexpression vector. In somecases, proteins were expressed from the vector POTS Nco 12, as described previously (Maune et al., 1992b).
For some mutants, the vector PET15b (Novagen) was used. In
these cases, mutant Nco I-Sal I cDNA fragments were transferred to the Nco I and Xho I sites of PET 15b.
Protein expression and purification
Wild-type Drosophila calmodulin and calmodulins containing
single binding site mutants were purified as described previously
using affinity chromatography on phenyl-Sepharose (Maune
et al., 1992b). For mutants expressed from PET 15b constructs,
the DE3 pLysS hostcells were grown in 2 L of “terrific broth”
(Tartof & Hobbs, 1987) at 30 “C to earlylmid
log phase and then
476
protein expression was induced by addition of IPTG to 30pM
followed by incubation at 30 "C for
a further 16-20 h. PhenylSepharose chromatography could notbe used for the multiple
binding site mutants and therefore an alternative purification
scheme was devised, based on that of Newtonet al. (1988). The
start point for the purificationwas a 100,ooO-g supernatant prepared after Frenchpress disruption of the bacterial suspensions
as described previously (Maune et al., 1992b). Ammonium sulfate fractionation was used as the first purification step. The
pellet formed between 60 and 80% ammonium sulfate saturation was dialyzed against 10 mM histidine buffer, H 5.6, containing 1 mM CaCl, (Buffer H). After determination of the
protein concentration of thesolubilized dialysate (Bio-Rad protein assay), it was adjusted to approximately10 mg/mL protein,
loaded ontoa DEAE-Sephacel column at4 "C, andeluted with
a linear salt gradient of 0-0.4 M NaCl, in Buffer H. Fractions
containing calmodulin were identified by SDS-PAGE, pooled,
and dialyzed against Buffer H containing 20% PEG (15,00020,000 MW) or Aquacide (Calbiochem) to concentrate the protein. After a further protein concentration determination, the
dialysate was adjusted to approximately 2 mg/mL protein for
HPLC on an anion exchange column (AX300, 250x10 mM).
For each HPLC run, about 20 mg of protein were used and
eluted with a 0-1.5 M NaCl gradient in Buffer H. Fractions containing calmodulin were identified by SDS-PAGE, pooled, and
dialyzed against 20 mM Tris-HC1, pH 7.0, 1 mM CaCI,, again
containing PEG or Aquacide. HPLC on the AX300 resin was
repeated, again using a 0-1.5 M NaCl gradient and the purity
of the calmodulin-containing fractions was assessed by SDSPAGE and absorbance in the 200-300-nm range. After these
four purification steps,all of the multiple mutants proteins were
purified to homogeneity, although yields were low (approximately 20 mg protein per 14 L of bacterial culture).
P. Mukherjea et al.
propriate buffers were used for baseline determinations and all
spectra were corrected for dilution and baseline effects. Data
wereprocessedusingtheUV2101/3101Kineticssoftware
package.
Near- U V CD
C D spectra were recorded at 22 "C using a Jasco 5-600 spectropolarimeter, with a sampling interval of 0.1 nm. Near-UV CD
spectra (250-310 nm) were recorded usinga pathlength of
10 mm. Mutant proteins at 100 pM concentration in 20 mM
Hepes/KOH buffer, p H 7.6, were used for the experiments and
spectra were recorded for theinitial decalcified samples and after addition of 2,4, and 10 equivalents of CaCl,. At least three
scans were averaged for each spectrum, with baseline corrections
for the buffer, light scattering (as measured at 310-350 nm), and
protein dilution. Data processing of the ASCII files was performed using the program PS plotof Micro-cal. As in previous
work (Maune et al., 1992a), data are presentedin terms of the
mean residue CD, Acaso,on the basis of a mean residue weight
of 112.7.
SK-MLCK assays
SK-MLCK activity was assayed by incorporation of 32Pinto
the regulatory light chain largely as described previously (Gao
et al., 1993). The reaction mixture (50 pL final volume) consisted
of 50 mM MOPS, pH 7.0, 100 pM CaCl,, 1 mM dithiothreitol,
30pM regulatorylight chain, 5 nM SK-MLCK, and1 mM y 3 * P
ATP (200-250 cpm/pmol). Calmodulin concentrations of0.05
nM-1 pM were used for thewild-type protein and 1.OO nM-5 pM
for the mutant calmodulins. Activationwas normalized as percent activation for the wild-type protein
(20 pmol of 32P/
midmg).
Decalcification and desalting of proteins
Buffer exchange and desaltingof proteins was achieved either
by repeated passage over (3-25 Sephadex (PD-10 columns, Pharmacia) or by extensive dialysis (Spectra-por tubing, MW cut-off
6,000-8,000). Proteins were decalcified by Chelex 100 treatment,
as described previously (Maune et al., 1992b), immediately before use and after exchange into the appropriate buffer.
U V extinction coefficient determinations
Like wild-type calmodulin, all of the multiple mutants studied
here contain no tryptophan or cysteine, a single tyrosine, and
nine phenylalanine residues. Extinction coefficients at the absorbance maximum for the tyrosine peak (at approximately
279 nm) could thus be determined using a 9:l mixture of the
N-acetyl methyl esters of phenylalanine and tyrosine, as described previously (Maune et al., 1992b).
UV-difference spectroscopy
UV absorbance spectra were collected on a Shimadzu UV2101-PC spectrophotometer duringsequential addition of equivalents of CaCl, to decalcified proteins at 100 pM concentration
in 1 0 0 mM KCI, 10 mM Hepes, pH 7.6. After incremental addition of four equivalents of CaCl,, six equivalents were added,
followed by a further six equivalents (16 equivalents total). Ap-
Acknowledgments
This work was supported by the following grants to K.B.: NIH grant
GM49155, grant 003604-028 of the Advanced Research/Advanced Technology Program of the Texas Higher Education Board, andWelch Foundation Grant C-1119. We thank Drs. James Stull andBeatrice Clack for
their generous help with the myosin light chain kinase assays described
here. We are grateful to Drs. John Olson and Fred Rudolph and the
members of their laboratories for helpful discussions and the use of many
facilities. Spectropolarimetry was performed on an instrument belonging to the Institute of Biosciences and Technology of the Texas A&M
University. We thank Jacquelynn Larson for her help with the use of
that instrument. We appreciate the helpful suggestions
of our colleagues
Bernard Andruss, Dr. Richard Atkinson, and Dr. Heidi Nelson, after
initial reading of the manuscript.
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