Structural characterization of Ca
2+
/CaM in complex with
the phosphorylase kinase PhK5 peptide
Atlanta G. Cook*, Louise N. Johnson and James M. McDonnell
Laboratory of Molecular Biophysics, Department of Biochemistry, Oxford University, UK
Phosphorylase kinase (PhK) is a Ca
2+
-regulated pro-
tein kinase that controls the breakdown of glycogen
through phosphorylation of glycogen phosphorylase
(reviewed in [1]). The enzyme is a large, 1.3-MDa hexa-
decameric complex consisting of four copies of four
subunits, a, b, c and d. The a and b subunits are regu-
latory and are the sites of phosphorylation and meta-
bolite binding and are also regulated by the binding
of extrinsic calmodulin (CaM). The c subunit is the
catalytic subunit and the d subunit is an intrinsic mole-
cule of CaM that binds to the enzyme even in the
absence of Ca
2+
[2]. The regulation of PhK through
Ca
2+
⁄ CaM enables the coordination of muscle contrac-
tion with the production of glucose through the action
of Ca
2+
on calmodulin and troponin C [3].
PhK is related to other Ca
2+

*Present address
EMBL, Meyerhofstrasse 1, D-69117
Heidelberg, Germany
(Received 7 December 2004, revised 23
January 2005, accepted 1 February 2005)
doi:10.1111/j.1742-4658.2005.04591.x
Phosphorylase kinase (PhK) is a large hexadecameric enzyme consisting of
four copies of four subunits: (abcd)
4
. An intrinsic calmodulin (CaM, the d
subunit) binds directly to the c protein kinase chain. The interaction site
of CaM on c has been localized to a C-terminal extension of the kinase
domain. Two 25-mer peptides derived from this region, PhK5 and PhK13,
were identified previously as potential CaM-binding sites. Complex forma-
tion between Ca
2+
⁄ CaM with these two peptides was characterized using
analytical gel filtration and NMR methods. NMR chemical shift perturba-
tion studies showed that while PhK5 forms a robust complex with
Ca
2+
⁄ CaM, no interactions with PhK13 were observed.
15
N relaxation
characteristics of Ca
2+
⁄ CaM and Ca
2+
⁄ CaM ⁄ PhK5 complexes were
compared with the experimentally determined structures of several

PhK directly [11]. Furthermore, separation of these
two chains is only possible through denaturation [12].
CaM is a ubiquitous Ca
2+
sensor that is found in
all eukaryotes and shows little sequence variation in
metazoans [13]. CaM undergoes large conformational
changes both on binding to Ca
2+
ions and on interact-
ing with its targets [14,15]. The protein consists of two
domains each encoding two EF-hand motifs separated
by a linker region that imparts a high degree of
conformational flexibility. In the presence of Ca
2+
ions
the protein undergoes conformation changes that alter
the surface properties of the two domains allowing
CaM to recognize its targets.
A number of structures of CaM in complex with
peptides derived from CaM target proteins have been
solved. In the classical case, these CaM binding pep-
tides have been identified as basic motifs of approxi-
mately 20 amino acids that are able to bind to CaM as
amphipathic helices [16]. Binding causes CaM to wrap
around the peptide helix forming a hydrophobic chan-
nel and a number of acidic residues on the surface of
the CaM domains typically form salt bridges with the
basic residues that are found in the peptide (Fig. 1).
Despite an overall similarity, the closed Ca

strating the ‘compact’ structure of a Ca
2+
⁄ CaM ⁄ peptide complex.
The N-terminal domain is in blue and the C-terminal domain is in
pink. The peptide is shown as a green helix and the termini are
indicated with N and C. Two side chains are depicted correspond-
ing to the two anchor residues of the smMLCK peptide, Trp800
and Leu813. The individual helices are labelled with roman numer-
als starting from the N terminus. The Ca
2+
ions are shown as blue
spheres and are labelled 1–4. Figure prepared with
PYMOL [42]. The
alignment shows peptide sequences from various Ca
2+
⁄ CaM ⁄ pep-
tide structures in single amino acid code. The residues in yellow
are anchor residues and basic residues are shown in blue. In the
two PhK peptides large hydrophobic and aromatic residues are
shown in green that have been predicted as potential anchor resi-
dues for these two peptides. Both PhK peptides have previously
been assigned a ‘1–12 motif’, a structurally uncharacterized motif.
Fig. 2. An overview of the PhKc domain structure. The first 296
residues of the subunit encode a protein kinase domain. The struc-
ture of the constitutively active kinase domain has previously been
solved, coordinates are taken from PDBid 2phk [43,44]. Proteolytic
treatment cleaves between the kinase domain and the 90-residue
C-terminal extension of the kinase. Two 25-mer peptides in this
region, PhK13 and PhK5, were identified as potential CaM binding
domains.

evidence for these two complexes is available. In this
paper we have used NMR methods to characterize
the structures. The NMR evidence shows that PhK5
does indeed form a classical collapsed complex with
CaM, but no interaction of CaM with PhK13 could
be detected. A method for identifying structural
similarity between Ca
2+
⁄ CaM ⁄ peptide complexes is
presented.
Results
The binding of PhK5 and PhK13 to Ca
2+
/CaM
High resolution analytical gel filtration was used to
identify the complexes of CaM with the two PhK pep-
tides. Because the previously reported K
I
values for the
peptides were in the low nanomolar range, the samples
were mixed together in a 1 : 1 molar ratio and gel fil-
tration was carried out in the presence of Ca
2+
.
Ca
2+
⁄ CaM, in the absence of peptides, elutes as a sin-
gle peak after a volume of 16.12 mL. As Ca
2+
⁄ CaM

ance increase suggests that PhK13 does not bind.
Furthermore, analysis of peak fractions by SDS ⁄
PAGE indicates that no peptide coelutes with
Ca
2+
⁄ CaM (Fig. 3 inset).
The lack of PhK13 binding was surprising, so we
sought a more definitive experiment to demonstrate
peptide binding. Using NMR spectroscopy, titrations
were carried out with unlabelled peptides into
15
N
labelled Ca
2+
⁄ CaM. Chemical shift changes in the
CaM backbone amides were monitored using
1
H-
15
N
HSQC. PhK5 causes a large number of chemical shift
changes in the Ca
2+
⁄ CaM HSQC spectrum indicative
of a large conformational change upon binding of
PhK5 (Fig. 4A). These changes are observed early in
the titration, at as little as 20% saturation changes are
readily apparent. Intermediate peaks between the
unbound and bound species are not observed indica-
ting that the binding of PhK5 to Ca

not to detect it by this method.
The
1
H-
15
N HSQC for free Ca
2+
⁄ CaM are essen-
tially identical to previously described spectra [25], and
so peaks were assigned by comparison. Of the 92
peaks that could be assigned in this way, only 13 in
the PhK5 titration do not show any alteration in
chemical shift (summarized in Table 1). These include
residues that are found in solvent exposed regions
(Leu4, Glu6, Asn42 and Glu45) that are unlikely to
change on complex formation as they do not interact
with peptide ligands. In addition, four glycine residues,
found in the second position of the four distinct EF-
hand motifs (Gly23, Gly59, Gly96 and Gly132) also
show no changes in chemical shift. Three further resi-
dues that are found buried between the two EF-hand
motifs (Thr62, Ile100 and Val136) are also found not
to have any chemical shift changes. These residues
form part of the hydrophobic packing between EF-
hand motifs and this explains their unaltered chemical
environment. Lastly two further residues, Met72 and
Glu82 are also unchanged. These two residues are
found on the connecting helices between the two
domains of Ca
2+

have indicated that PhK5 binds to CaM as a col-
lapsed complex that has similar properties to other
CaM ⁄ peptide complexes [24]. To determine whether
the binding of PhK5 does cause a conformational
change in CaM, the T1 and T2 constants were meas-
ured for each residue to allow a better understanding
of the hydrodynamic behaviour of Ca
2+
⁄ CaM and
Ca
2+
⁄ CaM ⁄ PhK5.
HSQC spectra were taken after a series of increasing
relaxation delays to measure the T1 and T2 relaxation
times. For each residue identified in the spectrum, a
single exponential fit to the peak intensities over the
series of spectra was used to calculate R1 (1 ⁄ T1) and
R2 (1 ⁄ T2). The R1 and R2 values were plotted against
Fig. 4. (Top) Overlay of two spectra from the titration of Ca
2+
⁄ CaM
with PhK5 peptide. The red spectrum shows the position of peaks
prior to the addition of PhK5 peptide. The blue spectrum shows a
spectrum taken when a 1 : 1.2 molar ratio of Ca
2+
⁄ CaM to PhK5
had been reached. The inset shows an expanded view of the indi-
cated area of the spectrum. (Bottom) An overlay of spectra from
the titration of Ca
2+

⁄ CaM ⁄ PhK5
forms a more compact structure than Ca
2+
⁄ CaM, and
that PhK5 binding does induce conformational
change.
The R2 plot for Ca
2+
⁄ CaM ⁄ PhK5 shows unusual
periodic increases in R2 over the length of helix I that
are not reflected in the R1 values. The periodicity of
the changes follow approximately the periodicity of the
helix and the residues with higher R2 values are found
on one face of helix I that is found to interact with
peptide ligands in CaM ⁄ peptide complexes. This effect
could be caused by conformational exchange on the
millisecond timescale (R
ex
) that affects only one side of
the helix.
Fig. 5. The R1 and R2 relaxation rates were
plotted against residue number. At the base
of each plot the structural elements of CaM
are indicated by the single line plot with heli-
ces labelled in Roman numerals in the first
plot. The average relaxation rate of each helix
is plotted as a black bar. The first helix of the
Ca
2+
⁄ CaM ⁄ PhK5 R2 is highlighted by a box;

molecule. Upon binding of peptide ligands, calmodulin
undergoes a dramatic alteration in structure marked
by changes in the relative orientations of its eight
a-helical elements. Different peptide ligands result in
subtle differences in the CaM conformations.
We did not have an experimental model of
Ca
2+
⁄ CaM ⁄ PhK5 structure, but a number of struc-
tures of CaM ⁄ peptide complexes are available that
reflect wide conformational diversity of the CaM mole-
cule. Therefore we used the program rotdif [29,30] to
calculate the diffusion tensor of a PDB-derived CaM
structure, back-calculate the R1 and R2 values for the
helical elements of the PDB structure and then com-
pare these values to the experimentally derived relaxa-
tion parameters for Ca
2+
⁄ CaM ⁄ PhK5 and, as a
control, for Ca
2+
⁄ CaM. For these calculations each
helix was treated as a structural element; R1 and R2
values for each helix were averaged based on four con-
secutive residues in the middle of the helix. For the
Ca
2+
⁄ CaM ⁄ PhK5 data, helix IV had to be excluded
because of insufficient data due to spectral overlap and
in the case of helix I, only the lower R2 values were

perp
ratio that was calculated for this com-
plex (Table 2). However, this structure was included
and treated as axially symmetric for the purposes of
this study.
Table 2 shows the target functions calculated by
comparison of experimental relaxation data with data
back-calculated from the PDB files of the input models.
As expected, poor fits were generally observed between
the Ca
2+
⁄ CaM data, where CaM is in the extended
conformation, and the various peptide complex models,
where CaM is in the folded conformation. The
Ca
2+
⁄ CaM ⁄ PhK5 dataset showed betters fits in
general and several structures (Ca
2+
⁄ CaM ⁄ smMLCK,
Ca
2+
⁄ CaM ⁄ endothelial nitric oxide synthase (eNOS)
and Ca
2+
⁄ CaM ⁄ CaMKI) appeared to fit the relaxation
data better than others. A structural alignment of these
three models shows that they are indeed close structural
Table 2. The quality of fits (described by the chi-squared per degrees of freedom) for a series of Ca
2+

⁄ CaM s
c
(ns) D
para
⁄ D
perp
smMLCK 1cdl 0.178 2.46 9.41 1.18
CaMKII 1cdm 0.239 2.67 9.41 1.21
CaMKI 1mxe 0.144 2.53 9.41 1.18
CaMKK 1iq5 0.697 3.21 9.37 1.13
eNOS 1niw 0.170 2.15 9.45 1.23
MARCKS 1iwq 0.329 2.68 9.40 1.20
Ca
2+
⁄ CaM (compact) 1prw 0.449 3.31 9.41 1.18
Ca
2+
⁄ CaM in complex with the PhK5 peptide A. G. Cook et al.
1516 FEBS Journal 272 (2005) 1511–1522 ª 2005 FEBS
relatives. The remaining structures, which fit the relaxa-
tion data less well, show a variety of relative helix ori-
entations (Fig. 6).
Discussion
The binding of PhK5 and PhK13 to Ca
2+
/CaM
PhKc interacts with the CaM Ca
2+
sensor through a
C-terminal extension to the kinase domain of % 90 res-

this peptide to inhibit the MLCK activity was well
established and it was demonstrated to bind to CaM
in a Ca
2+
-dependent manner in gel mobility assays
[18]. However the small angle X-ray and neutron
scattering data indicated that the PhK ⁄ Ca
2+
⁄ CaM
interaction is anomalous [23]. The CaM remained
extended on binding PhK13. PhK13 failed to protect
CaM against proteolysis while PhK5 did protect in a
manner similar to other CaM binding peptides [22].
Analysis of the PhKc C-terminal extension sequences
from a number of different organisms suggests that
the PhK5 region is likely to be the more important
in Ca
2+
signalling. The PhK5 region is conserved
(15 residues out of 25 identical) while the PhK13
region shows only two residues out of 25 identical
and does not exhibit a canonical CaM binding
sequence (Fig. 7). The differences observed under dif-
ferent experimental conditions for the PhK13 ⁄ CaM
interactions require further work and could best be
resolved by a crystal structure of full length PhKc
subunit with CaM, a structure that has so far been
elusive.
Does the PhK5 peptide cause conformational
changes in Ca

⁄ CaM ⁄ smMLCK is shown in pink once again as a reference. Ca
2+
⁄ CaM ⁄ CaMKK is in red (PDBid 1iq5),
Ca
2+
⁄ CaM ⁄ CaMKIIa is shown in green (PDBid 1cdm) and the Ca
2+
⁄ CaM ⁄ MARCKS structure (PDBid 1iwq) is shown in yellow. Figure pre-
pared with
AESOP (MEM Noble, unpublished work).
A. G. Cook et al. Ca
2+
⁄ CaM in complex with the PhK5 peptide
FEBS Journal 272 (2005) 1511–1522 ª 2005 FEBS 1517
consistent with a compact structure of the Ca
2+
⁄
CaM ⁄ PhK5 complex. The spectrum of Ca
2+
⁄ CaM ⁄
PhK5 is dramatically different from that of the
unbound Ca
2+
⁄ CaM with only 13 peaks out of 146
that show no alteration in chemical shift, indicating
that a large conformational change occurs on binding
peptide. Comparison of the R1 values for Ca
2+
⁄ CaM
and Ca

rotation of the molecule in solution. When a mole-
cule tumbles anisotropically, the residues with N–H
bond vectors aligned with the long axis of the mole-
cule will reorient more slowly compared with N–H
bond vectors that are orientated along the shorter
axes. This causes differences in the average R1 and
R2 properties of a given helix depending on how it
is oriented with respect to the long axis of the mole-
cule. The differing patterns of average R1 and R2
values along each helix for Ca
2+
⁄ CaM and the
Ca
2+
⁄ CaM ⁄ PhK5 indicate that the orientations of
the helices that make up the structure are different
in the two species. As these data apply only to the
main chain nitrogen atoms of the CaM structures
this shows that PhK5 has indeed induced a conform-
ational change in Ca
2+
⁄ CaM.
Fig. 7. Sequence alignment of the C-terminal extension of PhKc. Sequences were obtained for both vertebrate and nonvertebrates by using
sequence from 296 to 386 of PhKc from rabbit muscle. Identities are shown by the blue boxes with white text, while blue text and green
boxes indicate regions of sequence similarity. Numbering is taken from the rabbit muscle sequence. The regions corresponding to the PhK5
and PhK13 peptides are indicated by pink boxes. This figure was prepared using
ESPRIPT [45].
Ca
2+
⁄ CaM in complex with the PhK5 peptide A. G. Cook et al.

of CaM and its effects on the relaxation rates of
N–H bond vectors of the CaM backbone. Each of
the Ca
2+
⁄ CaM ⁄ peptide structures was used as an
input model in lieu of a Ca
2+
⁄ CaM ⁄ PhK5 model,
for the program rotdif that calculates hydro-
dynamic parameters based on a structure and its
relaxation data [29,30]. The hydrodynamic data were
consistent with a monomeric structure of % 20 kDa.
Out of the seven PDB files that were used as input
models, three structures showed better fits than the
rest. These three structures are the complexes with
the eNOS peptide, the smMLCK peptide and the
CaMKI peptide. All three of these are structural rel-
atives and show rmsds of the Ca atoms of 1.705 A
˚
comparing CaMKI to smMLCK, 1.972 A
˚
comparing
CaMKI to eNOS and 2.633 A
˚
comparing smMLCK
to eNOS. In addition, all three peptides have been
identified as binding with a 1–14 motif of anchor
residues while the remaining structures in the study
show 1–16 binding (CaMKK) and 1–10 binding
(CaMKIIa) [17]. The MARCKS peptide structure

that produces a classical compact Ca
2+
⁄ CaM ⁄ peptide
complex. Analysis of the NMR relaxation data sug-
gests that the Ca
2+
⁄ CaM ⁄ PhK5 complex is a close
structural relative of CaM complexes with eNOS,
smMLCK and CaMKI.
Experimental procedures
Preparation of CaM
The CaM cDNA from Xenopus leavis was a kind gift from
D. Owen (Oxford University, UK) and was cloned into the
pPROTet.E232 vector (Clontech, Oxford, UK). The plasmid
was transformed into BL21-PRO cells (Clontech) and
expressed at 37 ° C by induction with 100 ngÆmL
)1
anhydro-
tetracycline for 5 h. The cells were harvested by centrifuga-
tion and resuspended in 50 mm Tris ⁄ HCl pH 7.5, 2 mm
EDTA, 0.2 mm phenylmethanesulfonyl fluoride and were
stored at )20 °C. Purification of CaM was carried out using
the method of Hayashi et al. with minor modifications [33].
The cells were thawed and lysed by sonication and the sol-
uble fraction was collected by centrifugation at 100 000 g in
a Beckman L8-M ultracentrifuge for 1 h at 4 °C. CaCl
2
was
added to the supernatant to a final concentration of 5 mm
and the sample was then applied to a 50-mL phenyl seph-

⁄ CaM in complex with the PhK5 peptide
FEBS Journal 272 (2005) 1511–1522 ª 2005 FEBS 1519
concentrated to % 1mm and buffer exchanged at least four
times into 20 mm deuterated Tris ⁄ HCl pH 6.5.
PhK5 and PhK13 peptides
The PhK5 peptide (amino acid sequence LRRLIDAYAFRI
YGHWVKKGQQQNR) and the PhK13 peptide (amino
acid sequence GKFKIVCLTVLASVRIYYQYRRVKP)
were custom synthesized using solid phase fmoc chemistry
by G. Bloomberg (Bristol University, UK). The peptides
were further purified by reverse phase chromatography
using a 250 · 10 mm C5 column (Phenomenex, Maccles-
field, UK). The peptides were loaded in 0.1% trifluoracetic
acid (TFA) and eluted using a gradient from 0.1% TFA to
0.1% TFA and 50% acetonitrile over five column volumes.
The peptides were then lyophilized and reconstituted into
double deionized H
2
O and dialysed against water at 4 °Cto
remove salt impurities. MS of these purified peptides was
performed on a Micromass Platform-II ESI mass spectro-
meter (Waters, Elstree, UK) and gave expected molecular
mass values (3118 ± 3 and 3004 ± 4 Da, for PhK5 and
PhK13, respectively), thus confirming the composition of
the peptides that were used in subsequent experiments.
Analytical gel filtration
Analytical gel filtration was carried out using an SD200
high resolution sepharose column (Amersham-Pharmacia,
Uppsala, Sweden). The column was pre-equilibrated with
50 mm Tris ⁄ HCl pH 7.0, 10 mm CaCl

H carrier
frequency set to 4.74 p.p.m. and the
15
N carrier frequency
set to 120 p.p.m. For each experiment 32 scans were taken
with 128 increments in the nitrogen dimension. The
Ca
2+
⁄ CaM sample was at a concentration of 0.45 mm and
contained % 0.2 lmol protein. The PhK5 peptide was
reconstituted in NMR buffer to a concentration of 16 mm.
Successive additions of 0.02–0.04 lmol of peptide were
made to the CaM sample, to a final molar ratio of 1 : 1.4
CaM to peptide. The PhK13 peptide was treated in the same
way and spectra were taken over a similar range, up to a
ratio of 1 : 0.8 CaM to PhK13. Data were processed using
FELIX 2.3 (Biosym Inc.) and analysed with xeasy [34].
Analysis of
15
N relaxation data
NMR experiments were performed on spectrometers oper-
ating at
1
H frequencies of 600 MHz at 25 °C. Backbone
15
N relaxation parameters, comprising the rates of
15
N
transverse (R2) and longitudinal (R1) relaxation were
measured using previously described experimental protocols

peak intensities.
The longitudinal and transverse relaxation rates (R1 ¼
1 ⁄ T1 and R2 ¼ 1 ⁄ T2, respectively), were calculated by fit-
ting a single exponential to the peak intensities for different
time points using matlab 6.5. Four residues from each helix
in the structure were used and their R1 and R2 values were
averaged to produce orientation vectors for each helix for
the rotational anisotropy analysis. Fitting of the hydro-
dynamic parameters was carried out using rotdif written
by D. Fushman (University of Maryland, USA) [29,30]. For
the Ca
2+
⁄ CaM data all eight helices from the CaM structure
were represented, however, in the Ca
2+
⁄ CaM ⁄ PhK5 data
the fourth helix was discarded as spectral overlap resulted in
too few data points for meaningful analysis. As no hetero-
nuclear NOE measurements were taken for either data set,
the NOE values assigned for each residue used in the analy-
sis was 0.80 ± 0.04, to reflect typical values for residues in
stable secondary structure elements and are consistent with
previous measurements made for CaM [25,36].
Six Ca
2+
⁄ CaM ⁄ peptide structures were selected from the
PDB along with the collapsed, peptide-free structure of
Ca
2+
⁄ CaM (PDBid 1prw) [37] as input models for the

N. The calculation of R2¢ and
R1¢ subtracts contributions from the high frequency com-
ponents of local motions. The Levenberg–Marquardt algo-
rithm is used to minimize the target function:
v
2
¼
X
i
q
exp
i
À q
calc
i
r
qi

2
Where q
exp
i
is the ratio from the measured relaxation
parameters for each residue i, and q
calc
i
is the ratio calcula-
ted from the current model. The value r
i
is the error in q

7 Kobe B, Heierhorst J, Feil SC, Parker MW, Benian
GM, Weiss KR & Kemp BE (1996) Giant protein
kinases: domain interactions and structural basis of
autoregulation. EMBO J 15 , 6810–6821.
8 Kemp BE & Pearson RB (1991) Intrasteric regulation
of protein kinases and phosphatases. Biochim Biophys
Acta 1094, 67–76.
9 Wilmanns M, Gautel M & Mayans O (2000) Activation
of calcium ⁄ calmodulin regulated kinases. Cell Mol Biol
(Noisy-le-Grand) 46, 883–894.
10 Harris WR, Malencik DA, Johnson CM, Carr SA,
Roberts GD, Byles CA, Anderson SR, Heilmeyer LM
Jr, Fischer EH & Crabb JW (1990) Purification and
characterization of catalytic fragments of phosphorylase
kinase gamma subunit missing a calmodulin-binding
domain. J Biol Chem 265, 11740–11745.
11 Chan KF & Graves DJ (1982) Isolation and physico-
chemical properties of active complexes of rabbit muscle
phosphorylase kinase. J Biol Chem 257, 5939–5947.
12 Crabb J & Heilmeyer L Jr (1984) High performance
liquid chromatography purification and structural char-
acterization of the subunits of rabbit muscle phosphory-
lase kinase. J Biol Chem 259, 6346–6350.
13 Chin D & Means A (2000) Calmodulin: a prototypical
calcium sensor. Trends Cell Biol 10, 322–328.
14 Finn BE & Forsen S (1995) The evolving model of cal-
modulin structure, function and activation. Structure 3,
7–11.
15 Vetter SW & Leclerc E (2003) Novel aspects of calmo-
dulin target recognition and activation. Eur J Biochem

34566.
22 Juminaga D, Albaugh SA & Steiner RF (1994) The
interaction of calmodulin with regulatory peptides of
phosphorylase kinase. J Biol Chem 269, 1660–1667.
23 Trewhella J, Blumenthal DK, Rokop SE & Seeger PA
(1990) Small-angle scattering studies show distinct con-
formations of calmodulin in its complexes with two pep-
tides based on the regulatory domain of the catalytic
subunit of phosphorylase kinase. Biochemistry 29, 9316–
9324.
24 Yao Y & Squier TC (1996) Variable conformation and
dynamics of calmodulin complexed with peptides
derived from the autoinhibitory domains of target pro-
teins. Biochemistry 35, 6815–6827.
25 Ikura M, Kay LE & Bax A (1990) A novel approach
for sequential assignment of 1H, 13C, and 15N spectra
of proteins: heteronuclear triple-resonance three-dimen-
sional NMR spectroscopy. Application to calmodulin.
Biochemistry 29, 4659–4667.
26 Meador WE, Means AR & Quiocho FA (1993) Mod-
ulation of calmodulin plasticity in molecular recogni-
tion on the basis of x-ray structures. Science 262,
1718–1721.
27 Meador WE, Means AR & Quiocho FA (1992) Target
enzyme recognition by calmodulin: 2.4 A structure of a
calmodulin-peptide complex. Science 257, 1251–1255.
28 Tjandra N, Garrett DS, Gronenborn AM, Bax A &
Clore GM (1997) Defining long range order in NMR
structure determination from the dependence of hetero-
nuclear relaxation times on rotational diffusion aniso-

domain in solution: analysis of 15N relaxation with
monomer ⁄ dimer equilibrium. J Mol Biol 266, 173–194.
36 Ikura M, Kay LE, Krinks M & Bax A (1991) Triple-
resonance multidimensional NMR study of calmodulin
complexed with the binding domain of skeletal muscle
myosin light-chain kinase: indication of a conforma-
tional change in the central helix. Biochemistry 30 ,
5498–5504.
37 Fallon JL & Quiocho FA (2003) A closed compact
structure of native Ca
2+
-calmodulin. Structure (Camb)
11, 1303–1307.
38 Clapperton JA, Martin SR, Smerdon SJ, Gamblin SJ &
Bayley PM (2002) Structure of the complex of calmodu-
lin with the target sequence of calmodulin-dependent
protein kinase I: studies of the kinase activation
mechanism. Biochemistry 41, 14669–14679.
39 Kurokawa H, Osawa M, Kurihara H, Katayama N,
Tokumitsu H, Swindells MB, Kainosho M & Ikura M
(2001) Target-induced conformational adaptation of cal-
modulin revealed by the crystal structure of a complex
with nematode Ca
2+
⁄ calmodulin-dependent kinase kin-
ase peptide. J Mol Biol 312, 59–68.
40 Aoyagi M, Arvai AS, Tainer JA & Getzoff ED (2003)
Structural basis for endothelial nitric oxide synthase
binding to calmodulin. EMBO J 22, 766–775.
41 Yamauchi E, Nakatsu T, Matsubara M, Kato H &