Comparative studies of the two peptides in complex with Ca2+⁄ CaM with CD demonstrated that PhK5 was likely to form an a-helix when bound to CaM but that PhK13 showed no changes in secon
Trang 1the 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 Ca2+-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 Ca2+ [2] The regulation of PhK through
Ca2+⁄ CaM enables the coordination of muscle
contrac-tion with the produccontrac-tion of glucose through the accontrac-tion
of Ca2+on calmodulin and troponin C [3]
PhK is related to other Ca2+⁄ CaM-dependent protein
kinases including myosin light chain kinase (MLCK),
CaM kinases I, II and IV (CaMKI, CaMKII and CaMKIV, respectively), CaM kinase kinase (CaMKK), titin kinase, and death associated kinase [4] Structural studies on CaMKI [5], titin kinase [6] and twitchin kinase [7] have upheld the prediction that many CaM-dependent protein kinases are regulated through
an autoinhibitory mechanism [8] In these structures a C-terminal extension to the protein kinase folds back
on the kinase domain and interferes with the substrate binding sites In the case of the titin and twitchin kinases, the autoinhibitory sequence acts as a pseudo-substrate, occluding ATP binding and preventing protein substrates from binding (reviewed in [9]) PhK shows typical traits associated with such an autoinhibitory mechanism The sequence of the PhKc subunit encodes a C-terminal extension to the protein kinase domain and treatment of the kinase with
Keywords
calmodulin; kinase regulation; protein–
protein interaction; NMR spectroscopy
Correspondence
J M McDonnell, Laboratory of Molecular
Biophysics, Department of Biochemistry,
Oxford University, South Parks Road,
Oxford OX1 3QU, UK
Fax: +44 1865 275182
Tel: +44 1865 275381
E-mail: jim@biop.ox.ac.uk
*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 Ca2+⁄ 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
Ca2+⁄ CaM, no interactions with PhK13 were observed 15N relaxation characteristics of Ca2+⁄ CaM and Ca2+⁄ CaM ⁄ PhK5 complexes were compared with the experimentally determined structures of several
Ca2+⁄ CaM ⁄ peptide complexes Good fits were observed between
Ca2+⁄ CaM ⁄ PhK5 and three structures: Ca2+⁄ CaM complexes with pep-tides from endothelial nitric oxide synthase, with smooth muscle myosin light chain kinase and CaM kinase I We conclude that the PhK5 site is likely to have a direct role in Ca2+-regulated control of PhK activity through the formation of a classical ‘compact’ CaM complex
Abbreviations
CaM, calmodulin; CaMK, CaM kinase; CaMKK, CaM kinase kinase; eNOS, endothelial nitric oxide synthase; MLCK, myosin light chain kinase; PhK, phosphorylase kinase; TFA, trifluoracetic acid.
Trang 2proteases causes cleavage of this extension at residue
296 and 298 and loss of Ca2+-dependent activity [10]
(Fig 1) However, PhK also differs significantly from
other CaM-dependent protein kinases in that it binds
to CaM even in the absence of Ca2+ Studies on the
disruption of the holoenzyme produced two functional
complexes, the acd complex and the cd complex
dem-onstrating that CaM interacts with the kinase chain of
PhK directly [11] Furthermore, separation of these
two chains is only possible through denaturation [12]
CaM is a ubiquitous Ca2+ sensor that is found in
all eukaryotes and shows little sequence variation in
metazoans [13] CaM undergoes large conformational
changes both on binding to Ca2+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 Ca2+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 Ca2+⁄ CaM ⁄ peptide complexes show a variety of conformations with respect to domain orientation and conformation
of the EF hands The CaM binding domains in the different proteins have little sequence similarity They display a variable distance between the hydrophobic
‘anchor’ residues that bind into each domain of CaM and are classified on the basis of the distance between these two motifs [17]
Two 25-mer peptide regions from the C-terminal extension of PhKc were previously identified as poten-tial CaM binding sites using a series of overlapping synthetic peptides These two peptides, PhK5 (342– 367) and PhK13 (302–326), were able to inhibit CaM activation of MLCK [18] and Ca2+-dependent phos-phodiesterase [19] and were shown to have KI values
in the low nanomolar range Both peptides were dem-onstrated to have an inhibitory effect on PhK with PhK13 demonstrating competitive inhibition and PhK5 showing noncompetitive inhibition kinetics [20] PhK13, which lies towards the N terminus of the C-terminal extension (Fig 2), contains a sequence that
Fig 1 The Ca 2+ ⁄ CaM ⁄ smMLCK structure (PDBid 1cdl),
demon-strating the ‘compact’ structure of a Ca2+⁄ 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.
Trang 3suggested it could act as a pseudosubstrate mimicking
the substrate phosphorylase However, this role was
not supported by mutagenesis studies that converted a
potential pseudosubstrate cysteine residue to a serine
residue [21]
Comparative studies of the two peptides in complex
with Ca2+⁄ CaM with CD demonstrated that PhK5
was likely to form an a-helix when bound to CaM but
that PhK13 showed no changes in secondary structure
contributions on binding [22] Further studies with
small angle X-ray scattering [23] demonstrated that
PhK5 induced a compact conformation of CaM,
sim-ilar to that observed with MLCK peptides, a result
that was further confirmed by fluorescence anisotropy
studies [24], but that the interactions of PhK13 were
anomalous and in the complex CaM adopted an
exten-ded conformation
While these studies indicate that there are marked
differences in the Ca2+⁄ CaM ⁄ PhK5 complex vs the
Ca2+⁄ CaM ⁄ PhK13 complexes, no direct structural
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 Ca2+⁄ CaM ⁄ peptide complexes is
presented
Results
The binding of PhK5 and PhK13 to Ca2+/CaM
High resolution analytical gel filtration was used to
identify the complexes of CaM with the two PhK
pep-tides Because the previously reported KIvalues 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 Ca2+
Ca2+⁄ CaM, in the absence of peptides, elutes as a
sin-gle peak after a volume of 16.12 mL As Ca2+⁄ CaM
does not contain any tryptophan residues the
absorb-ance at 280 nm is entirely contributed by tyrosine
residues and is therefore relatively low When the
Ca2+⁄ CaM ⁄ PhK5 complex was loaded onto the
col-umn, the peak moved to 16.31 mL and showed a
higher absorbance at 280 nm PhK5 has one
trypto-phan residue and two tyrosine residues that accounts
for the increase in absorbance The formation of a
Ca2+⁄ CaM ⁄ PhK5 complex was confirmed by analysis
of the peak fractions by tris-tricine SDS⁄ PAGE that
shows a smaller band, of 3 kDa, that coelutes with
CaM That the peak shows an increase in peak elution
volume indicates that complex Ca2+⁄ CaM ⁄ PhK5 has
a smaller radius of gyration than Ca2+⁄ CaM and this is consistent with a more compact structure When a similar analysis was carried out with the
Ca2+⁄ CaM ⁄ PhK13 mixture no alteration in the peak intensity or the peak volume was observed Although PhK13 has no tryptophan residues, it does contain sev-eral tyrosine residues and the absence of an absorb-ance increase suggests that PhK13 does not bind Furthermore, analysis of peak fractions by SDS⁄ PAGE indicates that no peptide coelutes with
Ca2+⁄ 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 15N labelled Ca2+⁄ CaM Chemical shift changes in the CaM backbone amides were monitored using 1H-15N HSQC PhK5 causes a large number of chemical shift changes in the Ca2+⁄ 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 Ca2+⁄ CaM has slow exchange kinetics and this is consistent with the nano-molar KI values that had previously been reported
Fig 3 Gel filtration analysis of Ca 2+ ⁄ CaM and its complexes with PhK5 and PhK13 In all runs the same concentration of Ca 2+ ⁄ CaM was used and a 1 : 1 molar ratio of peptide was added The inset shows a Tris ⁄ tricine SDS ⁄ PAGE gel analysis of the peak fractions from the gel filtration run In all cases CaM is seen as a band of
17 kDa Only when Ca 2+ ⁄ CaM is mixed with PhK5 is a smaller peptide band also observed.
Trang 4When PhK13 was titrated into Ca2+⁄ CaM no chemical
shift changes are observed (Fig 4B No binding
inter-actions are observed between PhK13 and CaM; at the
concentrations we performed this experiment the
affin-ity of this interaction would need to be 10 mm for us
not to detect it by this method
The 1H-15N HSQC for free Ca2+⁄ 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 Ca2+⁄ CaM While Glu82 is either solvent exposed or disordered in Ca2+⁄ CaM peptides com-plexes, Met72 is found to interact with peptide ligands
in some structures such as the complex with CaMKIIa peptide [26], but not in others, for example the com-plex with smMLCK [27]
T1 and T2 relaxation times for Ca2+/CaM and
Ca2+/CaM/PhK5 The large number of chemical shift changes that occur on binding of PhK5 to Ca2+⁄ CaM suggest that this binding event is not a localized phenomenon and causes large changes in the structure throughout the molecule This is consistent with a large conforma-tional change occurring on binding of the peptide and could indicate that the binding of PhK5 to
Ca2+⁄ CaM is similar to that observed for Ca2+⁄ CaM binding to isolated peptides from other protein kin-ases Previous studies using fluorescence anisotropy 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 Ca2+⁄ CaM and
Ca2+⁄ 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 Ca2+⁄ 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 Ca2+⁄ 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 Ca2+⁄ CaM with PhK13 peptide showing in red the
spectrum prior to titration and in blue, the spectrum after a 1 : 0.8
molar ratio of Ca 2+ ⁄ CaM to PhK13 had been reached The inset
shows an expanded view of part of the spectrum equivalent to that
shown in (A) No chemical shift changes were observed on titration
with PhK13.
Table 1 CaM residues unaffected by PhK5 binding.
Residues that
Leu4, Glu6, Asn42, Glu45 Solvent exposed Gly23, Gly59, Gly96, Gly132 Second glycine in EF-motif Thr62, Ile100, Val136 Buried between EF-motifs
binds ligands in some CaM structures
Trang 5residue number, and average values for R1 and R2
over each helix were calculated (Fig 5) R1 and R2
are expected to have a reciprocal relationship for
movements on the nanosecond to picosecond
time-scale, i.e the timescale for molecular reorientation
The pattern of the average helical R1 and R2 values
clearly indicate that the complex of Ca2+⁄ CaM ⁄ PhK5
forms a more compact structure than Ca2+⁄ CaM, and
that PhK5 binding does induce conformational
change
The R2 plot for Ca2+⁄ 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 (Rex) 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
Ca2+⁄ CaM ⁄ PhK5 R2 is highlighted by a box;
the periodicity of the R2 effects suggests Rex
phenomena in this helix Residues 21, 57, 94
and 130 (indicated), which occupy identical
positions in each EF-hand motif and are
involved in chelating the Ca 2+ ions, each
show markedly reduced R2 values.
Trang 6Comparison of relaxation data with other
CaM/peptide complexes
In anisotropic proteins,15N relaxation rates depend on
the orientation of the N–H bond vectors relative to
the principal axis frame of the rotational diffusion
ten-sor For residues that are known to be structurally
rigid relaxation anisotropy can be used to derive
orien-tational constraints for structure calculations [28] The
analysis of orientational information from the
relaxa-tion characteristics of amide bond vectors is made
more straightforward in the case of a-helical proteins
Because the amide bond vectors of an a-helix all point
in the same direction, the average R1⁄ R2 ratio for
resi-dues in that helix provides information about its
orien-tation relative to the overall diffusion tensor of the
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
Ca2+⁄ 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 Ca2+⁄ CaM ⁄ PhK5 and, as a
control, for Ca2+⁄ 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
Ca2+⁄ 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 used to exclude the Rexeffects
Isotropic, axially symmetric (z¼ long axis, x ¼ y ¼ short axes) and fully anisotropic models (z > y > x axis) were all considered for fitting the relaxation data The data fit much better to axially symmetric models than to isotopic models (P < 1· 10)4, unless noted) The improvement in fit going from an axially symmet-ric model to the fully anisotropic model was generally not statistically significant (the likelihood that the improvements in fit occurred by chance showed a range of P¼ 4.58 · 10)1 to P¼ 2.12 · 10)2) and therefore the data presented here are for an axially symmetric model only One case, CaMKK, was noted
to be borderline as the improvement in fit using an axially symmetric model over an isotropic model gave
a poorer probability score (P¼ 2.29 · 10)2 for the anisotropic vs isotropic models) and this is reflected in the Dpara⁄ Dperpratio 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 Ca2+⁄ CaM data, where CaM is in the extended conformation, and the various peptide complex models, where CaM is in the folded conformation The
Ca2+⁄ CaM ⁄ PhK5 dataset showed betters fits in general and several structures (Ca2+⁄ CaM ⁄ smMLCK,
Ca2+⁄ CaM ⁄ endothelial nitric oxide synthase (eNOS) and Ca2+⁄ 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 ⁄ peptide complexes against the relaxation data for Ca2+⁄ CaM ⁄ PhK5 and Ca 2+ ⁄ CaM While all structures show relatively poor fits with the unbound Ca 2+ ⁄ CaM structure, including the compact form of Ca 2+ ⁄ CaM, much better fits are observed with the peptide bound structures, particularly the complexes with smMLCK peptide, CaMKI peptide and eNOS peptide The s c value, the molecular correlation time, is calculated from the fit and can be defined as the time taken for the molecule to rotate through 1 radian or to translate through its own length D para ⁄ D perp is the ratio between the length of the longs axis, z, and the length of the shorter, equal x and y axes.
CaM ⁄ peptide complex PDB file
v 2
Ca 2+ ⁄ CaM ⁄ PhK5
v 2
Ca 2+ ⁄ CaM sc(ns) Dpara⁄ D perp
Trang 7relatives 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 Ca2+/CaM
PhKc interacts with the CaM Ca2+ sensor through a
C-terminal extension to the kinase domain of 90
res-idues Previous studies on PhKc identified two regions
in this C-terminal extension, PhK5 and PhK13, that
were able to inhibit Ca2+⁄ CaM-mediated activation of
MLCK Further studies also indicated that there may
be substantial differences in the binding of PhK5 and
PhK13 to CaM based on small angle X-ray scattering
studies and CD measurements The present studies on
the PhK13 and PhK5 peptide complexes have
substan-tiated these significant differences in their interactions
with CaM Analytical gel filtration and NMR chemical
shift perturbation studies indicated no detectable
inter-action between Ca2+⁄ CaM and PhK13 In contrast,
PhK5 formed a robust complex with Ca2+⁄ CaM under
conditions of gel filtration and the addition of PhK5
caused a large number of NMR chemical shift changes
in Ca2+⁄ CaM
Taken together, these data demonstrate that the
region of PhKc that is delineated by the PhK13
pep-tide does not interact directly with Ca2+⁄ CaM under
the conditions used in this study The ability of
this peptide to inhibit the MLCK activity was well
established and it was demonstrated to bind to CaM
in a Ca2+-dependent manner in gel mobility assays [18] However the small angle X-ray and neutron scattering data indicated that the PhK⁄ Ca2+⁄ 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 Ca2+ 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 Ca2+/CaM?
Both X-ray scattering and fluorescence anisotropy studies of the Ca2+⁄ CaM ⁄ PhK5 complex have indica-ted that this complex has a similar structure to the complexes of Ca2+⁄ CaM with peptides derived from other CaM-dependent enzymes Our analytical gel fil-tration studies and NMR tifil-tration experiments are also
Fig 6 (A) A comparison the three structures that show highest correlation with Ca2+⁄ CaM ⁄ PhK5, that includes Ca 2+ ⁄ CaM ⁄ eNOS in blue (PDBid 1niw), Ca 2+ ⁄ CaM ⁄ smMLCK in pink (PDBid 1cdl) and Ca 2+ ⁄ CaM ⁄ CaMKI in orange (PDBid 1mxe) The Ca 2+ ions are indicated as cyan spheres and the smMLCK peptide is shown as a white helix Structures are aligned using the C-terminal domain of CaM (residues 86–146) The orientation presented here is similar to that shown in Fig 1 (B) A similar alignment showing structures that show poorer correlations with Ca 2+ ⁄ CaM ⁄ PhK5 Ca 2+ ⁄ 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).
Trang 8consistent with a compact structure of the Ca2+⁄
CaM⁄ PhK5 complex The spectrum of Ca2+⁄ CaM ⁄
PhK5 is dramatically different from that of the
unbound Ca2+⁄ 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 Ca2+⁄ CaM
and Ca2+⁄ CaM ⁄ PhK5 show a significant decrease
upon PhK5 binding as well as a reduction in the
vari-ation of R1 in secondary structural elements,
suggest-ing that the two domains no longer tumble
independently and that CaM assumes a more compact
structure in the bound form
Analysis of the R1 and R2 relaxation times for
each assigned peak in the Ca2+⁄ CaM and Ca2+⁄
CaM⁄ PhK5 samples supported the notion of a
significant conformational change The R1 and R2
relaxation times for a particular nucleus are
depend-ent on its reoridepend-entation in solution and can give
information about the conformational flexibility of
different protein regions For relatively rigid residues
the relaxation times will reflect the overall motion
of the molecule as it tumbles in solution These
motions, on the nanosecond to picosecond timescale, will have reciprocal effects on the R1 and R2 relaxa-tion times, although this relarelaxa-tionship breaks down when the residues are subject to conformational exchange (Rex) [29,30] Thus for residues in secon-dary structure elements, such as a-helices, the R1 and R2 relaxation times can give information on the 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 Ca2+⁄ CaM and the
Ca2+⁄ 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 Ca2+⁄ 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].
Trang 9How similar is the Ca2+/CaM/PhK5 complex to
other Ca2+/CaM/peptide complexes?
Classical Ca2+⁄ CaM ⁄ peptide complexes are largely
similar in their formation of a closed, compact CaM
structure around a helical amphipathic peptide
How-ever, many of these complexes also show distinct
con-formations, not only in the mode of ligand binding
but also in the relative orientation of the two CaM
domains These differences are facilitated by the
flexi-bility of the linker between the two domains as well as
a certain degree of conformational flexibility in the
two EF hand motifs of each CaM domain The
pres-ence of a relatively large number of Ca2+⁄ CaM ⁄
pep-tide complexes in the PDB opens up the possibility of
using these structures to perform structural
compari-sons against unknown structures Indeed, comparative
studies have previously been performed using NMR
NOE data or residual dipolar couplings [31,32] to
identify structural similarities between different CaM⁄
peptide complexes
The study presented here uses rotation anisotropy
of CaM and its effects on the relaxation rates of
N–H bond vectors of the CaM backbone Each of
the Ca2+⁄ CaM ⁄ peptide structures was used as an
input model in lieu of a Ca2+⁄ 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
shows an unusual pattern of binding in that its
anchor residues are separated by only one residue A
1–14 motif is compatible with the PhK5 sequence,
with L345 and V358 serving as anchor residues
With this predicted motif a strong similarity between
the PhK5 and CaMKI peptide sequences becomes
more apparent (Fig 1) Of the PDB files tested, the
CaM⁄ CaMKI structure was the best fit to the
CaM⁄ PhK5 relaxation data (Table 2) On the other
hand, PhK5 does contain one large hydrophobic
residue, Trp357, that is perhaps more likely to serve
as anchor reside than Val358 Interestingly, the three peptides from MLCK, CaMKI and eNOS each have
a large aromatic residue in the N-terminal part of the peptide that binds in the with the C-terminal domain in CaM However in PhK5 Trp357 is found
at the C-terminal end of the peptide This suggests that either PhK5 might bind with Trp357 in the N-terminal site in CaM, or perhaps could bind in the opposite orientation to incorporate the Trp into the larger C-terminal domain binding site
In summary, PhKc is known to bind to CaM via its C-terminal extension The data presented here demon-strate that PhK5 interacts directly with Ca2+⁄ CaM and that PhK13 does not The PhK5 interaction causes
a large conformational change to occur in Ca2+⁄ CaM that produces a classical compact Ca2+⁄ CaM ⁄ peptide complex Analysis of the NMR relaxation data sug-gests that the Ca2+⁄ 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 37C 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 4C CaCl2was added to the supernatant to a final concentration of 5 mm and the sample was then applied to a 50-mL phenyl seph-arose column pre-equilibrated in 50 mm Tris⁄ HCl pH 7.5,
5 mm CaCl2, 100 mm NaCl Two steps were carried out to remove the majority of contaminants from the protein sam-ple, a low Ca2+wash with 50 mm Tris⁄ HCl pH 7.5, 0.1 mm CaCl2, 100 mm NaCl and then a high salt wash with 50 mm Tris⁄ HCl pH 7.5, 0.1 mm CaCl2, 0.5 m NaCl Finally, CaM was eluted from the column using a Ca2+-free buffer con-taining 0.2 mm EDTA and 50 mm Tris⁄ HCl pH 7.5
To produce 15N labelled protein for NMR experiments, the cells were grown on M9 medium instead of Luria– Bertani broth and supplemented with 1.5 lm thiamine and
15
NH4Cl as sole nitrogen source Purification was carried out using the standard protocol and the pure protein was
Trang 10concentrated to 1 mm 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 H2O and dialysed against water at 4C to
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 CaCl2, 100 mm KCl The
concentration of CaM and the peptides was determined by
absorbance at 280 nm using calculated e-values Peptides
were mixed with 30 nmol CaM in a 1 : 1 molar ratio and
then analysed by gel filtration chromatography The peaks
were concentrated using 3 lL of Strataclean protein
bind-ing beads (Stratagene, Amsterdam, the Netherlands) and
then analysed on a 10–20% gradient Tris⁄ tricine
polyacryl-amide gel (BioRad, Hercules, CA, USA)
CaM/peptide titrations
Samples for NMR titrations contained 5 mm CaCl2, 20 mm
deuterated Tris⁄ HCl pH 6.5, 5% (v ⁄ v) D2O in a 400 lL
vol-ume Titrations were carried out using a spectrometer with a
magnet (Oxford Instruments) operating with a1H frequency
of 500 MHz The sample was maintained at a temperature
of 25C Gradient enhanced HSQC spectra were collected
with a sweep width of 16 p.p.m in the 1H dimension
and 40 p.p.m in the 15N dimension with the 1H carrier
frequency set to 4.74 p.p.m and the15N carrier frequency
set to 120 p.p.m For each experiment 32 scans were taken
with 128 increments in the nitrogen dimension The
Ca2+⁄ 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 of15N relaxation data
NMR experiments were performed on spectrometers oper-ating at 1H frequencies of 600 MHz at 25C Backbone
15
N relaxation parameters, comprising the rates of 15N transverse (R2) and longitudinal (R1) relaxation were measured using previously described experimental protocols [35] 15N R1 and R2 relaxation data for Ca2+⁄ CaM and for the Ca2+⁄ CaM ⁄ PhK5 complex were obtained by recording a series of gradient enhanced two-dimensional HSQC spectra with a series of T1 (20, 400, 600, 800, 1000,
1200 and 1400 ms) and T2 (8.6, 60.5, 86.4, 103.7, 129.6, 172.8 and 216 ms) delays For the Ca2+⁄ CaM sample, a 1.4 mm sample of CaM was made up in 20 mm deuterated Tris⁄ HCl pH 6.5, 100 mm KCl, 10 mm CaCl2 and 5% (v⁄ v) D2O For the Ca2+⁄ CaM ⁄ PhK5 complex 350 lL of 1.4 mm CaM was diluted into 6 mL of the NMR buffer and mixed with 0.5 lmol PhK5 peptide The complex was concentrated using a centricon filter unit with a 3 kDa cut-off to a final concentration of 0.75 mm Data were processed with felix 2.3 The peaks were assigned by com-parison with the previous assignments for CaM [25] using the program sparky for assignment and measurement of peak intensities
The longitudinal and transverse relaxation rates (R1¼
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 Ca2+⁄ CaM data all eight helices from the CaM structure were represented, however, in the Ca2+⁄ 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 Ca2+⁄ CaM ⁄ peptide structures were selected from the PDB along with the collapsed, peptide-free structure of
Ca2+⁄ CaM (PDBid 1prw) [37] as input models for the calculations in rotdif The structures included complexes