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J. Biol. Chem., Vol. 277, Issue 15, 12988-12997, April 12, 2002
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From the
Received for publication, July 20, 2001, and in revised form, December 4, 2001
Protein kinase C (PKC) isozymes
comprise a family of related enzymes that play a central role in many
intracellular eukaryotic signaling events. Isozyme specificity is
mediated by association of each PKC isozyme with specific anchoring
proteins, termed RACKs. The C2 domain of Protein kinase C (PKC)1
is a family of protein kinases that undergo translocation from one
intracellular compartment to another when activated by
neurotransmitters, hormones, and growth factors. Most members of this
family are activated by phosphatidylserine (PS), diacylglycerol (DG),
and, to different extents, by other lipid second messengers and
Ca2+ ions (1, 2). There are at least three subfamilies of
PKCs, classified according to their homology and sensitivity to
activators (2-4). The C2 domain is found also in proteins other than PKC (4). Because
many of these proteins bind lipids and particularly PS in a
calcium-dependent manner, it was obvious to suggest that the calcium- and PS-binding sites reside within this domain (6). Sequence alignment studies (4) revealed that the C2 domains exhibit two
types of topologies (type I and II), but all have a We found that the C2 domain of cPKC mediates at least some of the
direct protein-protein interactions between cPKC and RACKs (15). The
RACK1-binding site of Structures of the C2 domain obtained by x-ray diffraction and NMR
spectroscopy (18, 19) demonstrate that these RACK-binding sequences in
Using the same logic, we suggested that translocation
activators should be agonists of PKC function, independent of the
amount of second messengers that normally activate PKC (21). We
predicted that such peptide agonists would bind the unstable transition state between inactive and activated PKC, causing exposure of the
catalytic site and the RACK-binding site and thus enabling the
anchoring of the enzyme to RACKs (Fig. 1). Indeed, a
Because All calculations were carried out using AMBER 5.0 (29), a
program commonly used for molecular dynamics simulations of proteins (27, 30-34). The standard AMBER 1994 force field (35) was used. The
x-ray structure of the C2 domain of rat The solvent molecules were equilibrated initially by energy
minimization and subsequently by performing 15 ps of MD. After energy
minimization of the whole system (protein + water + counterions), MD
trajectories were calculated. Temperature was initially increased from
0 to 300 K (performing three MD runs of 3 ps each at 100, 200, and 300 K, respectively) and then maintained constant for the whole simulation
time, coupling the protein to a thermal bath (40). The SHAKE algorithm
(41-43) was applied on all bonds during all the MD runs. However, a
time-step of 1.5 fs could be used only in the calculation of the
trajectory for
Molecular Dynamics Characterization of the C2 Domain of Protein
Kinase C
*
§,¶,
, and
Centro di Risonanze Magnetiche, University
of Florence, 50019 Sesto Fiorentino, Florence, Italy and the
Department of Molecular Pharmacology, Stanford University School
of Medicine, Stanford, California 94305
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
PKC contains at least part
of the RACK-binding sites. Because the C2 domain contains also a
RACK-like sequence (termed pseudo-RACK), it was proposed that this
pseudo-RACK site mediates intramolecular interaction with one of the
RACK-binding sites in the C2 domain itself, stabilizing the inactive
conformation of PKC. PKC depends on calcium for its activation,
and the C2 domain contains the calcium-binding sites. The x-ray
structure of the C2 domain of PKC shows that three Ca2+
ions can be coordinated by two opposing loops at one end of the domain.
Starting from this x-ray structure, we have performed molecular
dynamics (MD) calculations on the C2 domain of PKC bound to three
Ca2+ ions, to two Ca2+ ions, and in the
Ca2+-free state, in order to analyze the effect of calcium
on the RACK-binding sites and the pseudo-RACK sites, as well as on the loops that constitute the binding site for the Ca2+ ions.
The results show that calcium stabilizes the -sandwich structure of
the C2 domain and thus affects two of the three RACK-binding sites
within the C2 domain. Also, the interactions between the third
RACK-binding site and the pseudo-RACK site are not notably modified by
the removal of Ca2+ ions. On that basis, we predict that
the pseudo-RACK site within the C2 domain masks a RACK-binding site in
another domain of PKC, possibly the V5 domain. Finally, the MD
modeling shows that two Ca2+ ions are able to interact with
two molecules of O-phospho-L-serine. These data
suggest that Ca2+ ions may be directly involved in PKC
binding to phosphatidylserine, an acidic lipid located exclusively on
the cytoplasmic face of membranes, that is required for PKC activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
PKC belongs to the so-called classic PKC
subfamily, or cPKC (
, I, II, and 
kinases). The members of
this class contain four conserved domains (C1, C2, C3, and C4)
inter-spaced with isozyme-unique (variable or V) domains. The I and
II PKC isozymes are splice products of the same gene and therefore
differ only in their C-terminal V5 domain (5).
-sandwich fold
composed of four -strands in each face of the structure (7). Based
on this homology, the C2 domain was suggested to contain the calcium
switch required for localization to the membrane (8). However,
immunofluorescence studies did not agree with simple localization of
the activated PKC isozymes to the plasma membrane. We found that
activated PKC isozymes are each localized to unique intracellular sites
and, therefore, suggested that this unique localization is mediated by
their selective interactions with specific anchoring proteins, termed
Receptors for Activated C-Kinase (RACKs, see Fig. 1) (9-14). We
subsequently demonstrated that the unique cellular functions of PKC
isozymes are indeed dependent on the binding of each isozyme to its
corresponding RACK, bringing the active PKC within a close proximity to
particular subsets of substrates and away from others (3).
PKC-C2 domain (Fig. 2) was identified by
sequence homology analysis with synaptotagmin, another C2
domain-containing and calcium-dependent PS-binding protein, also called p65 (15). We reasoned that, because the C2 domain of
synaptotagmin also binds to RACK1 (albeit with a 100-fold lower affinity) (15), sequences most conserved between the two domains in the
PKC-C2 domain would contain the RACK1-binding sites. Peptides corresponding to amino acids 186-198 (MDPNGLSDPYVKL, C2-2 site; red in Fig. 2), 209-216 (KQKTKTIK, C2-1 site;
orange in Fig. 2), and 218-226 (SLNPEWNET, C2-4 site;
blue in Fig. 2) were predicted to contain the RACK-binding
sites (16, 17).
PKC-C2 are located on three exposed -strands in the domain (Fig.
2B). Peptides corresponding to these three -strands specifically inhibit activation-induced translocation of the
C2-containing cPKC isozymes and their functions in cells (16, 20). As
predicted, a peptide derived from a non-conserved region of the C2
domain (C2-3, amino acids 201-207, IPDPKSE; yellow in
Fig. 2), which is not adjacent to the RACK-binding strands, had no
effect on PKC binding to RACK, or PKC translocation and function in
cells (16). It therefore appears that the C2 domain of PKC has a dual role. Upon activation, the domain binds PS in a
calcium-dependent manner, which results in membrane binding
of the enzyme. In addition, this domain participates in specific
protein-protein interactions with the corresponding RACK,
bringing the activated isozyme to a close proximity with a subset of
substrates and away from others, thus mediating functional specificity
of this family of enzymes.
PKC-derived peptide, termed pseudo-RACK1 peptide or
RACK because of its homology to RACK1 (amino acids 241-246
within the C2 domain, SVEIWD, green in Fig. 2), binds to
PKC, activates it in the absence of PS and calcium in
vitro, and acts as a selective agonist of PKC function in
vivo (22). We proposed that the RACK site in PKC is an
autoregulatory site (17, 21). When PKC is in an inactive conformation, the RACK site interacts with the RACK1-binding site; activation of PKC exposes the RACK1-binding site, enabling the
association of the enzyme with its anchoring RACK (22). A model for
this interaction is shown in Fig. 1, and
the relative position of the RACK1-binding sites within C2 and the
RACK site are indicated in the primary structure and in the
secondary structure (Fig. 2A)
as well as in the tertiary structure of the domain (Fig. 2B).
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Fig. 1.
Model of PKC activation.
Inactive PKC is depicted as a folded rod with the pseudo-substrate
autoinhibitory sequence associated with the substrate site in the
catalytic domain, as well as with the pseudo-RACK sequence associated
with the RACK-binding site. In the presence of PKC activators
(PS/DG/calcium), the rod unfolds and the RACK-binding and
substrate-binding sites become exposed, resulting in binding of PKC to
its RACK and to its substrate. The pseudo-RACK peptide is thought to
bind the unstable transition state between the inactive and active
forms, shifting the equilibrium between the two conformations toward
the active (open) one.
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Fig. 2.
Sequence and secondary structure
( strands, green; helices,
orange) (A) and ribbon diagram
(B) of PKC-C2 from x-ray
structure showing the position of C2-2
(red), C2-3
(yellow), C2-1
(orange), C2-4
(blue), and
RACK (green)
sites. The Ca2+ ions are depicted as
orange (site II), red (site III), and
purple (IV) spheres. The main loops of the domain
are labeled.
PKC binds to RACK1 upon activation with calcium and
PS, we studied the conformational changes in the C2 RACK-binding sites
in the absence and presence of these factors using computer simulation
techniques of molecular dynamics (MD). MD can provide an insight into
the structure and dynamics of proteins. Although such simulations are
limited by the number of conformational spaces that can be sampled,
these techniques are quite effective in monitoring even subtle
structural changes and variations in residue-residue interactions, also
when the comparison of very similar systems is concerned (23-27); in
particular, the effect of calcium ions on protein structure was
successfully evaluated on peroxidases (28). Here, using MD simulations
of the C2 domain of PKC, we examined the effects of calcium binding
on the RACK1-binding sites and RACK site within the C2 domain.
Activated PKC has been reported to bind two (18) or three (19)
Ca2+ ions in the C2 domain. Therefore, MD calculations were
performed on the C2 domain of PKC with three Ca2+ ions
(hereafter referred to as PKC-3Ca), with two Ca2+ ions
(PKC-2Ca hereafter), and with no Ca2+ ions (PKC
hereafter), using the available x-ray structure as the starting model
(19). Because a direct correlation between calcium and PS binding has
also been demonstrated, a simple model was built to examine the
possibility that Ca2+ ions bound to the domain are directly
involved in PS binding. Three molecules of
O-phospho-L-serine, which mimic the headgroup of
PS, were positioned near the Ca2+ ions in the MD structure
of PKC-3Ca. An MD simulation was also performed on this complex
(PKC-3Ca·PS hereafter). Our MD studies suggest that, although
conformational changes occur within the C2 domain due to calcium and PS
binding, they are unlikely to mediate the disruption of interaction of
the RACK site with a RACK-binding site within the C2 domain.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
PKC complexed with three
Ca2+ ions (2.7-Å resolution, PDB entry 1A25) was used to
model the starting structures (19). Although the crystal structure contains two molecules of PKC-C2 related by a dyad axis in the asymmetric unit (19), the modeling was conducted on a single molecule.
This domain contains 132 residues (157-288 of the complete sequence),
corresponding to 1088 heavy atoms. The water molecules found in the
crystal structure were excluded, following the standard procedure for
protein simulations in explicit water described in the AMBER manual
(29). Hydrogen atoms, which are missing in the 1A25 PDB file, were
added through the EDIT module of AMBER, resulting in 2173 atoms in the
native structure. The Ca2+ ions were added to the standard
AMBER residue data base by the PREP module of AMBER: They were treated
as divalent cations, with a van der Waals radius of 1.60 Å, and 
(i.e. 6-12 potential well depth) of 0.1 kcal
mol
1, based on previous MD calculations dealing with the
effect of calcium ions on the structure of peroxidases (28). The
starting positions of the Ca2+ ions were set according to
their coordinates in the x-ray structure and were labeled II, III, and
IV, following Sutton and Sprang notation (19). For PKC-2Ca, calcium
II and III were chosen, on the basis of the model of Ca2+
binding by C2 domains depicted by Shao et al. in Ref. 18: In that work, two calcium ions were found to be bound to the C2 domain through NMR spectroscopy. No direct bond was set between the calcium ions and any protein groups, and no distance constraints were introduced to prevent a bias in the modeling, i.e. the
calcium ions are free to leave the protein. A shell of TIP3P
(36) water molecules extending 10 Å in every direction from the
protein surface was created using the SOL option of the EDIT module of
AMBER, resulting in the introduction of about 2100 water molecules for all the model systems. Proper counterions were generated by the CION
program of AMBER and positioned near free charged surface residues of
the protein to achieve an overall charge of zero on each system,
ensuring that electrostatic interactions were not broken. A 10-Å
cut-off for the evaluation of the non-bonded interactions was used,
resulting in the evaluation of 2.5-2.6 × 106 pair
interactions. Because proteins are systems where long-range electrostatics are expected to play an important role in determining molecular conformational energies and structures, the choice of the
cut-off for non-bonded interactions is significantly important for the
quality of the simulation. The value of 10 Å adopted here was shown
(28, 32, 33, 37-39) to be a good compromise between the requirement
for accurate treatment of long-range electrostatics and the requirement
for a reasonable calculation time, of which the evaluation of
non-bonded interactions is by far the determinant part.
PKC, whereas a time-step of 1.0 fs was used both for
PKC-3Ca and PKC-2Ca, due to instability of the trajectory in the
initial steps of the simulations. The pair list was updated every 20 steps, and coordinates and energy values were collected every 100 steps
for further analysis. The simulations were performed for 1060 ps, and
the final 1000 ps were taken for the analysis. This time frame was
chosen because PKC system reached stabilization, i.e. the
structure reached an average constant
r.m.s.d.2 with respect to the
starting structure, after the initial 60 ps (Fig. 3). PKC-3Ca and
PKC-2Ca equilibrated faster, in about 10 ps (Fig.
3). The average structures were
calculated by averaging the coordinates at the various steps of the
trajectories. The average structures were then subjected to energy
minimization and subsequently analyzed by the program PROCHECK (44) to
confirm the stereochemical quality of the model structures.
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Fig. 3.
r.m.s. deviations of the backbone atoms with
respect to the starting structure as a function of simulation time
for PKC-3Ca (light
gray), PKC-2Ca (dark
gray), and PKC
(black).
In the PKC-3Ca·PS model, three molecules of
O-phospho-L-serine were built by the PREP module
of AMBER and initially positioned in proximity of the Ca2+
ions of PKC-3Ca average structure, to evaluate the possibility of a
direct calcium·PS interaction (45). Nevertheless, the initial calcium·PS distance was set larger than coordination distance, to
prevent a bias in the modeling of such interaction. The internal coordinates and the parameters for
O-phospho-L-serine were obtained from data for
serine and PO2 groups already present in AMBER libraries. The procedure for MD modeling of this complex was performed as described above, consisting of solvation, energy minimization, heating,
and dynamics. The simulation time was 600 ps, with 1.0 fs as the
time-step.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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PKC-3Ca--
The MD average structure, which is shown in Fig.
4A, is very similar to the
original x-ray structure (19), with average r.m.s.d. values of 0.85 Å for the backbone and 1.46 Å for the heavy atoms. The r.m.s.d. values
per residue are shown in Fig. 5A. All the secondary
structure elements found in the crystal structure are also present
after MD, as shown in Fig. 6 (top
scheme). The average fluctuation is 0.45 ± 0.05 Å for the
backbone and 0.52 ± 0.21 Å for the heavy atoms. These values,
which indicate the range of fluctuations of the protein over the time
of simulation, are rather low and suggest that the molecule is quite
rigid. The plot in Fig. 6 reports the average heavy atom fluctuation
per residue; the residues with the highest relative mobility correspond to the main loops of the domain, i.e. loop 2-3 (residues
182-193), loop 3-4 (residues 202-209), loop 4-5 (residues
212-221), and loop 6-7 (residues 247-253). -Helix 2 (residues
280-283) also shows relatively large fluctuations.
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The coordination pattern of Ca2+ ions in the MD structure
of PKC-3Ca is shown in Fig. 7 and is
in good agreement with the x-ray structure (19), validating our
modeling procedure. That coordination is maintained during the whole
PKC-3Ca simulation. Because neither direct bonds nor distance
constraints were set between Ca2+ ions and any protein
groups, we infer that these binding sites for calcium are highly
stable. The coordination sphere is completed with water molecules. Site
IV, which is hexacoordinate in the crystal structure (19), appears
heptacoordinate in the present model, with the carbonyl oxygen of
Asn253 as the seventh ligand.
|
PKC-3Ca·PS--
Phosphatidylserine (PS) is an acidic lipid
located exclusively on the cytoplasmic face of biological membranes and
is required for PKC activation (1, 2). The exact mode of interaction of
PS with the enzyme is unknown; however, it has been proposed that
Ca2+ ions bound to the protein may be involved in PS
binding by providing a positively charged site for the negatively
charged headgroup of PS (45). To mimic PS, we have chosen a simpler
molecule bearing the same headgroup as PS, i.e.
O-phospho-L-serine, which was also used in the
determination of the crystal structure (19). As described under
"Materials and Methods," in this simulation three molecules of
O-phospho-L-serine (PS1, PS2, and PS3) were
initially positioned close to the Ca2+ ions in the average
structure of PKC-3Ca. After 50 ps, the structure became stable for
the remainder of 550 ps of simulation. The snapshots along this
simulation (see Fig. 8) show that two
molecules of O-phospho-L-serine (PS1 and PS2)
are able to substitute the water molecules in the coordination sphere
of calcium III and IV (see Fig. 7). In particular, PS2 binds calcium IV
in the equatorial plane through an oxygen atom of its phosphate group
and PS1 binds to both calcium III and calcium IV in an axial position,
through the oxygen atoms of its carboxylate group. Conversely, the
third molecule of O-phospho-L-serine (PS3),
which binds calcium II for the initial 450 ps of the simulation,
eventually moves away, suggesting that this interaction is not stable.
Furthermore, the three PS-like molecules establish instantaneous,
probably nonspecific, interactions with some of the residues belonging
to loop 6-7, i.e. Leu249, Thr250,
Ser251, and Arg252. Though none of these
interactions are stable along the entire trajectory, altogether they
appreciably reduce the mobility of loop 6-7: the average value of the
fluctuations for residues 247-253 is 0.64 Å, compared with 1.00 Å for the same segment in the PKC-3Ca system, therefore, in the
absence of PS. Although this simulation does not completely mimic the
PKC interaction with the membrane bilayer, it further supports the
previous findings that calcium may be directly involved in binding of
PS (45, 46).
|
PKC-2Ca--
The average MD structure of PKC with two
calcium ions is shown in Fig. 4B. Removal of calcium IV from
PKC-3Ca increases the average r.m.s.d. relative to the crystal
structure, with 0.98 Å of r.m.s.d. for the backbone and 1.75 Å for
the heavy atoms, respectively. The values per residue are shown in Fig.
5B. Comparison of Figs. 4A and 4B
shows that the effect of removing this Ca2+ ion is mainly
observed on loop 6-7, which is involved in calcium coordination, and
on loop 3-4, probably due to the shortening of -strand 4 (from
209-211 to 210-211). Additional changes in secondary structures were
noted: -strands 5 and 8 are shortened (from 222-230 to 225-230 and
from 271-276 to 273-276, respectively), and the first helix (amino
acids 233-238) is missing after removal of calcium IV. In contrast,
the second helix (1) becomes longer (from 265-268 in PKC-3Ca to
263-268 in PKC-2Ca; see Fig. 6, top). The average
fluctuation is 0.51 ± 0.09 Å for the backbone and 0.58 ± 0.27 Å for the heavy atoms. Fig. 6 reports the average heavy atom
fluctuation per residue. That figure and analysis of Fig. 4 suggest
that the slight increase in the mobility of PKC-2Ca as compared with
PKC-3Ca is due to a larger flexibility of loops 6-7 and 3-4. The
removal of calcium IV results in an increased flexibility of loop 6-7,
because this calcium is coordinated exclusively by residues in loop
6-7 (see Fig. 7). The removal of calcium also affects loop 3-4
indirectly, because it results in a shortening of -strand 4.
PKC--
The MD average structure of the calcium-free form is
reported in Fig. 4C. This figure shows that, also in the
absence of Ca2+ ions, the global fold of the domain is
maintained. However, conformations of certain regions are notably
different from the calcium-bound structures.
The average r.m.s.d. of PKC as compared with the crystal structure
is higher than in the calcium-bound states; it is 1.65 Å for the
backbone and 2.44 Å for the heavy atoms. The largest r.m.s.d. values
are found for loops 3-4 and 6-7 (see Fig. 5C), supporting
the observation that these loops are the regions most affected by the
progressive removal of calcium. The most striking effect of calcium
removal is the disappearance of the short -strand 4 (Figs. 4 and 6,
top). Other secondary structure elements that differ from
PKC-2Ca (see Fig. 6, top) include the interruption of
-strand 3 in the middle (residues 198-199), shortening of -strand 7 (from 254-262 to 259-262) and lengthening of -strand 8 to the length found in the PKC-3Ca state (271-276). An increase of mobility for loop 3-4 is also induced, as shown in Fig. 6. However,
in the absence of calcium, the domain remains quite rigid: The average
fluctuation is 0.57 ± 0.08 Å for the backbone and 0.63 ± 0.32 Å for the heavy atoms (these values are only slightly larger than
the values found for PKC-2Ca and PKC-3Ca).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The present MD simulations allowed the analysis of the effects of
Ca2+ ions on the structure of the C2 domain of PKC. We
focused mainly on the regions of this domain that have a major role in
previously reported biological functions (16-18, 20, 22). These
regions are indicated in Fig. 2 and Table
I.
|
The simulations were performed on the C2 domain of PKC bound to
three Ca2+ ions (with and without the PS-like headgroups),
to two Ca2+ ions, or in the Ca2+-free state.
Using this analysis, we evaluated (i) the effect of Ca2+ on
pseudo-RACK1 and RACK1-binding sites, (ii) the effect of
Ca2+ on the regions of the domain involved in
Ca2+ coordination, and (iii) the possible role of calcium
in PS binding by the C2 domain.
Effect of Calcium on Pseudo-RACK1 and C2-2 Sites--
As
described in the introduction, the pseudo-RACK1 sequence was predicted
to be an autoregulatory site for PKC (21). We suggested that, when
PKC is in an inactive conformation, pseudo-RACK1 site interacts with
the RACK1-binding site within the enzyme (Fig. 1). In the active form,
this intramolecular interaction is interrupted, rendering the
RACK1-binding site available for the interaction with the RACK ((22)
and Fig. 1). According to the structure of the C2 domain (see Fig.
2B), it is evident that, among the three identified
RACK1-binding sites (16), only the C2-2 (red) could interact with the pseudo-RACK1 site (green). The
pseudo-RACK1 site is part of strand 6, and the spatial organization of
the -sheet to which it belongs places it next and anti-parallel to residues 194-199 of strand 3. In this position, three pairs of hydrogen bonds are present between the amide proton and the carbonyl oxygen of residues Lys199-Ser241,
Lys197-Glu243, and
Tyr195-Trp245 (Fig.
9).
|
Regardless of the presence or absence of calcium, the distances between
the -strands 194-199 and 241-246, the two central strands of a
four-stranded -sheet, remain constant in the three modeled
structures. The only exception to this general behavior is the distance
between Lys197 and Glu243. In PKC-3Ca and in
PKC-2Ca this distance is constant, whereas in the absence of calcium
(PKC) this distance fluctuates. These fluctuations, which are in the
range of 1-1.5 Å, could have some functional relevance, because
Glu243 constitutes the crucial difference between the
pseudo-RACK1 sequence in PKC (SVEIWD) and the
corresponding site in RACK1 (SIKIWD). The presence or
absence of Ca2+ ions does not significantly affect the
hydrogen bonds between the C2-2 and the pseudo-RACK1 segments: All
the hydrogen bonds are maintained during the MD simulations on the
various trajectories, thus stabilizing the interaction between these
strands in the -sandwich.
Interesting information is obtained by monitoring the behavior of the
side chains (Fig. 10). The fluctuations
of several distances during the simulations suggest that, in the
presence of three Ca2+ ions, there is a stable conformation
in which no interaction is present between the side chains of the
-strands 3 and 6. In particular, the amine group of
Lys199, which in PKC-2Ca and in PKC interacts with
the carboxylic group of Glu243 and, weakly, with the
hydroxyl group of Ser241, does not interact with these
groups in PKC-3Ca. Furthermore, an H-bond is formed between
Glu243 and the amide group of Gln280, which
belongs to helix 3; the distance between
Glu243-Gln280 is quite constant in the presence
of three Ca2+ ions, whereas it undergoes large fluctuations
in PKC-2Ca and in PKC. These findings may indicate that the third
Ca2+ ion stabilizes, at least partially, helix 3 in a
position close to pseudo-RACK1, whereas two Ca2+ ions are
not sufficient to do that. Fig. 10 shows a comparison between the
average structures in which the changes involving residues
Lys199, Glu243, and Gln280, induced
by calcium binding, are shown.
|
In conclusion, the interaction between pseudo-RACK1 and C2-2 sites
is a very stable strand·strand interaction in the -sandwich, which
is not markedly altered by the removal of Ca2+ ions. If the
activation of PKC requires the dissociation of the intramolecular
interaction between these two sites, it seems highly unlikely that this
dissociation involves the disruption of the -sandwich structure, and
calcium binding is unlikely to result in such a dramatic conformational
change. Instead, the intramolecular interaction between the
pseudo-RACK1 site and a RACK-binding site may involve side-chain to
side-chain interactions; removal of one Ca2+ ion induces an
interaction between Glu243 (part of pseudo-RACK1) and
Lys199 (immediately after C2-2). Therefore, it is
possible that calcium binding to the third site results in disruption
of intramolecular interactions between the RACK-binding site and the
pseudo-RACK1 site. In addition, the surface exposed to the solvent is
unaltered (Table II), suggesting that
calcium binding does not change the surface characteristics of this
site, and the potential interaction with RACK1. Together, these data
suggest that the interaction between C2-2 and pseudo-RACK1 do not
constitute a major intramolecular interaction site that is disrupted in
PKC activation by calcium. Instead, we favor the possibility that the
intramolecular interaction involves the pseudo-RACK1 site with a
RACK-binding site outside the C2 domain. This possibility is further
supported by the finding of a RACK1-binding site within the V5 region
of PKC (47). Examination of this possibility awaits the availability
of structural information on the intact enzyme.
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Effect of Calcium on the Other RACK1-binding Sites--
The
C2-1 segment, which contains a part of the RACK1-binding site (16)
is a very basic region, on which the removal of Ca2+ ions
has the largest effect. As noted above, the short -strand 4, which
is formed by residues 209-211 in PKC-3Ca, is shortened (210-211)
in PKC-2Ca and absent in PKC (Fig. 6, top). This loss in the secondary structure is associated to an appreciable increase in
the fluctuations around this region (Fig. 6). Thus, the stabilization of a certain conformation of the C2-1 segment is clearly a
calcium-induced effect and may be related to the activation process.
The presence or the absence of calcium does not affect the extent of
the solvent-exposed surface (Table II) but has significant effect on
its order as a consequence of reduced mobility. This increased rigidity
may be presumably necessary for the recognition by the RACK.
Similar to C2-1, the removal of Ca2+ ions determines a
decrease in the secondary structure elements of the C2-4 segment
(Fig. 6, top), although the surface exposed to the solvent
is not significantly modified (Table II). Removal of just a single
Ca2+ ion produces the shortening of -strand 5, where the
first three residues (222-224) are in a random coil conformation both
in the presence of two Ca2+ ions (PKC-2Ca) and in the
calcium-free form (PKC). Nevertheless, the fluctuations in these
regions are limited in all three cases (Fig. 5). From these structural
changes, we can infer that the C2-4 segment requires the binding of
three Ca2+ ions to stabilize the proper conformation for
binding to RACK.
Effect of Calcium on Loops 2-3 and 6-7--
Loops 2-3 and 6-7
are involved in calcium binding in the C2 domain of PKC (19, 48). As
previously predicted (48), the main effect of removal of
Ca2+ ions is to increase the distance between these loops.
The removal of one Ca2+ ion does not significantly modify
this distance, whereas in the calcium-free state (PKC) the
electrostatic repulsion among the acidic residues, deputed to bind
calcium, makes loop 6-7 move away from loop 2-3. Comparison of
PKC-2Ca and PKC structures (Fig. 4, B and
C) shows that, whereas loop 2-3 does not change significantly its position, the position of loop 6-7 is notably different. This is consistent with the larger fluctuations of loop 6-7
(1.5-2.1 Å, see Fig. 6) as compared with loop 2-3 (0.5-0.8 Å, see Fig. 6). The motions of loop 6-7 produce the disruption of the
first half of strand 7 and its shortening from 254-262 in PKC-2Ca
to 259-262 in PKC. Furthermore, the separation of the two loops is
associated with a reorganization of some hydrogen bonds in this region.
Specifically, the H-bonds between the backbone hydrogen of
Asp193 and a carboxylic oxygen of Asp246 and
between the backbone oxygen of Asp193 and the backbone
hydrogen of Trp247 are present only in PKC-3Ca, whereas
the H-bond between the backbone hydrogen of Asp246 and the
backbone oxygen of Asp254 is present in PKC-3Ca as well
as in PKC-2Ca. On the other hand, only in PKC does
Asp187 form two H-bonds with Asn189 (backbone
and side-chain hydrogen) with one of its carboxylic oxygens. These
differences between PKC-3Ca and PKC structures are shown in Fig.
11.
|
On the basis of NMR studies (18), it was found that PKC-C2 binds two
calcium ions (corresponding to sites II and III
in Fig. 7), whereas a third ion may bind with a lower affinity.
However, the crystal structure of the same domain (19) shows three
bound calcium ions per domain. In that crystal structure, one donor atom to the third calcium ion (site IV in Fig. 7) is
provided by a residue from the second C2 molecule in the asymmetric
unit of the crystal; in solution, this donor atom is replaced by a well-ordered, highly rigid water molecule (the axial water in Fig. 7).
Furthermore, Asn253 remains at a coordination distance for
the entire trajectory and completes heptacoordination of calcium IV,
together with another water molecule in the equatorial plane (see Fig.
7). Therefore, binding site IV is already well-organized to bind a
calcium ion in the crystal structure, which we used as the starting
conformation in PKC-3Ca simulation, and it remains very stable as
long as the coordination sphere is complete, as it happens in our
simulation: The energetics of this system is such that repulsion of
calcium IV does not occur, at least on the MD trajectory time-scale,
i.e. about 1 ns.
How Is Calcium Involved in PS Binding?--
It is well known that
calcium increases the affinity of cPKC for negatively charged lipids
(49, 50). Newton and collaborators originally suggested that the
positively charged surface of sheet B (-strands 3, 4, 6, and 7),
determined by a cluster of lysine residues at positions 197, 199, 209, 211, and 213, may provide the binding site for PS. The decreased
distance between loops 2-3 and 6-7 due to calcium binding could
orient this basic face to interact with the lipids' headgroups (51).
This hypothesis is in agreement with the x-ray structure by Sutton and
Sprang (19) that was determined using crystals grown in the presence of
O-phospho-L-serine as a headgroup analog of PS.
Weak electron density was found at the surface of sheet B, such that
the phosphate group was in contact with lysines 197, 199, and 211, even
though the absence of strong density for the putative seryl group and specific protein contacts with it suggested that the interaction might
be nonspecific (19).
Our results show that the accessibility of this basic cluster is not
reduced in the absence of calcium as compared with the calcium-bound
states (see Table II). Therefore, it does not seem likely that the
calcium-induced increased affinity of PKC for negatively charged lipids
is due to a larger exposure of the basic cluster on the surface of
sheet B. Indeed, subsequent experimental data from Newton's group show
that substitution of four lysine residues in the cluster with neutral
residues does not inhibit association of PKC with PS-enriched
vesicles (52). Instead, our data suggest that this sequence provides
part of the docking site for RACK1 (16), and the stabilization of the
-sandwich structure induced by calcium binding may be required for
this protein·protein interaction (52).
As an alternative explanation, it has also been suggested that the
Ca2+ ions bound to PKC maintain some of their coordination
open to interact with the negatively charged headgroup of phospholipids (45). We have examined this possibility by simulating the interaction of three O-phospho-L-serine molecules with the
Ca2+ ions in the structure of PKC-3Ca. This MD
simulation shows that only two molecules of
O-phospho-L-serine are able to coordinate the
three Ca2+ ions through the oxygen atoms of the phosphate
or of the carboxylate group, by substituting the water molecules in the
coordination sphere of the calcium ions (Figs. 7 and 8). These data
support the possibility that Ca2+ ions are indeed directly
involved in PS binding by acting as a bridge.
The Ca2+ bridge model is supported by an x-ray structure of
the PKC-C2·(Ca2+)2·PS complex by
Verdaguer et al. (53), showing that one molecule of
1,2-dicaproyl-sn-phosphatidyl-L-serine is
specifically coordinated to a Ca2+ ion and other residues
in the Ca2+-binding loops. In that work, a membrane-binding
mechanism of the PKC-C2 domain is suggested in which two calcium
ions play different roles in membrane binding. Recent studies of the C2 domains of PKC (54) and cytosolic phospholipase A2 (55), both of which bind two Ca2+ ions, also showed that two
Ca2+ ions play distinct roles, with one primarily involved
in inducing conformational changes and the other in Ca2+
bridging. On this basis, we can infer similar differential roles for
Ca2+ ions in the membrane targeting of PKC-C2, because
PKC-3Ca·PS simulation data show that only calcium III and IV are
coordinated in a stable way by
O-phospho-L-serine, whereas calcium II is not. On the other hand, comparison of data from PKC-3Ca, PKC-2Ca, and
PKC simulations (see above) show that all three Ca2+
ions are involved in inducing conformational changes in the domain, especially in loop 6-7. Therefore, we suggest that calcium III and IV
play a dual role in the membrane targeting of PKC-C2, providing a
bridge between the C2 domain and phospholipids as well as inducing
conformational changes. Conversely, calcium II is not involved in
bridging and its role appears limited to the stabilization of the
domain structure.
In addition, in the course of PKC-3Ca·PS simulation,
O-phospho-L-serine molecules appear to interact
also with protein residues of loop 6-7, reducing the mobility of that
loop. This suggests that phospholipid binding may determine a further
increase in the order of this region of the PKC-C2, thus
establishing a positive cooperation with Ca2+ binding to
achieve a certain conformation of the domain.
This possibility is supported by a recent mutation analysis of the C2
domain of PKC (54). In particular, in agreement with our findings,
interactions along loop 6-7 indicate that at least two residues
(Arg249 and Arg252) participate in
electrostatic interaction with anionic lipids and two others
(Trp245 and Trp247) participate in penetration
into the membrane. Also in the above-mentioned x-ray structure of
PKC complex with
1,2-dicaproyl-sn-phosphatidyl-L-serine (53),
this PS-mimicking molecule is found to bind to the C2 domain of PKC
both via the calcium coordination and by direct interaction with
Trp247 and Arg249. On the other hand, in the
same work (53) the authors emphasize that phospholipid analogs, such us
O-phospho-L-serine, used to analyze the binding
of phospholipids to PKC (19), lack the possibility of some of the
interactions seen in the structure of
PKC-C2·(Ca2+)2·PS complex. Therefore,
the unstable interactions between PKC-C2 and
O-phospho-L-serine we identified in the
simulation may in fact mimic the direct association of the enzyme with
the lipids, and the instability could be due to the inadequacy of
O-phospho-L-serine in mimicking PS. The docked
model of the PKC-C2·(Ca2+)2·PS complex
onto a model membrane proposed by Verdaguer et al. (53)
suggested to us that the molecule of PS3 in PKC-3Ca·PS simulation
(see Fig. 8) could be actually replaced by a longer hydrophobic tail of
PS1. This model is, of course, mainly speculative.
Finally, Nalefski and collaborators (56) compared the equilibrium and kinetic parameters of C2 domains binding to calcium and lipids. This study demonstrates that there are at least two steps in the docking of the C2 domain to membranes: A rapid calcium binding followed by a slower membrane binding. These authors further demonstrated that, although the C2 domains of various proteins share sequence homology and similar architecture, they exhibit unique coordination of calcium, resulting in different kinetics of membrane docking (56). Our previous work on the role of RACKs in docking of activated PKC adds a third step in this process of docking, providing further mechanism to ensure specific subcellular localization of the activated enzyme and possibly increased stability for this docking.
Final Considerations--
The present MD simulations indicated
that the progressive reduction of the number of Ca2+ ions
bound to the C2 domain of PKC causes a decrease in the number and
the length of secondary structure elements, as well as an increase in
the average fluctuations over the time of simulation. On this basis, we
infer that calcium binding determines a stabilization of the
-sandwich structure of the C2 domain. In particular, we observed
that this stabilization affects two regions of the C2 domain, which are
involved in binding of the activated form of PKC to its receptor,
RACK1. These regions correspond to C2-1 and C2-4. This
observation suggests that calcium cooperates in PKC activation by
driving the RACK1-binding sites within C2 toward the most favorable
conformation for the interaction with RACK1.
In addition to the effect of calcium on the structure of C2, calcium
plays an electrostatic role in PKC activation: The binding of two or
three Ca2+ ions is expected to produce a drastic change in
the electrostatic potential field around their binding sites,
neutralizing the negative charge of the acidic cluster of aspartates on
C2. This change in electrostatic field has been suggested to represent
a molecular switch (50, 57) that contributes to shift the enzyme to its activated form. We propose here two possible but not incompatible mechanisms for the working of this switch. In the former mechanism, Ca2+ ions are directly involved in PKC binding to PS: If
this occurs, the same region of the C2 domain would bind both calcium
and PS. This region, i.e. loops 2-3 and 6-7, would be able
to interact with PS only after the reversal of its electrostatic
properties induced by binding of Ca2+ ions. In the latter
mechanism, the changes induced by calcium binding affect the
domain·domain interactions between the C2 domain and other domains in
PKC, which may contribute to maintaining PKC in the inactive state
(58). As mentioned earlier, one possible domain participating in this
intramolecular interaction is the V5 domain. It is located in the last
50 amino acids of PKC and constitutes the only difference between I
and IIPKC isozymes (3). Our recent study showed that at least part
of the RACK1-binding site in IIPKC is located within the V5 domain
of the enzyme (47). The IIPKC V5 domain binds directly to RACK1 with
an affinity similar to that of the isolated C2 domain enzyme (47). In
addition, peptides derived from the IIV5 domain inhibit IIPKC
binding to RACK1 in vitro and inhibit IIPKC translocation
and function in cells (47). The presence of relatively limited
interactions between the pseudo-RACK1 sequence with RACK1-binding sites
within the C2 domain in our modeled structures suggests that
pseudo-RACK1 site interacts with RACK-binding sites on other PKC
domains. Because the V5 domain directly binds to RACK1, it may also
interact with the pseudo-RACK1 sequence in the C2 domain, to mask it in
the inactive state. Such V5·C2 interactions have been previously
suggested (59, 60). The first reports indicating interaction between the V5 and C2 domains were provided by the work of Newton et
al., showing that calcium binding to the C2 domain of PKC
isozymes is affected by their V5 domains (reviewed in Ref. 59). In
addition, the phosphorylation of Ser660 in the V5 region
increases the affinity of the enzyme to both phospholipids and calcium,
presumably due to allosteric effects of the V5 domain on the C2 domain
(60). The possibility of inducible intra-domains interactions in PKC,
such as these suggested here between the C2 and V5, could be directly
addressed when the complete structure of PKC becomes available.
Finally, it is still not known what are the steps that lead inactive
cytosolic PKC to anchor to a particular site within the cells. There
could be at least three steps, including calcium binding, lipid
binding, and RACK binding. The final step leads to maximal
stabilization of the activated isozymes, which occurs during
protein·protein binding of the PKC via the C2 domain and the V5
domain to the pre-anchored RACK1. Future studies including co-crystallographic kinetic analysis with purified components will help
elucidate the question.
| |
FOOTNOTES |
|---|
* This work was supported in part by National Institutes of Health Grant HL52141 (to D. M.-R.) and by Ministero dell'Università e della Ricerca Scientifica, Progetto EX 40% (to L. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Centro di Risonanze Magnetiche, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino (Florence), Italy. Tel.: 39-055-457-4263; Fax: 39-055-457-4253; E-mail: banci@cerm.unifi.it.
¶ A Ph.D. student of the International Doctorate in Structural Biology instituted by CERM (University of Florence), in collaboration with Biozentrum (University of Frankfurt) and Bijvoet Center (University of Utrecht).
Published, JBC Papers in Press, January 8, 2002, DOI 10.1074/jbc.M106875200
2
r.m.s.d. (root mean square deviation) is defined
as
(
i
r
ri is the displacement of an atom in a
structure with respect to a reference structure, and the sum is
performed over the atoms of the backbone, or over the heavy atoms,
i.e. all the atoms excluding hydrogen.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: PKC, protein kinase C; RACK, receptor for activated C kinase; MD, molecular dynamics; PS, phosphatidylserine; DG, diacylglycerol; cPKC, classic PKC subfamily; r.m.s.d., root mean square deviation; H-bond, hydrogen bond.
| |
REFERENCES |
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|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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