 |
INTRODUCTION |
The two ubiquitous calpains, µ- and m-calpain, are cytosolic
thiol proteases entirely dependent on Ca2+ for their
activity. These two calpains consist of a large or catalytic subunit
(80 kDa) (from the genes capn1 and capn2,
respectively) and a common small or regulatory subunit (28 kDa) (from
capn4). The µ- and m-isoforms differ also in the
concentration of Ca2+ required for half-maximal activation
in vitro. Both enzymes require a Ca2+
concentration (~50 and ~300 µM for µ- and
m-calpain, respectively) that is significantly higher than that
available in vivo (<1 µM). Autolysis causes a
drop in Ca2+ requirement, but calpain activation in
vivo must involve additional factors, such as membrane-binding and
activator proteins. Although the physiological roles of these
calpains remain unclear, there is much evidence suggesting that they
contribute to many cellular processes, including signal transduction,
apoptosis, cell cycle regulation, and cytoskeletal reorganization
(1-5). Their physiological importance is exemplified by the recent
demonstration that transgenic mice lacking the classical calpain
isoforms die during embryonic development (6). Excessive proteolysis by
these enzymes in response to altered Ca2+ homeostasis has
been observed in several neuropathological states, including
Alzheimer's disease (7, 8). With regard to other forms of calpain,
defects in calpain-3 lead to the development of limb-girdle
muscular dystrophy 2A (9), calpain-10 is linked to type II
diabetes (10), and in Caenorhabditis elegans, the protease
activity of the calpain homologue TRA-3 is required for sex
determination (11, 12).
The x-ray structures of rat (13) and human (14) m-calpain in the
absence of Ca2+ show that the large subunit consists of a
short
-helix at the N terminus; domains I and II, which constitute
the protease function; domain III, which resembles a
C2-domain; and domain IV, which contains several EF-hands.
The small subunit contains domain V, which is glycine- and proline-rich
and was either not present (13) or not detectable (14) in the x-ray
structure, and domain VI, which contains several EF-hands and is
structurally very similar to domain IV. Importantly, the structure
revealed that the protease active site is not assembled in the absence
of Ca2+, a feature not previously observed in any other
cysteine protease. The protease domains (domains I and II) are held
apart in the Ca2+-free conformation by interactions within
the molecule, which prevent the catalytic triad residues (Cys-105 in
domain I and His-262 and Asn-286 in domain II) from assuming the
correct conformation to hydrolyze substrates. Activation by
Ca2+ must somehow relieve these constraints, permitting
domains I and II to move toward each other and form a competent active
site. A Ca2+-bound structure of calpain might answer
several questions concerning the Ca2+-induced activation
mechanism, but this structure has not yet been determined
because of significant technical difficulties.
Based on the primary sequence alone, it was for a long time assumed
that the EF-hand-containing domains IV and VI were the major
determinants of the Ca2+ requirement of calpain. Recent
studies have shown that Ca2+ binding at EF-hand 3 makes the
largest contribution to calpain activation, EF-hand 2 may make some
contribution, and the other EF-hands apparently have no direct role in
Ca2+ regulation (15). Additionally, it has recently been
demonstrated that TRA-3 has Ca2+-dependent
protease activity although it lacks the EF-hand domain (12). Thus it is
likely that the observed Ca2+ requirement of the whole
enzyme is not determined solely by the EF-hands and is greatly
affected by interactions elsewhere in the molecule.
The x-ray structures revealed two interesting features in domain III
that may be very important in this regard (Fig. 1A). First, domain III was found to be
structurally similar to the Ca2+-dependent
C2 domains, which are known to promote the phospholipid binding of C2-containing proteins (16, 17). This is
consistent with the following two considerations: 1) calpains are
thought to translocate to the cell membrane when activated by
Ca2+ and 2) the Ca2+ requirement of calpain is
greatly reduced in the presence of phospholipids, which appears to be a
part of the in vivo activation mechanism of calpain. Second,
at the interface between domains II and III, there is a set of
electrostatic interactions involving a remarkably acidic loop composed
of Glu-392-Asp-400 and Glu-504 in domain III and clustered lysine
residues Lys-226, Lys-230, and Lys-234 in domain II (Fig.
1B). All these residues are highly conserved within the
known m-calpain sequences. We hypothesize therefore that this salt
bridge region exerts a conformational constraint on the movement of
domain II and therefore on the assembly of the active site, which will
tend to elevate the Ca2+ requirement of the enzyme. By
disrupting these salt bridges, the mutations described here were
designed to investigate the effects on the Ca2+ requirement
of calpain. Our experiments would further address the question of
whether these electrostatic interactions directly contribute to
maintaining the inactive conformation of calpain.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 1.
Interactions between the
C2-like domain III and protease domain II.
A, overall view of the m-calpain structure in the absence of
Ca2+. The domain classification is taken from Ref. 13. The
region contained within the black circle is amplified for
clarity in B. B, electrostatic interactions
between domains II and III. Lysine residues (dark blue)
Lys-226, Lys-230, and Lys-234 on a domain II -helix
(cyan), as well as Lys-354, form several interdomain salt
bridges with Glu-504 and several other acidic residues (red)
on one loop (residues 392-400) of domain III. Dotted lines,
possible interactions with Glu-504. In this study, Lys-226, Lys-230,
Lys-234, and Glu-504 were mutated to determine the effects that
disruption of these interdomain interactions might have on the
activation of m-calpain by Ca2+. This figure was prepared
using MOLSCRIPT (23) and RASTER3D (24).
|
|
 |
EXPERIMENTAL PROCEDURES |
Site-directed Mutagenesis and Protein
Expression--
Site-directed mutagenesis was performed on
single-stranded DNA derived from pET-24-m-80k-CHis6, using
the following antisense primers:
5'-P-cttctggatgatcgagaagagattgggaggagg-3' (K226S),
5'-P-gagaacctttctcgagagccgactggatgatcttg-3' (K230S),
5'-P-gagaacctttctcgagagcctcctggatgatcttg-3' (K230E), 5'-P-aagcagagaacctgactcgagagccttctggatg-3' (K234S),
5'-P-agcagagaaccttcctcgagagccttctggat-3' (K234E), and
5'-P-agtcagccttcttgctcgagaagactcgg-3' (E504S). In all cases, the
nucleotide sequence around the mutated site was confirmed by DNA sequencing.
The expression and purification of wild-type rat m-calpain and mutant
enzymes were performed as previously described (18). Coexpression of
the constructs pET-24-m-80k-CHis6 (encoding the m-calpain
80-kDa subunit with a C-terminal histidine tag) and pACpET-21k
(encoding the C-terminal 184 residues of the regulatory subunit,
referred to as domain VI or the 21-kDa subunit) in Escherichia coli strain BL21(DE3) gives rise to an active heterodimer.
Enzymes were purified through several columns and finally concentrated by centrifugation using a BioMax 30-kDa molecular mass exclusion device
to ~10 mg/ml in a buffer containing 10 mM
dithiothreitol, 50 mM Tris-HCl, pH 7.6, 100 mM NaCl, and 200 µM EDTA. Small aliquots of
the enzyme samples (typically 50 µl) were flash frozen in liquid nitrogen and stored at
70 °C.
Titration of Ca2+ Requirement--
For the
measurement of the Ca2+ dependence of calpain activity, a
modification of the standard casein assay was used. The duplicate assays contained 4 mg/ml casein, 0.2 M NaCl, 10 mM 2-mercaptoethanol, and 50 mM Tris-HCl, pH
7.6, in a final volume of 100 µl. Net final CaCl2
concentrations ranged from 0 µM to 5.0 mM.
The reaction was initiated with 4 µl of enzyme sample (see below),
and the mixtures were incubated at 25 °C for 30 min before the
reaction was terminated by the addition of 70 µl of ice-cold 10%
trichloroacetic acid. The resultant mixture was placed on ice for 10 min and centrifuged at 15,000 rpm for 15 min, and the absorbance values
of the supernatants were recorded at 280 nm. Immediately prior to the
Ca2+ titration, the enzyme aliquots were freshly thawed
from
70 °C and diluted in 50 mM Tris-HCl, pH 7.6, to a
final enzyme concentration of 0.2 mg/ml (wild type, E504S, K226S,
K230S, K234S) or 2 mg/ml (K230E, K234E). The Ca2+
concentration required for half-maximal activity with casein as
substrate is given as [Ca2+]0.5. This value
was calculated by fitting the normalized activity data to the Hill
equation y = xn/(kn + xn)
where y is the fraction of maximum activity,
k = [Ca2+]0.5, n
is the Hill constant, and x is [Ca2+].
Crystallization, Diffraction Data Collection, and
Processing--
Crystals of the m-calpain mutant enzymes were grown in
conditions very similar to those for wild-type m-calpain (19). X-ray data from the K230E mutant were collected at 1.54 Å at an in-house facility that consists of an MAR Research imaging plate and a Rigaku
RU-200 x-ray generator operated at 50 kV and 100 mA. Data for the E504S
mutant were collected at beamline A-1 at the Cornell High Energy
Synchrotron Source, using monochromatic radiation at 0.925 Å and a
Quantum-4 CCD camera from Area Detector Systems Corporation (San Diego,
CA). All crystals were frozen in liquid propane, and data sets were
collected at 100 K. Raw x-ray data were processed using the HKL program
suite (20). Both mutants and wild-type m-calpain crystallized
isomorphously in space group P21. Cell
dimensions for the K230E mutant were a = 51.62 Å,
b = 156.13 Å, c = 64.00 Å,
= 95.21° whereas the dimensions for the E504S mutants were
a = 51.82 Å, b = 157.80 Å,
c = 64.49 Å,
= 95.18°.
Structure Solution and Refinement--
Because the mutant
crystals were virtually isomorphous to the wild-type protein, we were
able to incorporate the coordinates of wild-type m-calpain (PDB code
1DF0) directly for the initial refinement of the mutant enzymes. All
refinement was carried out using the CNS package (21) with 10%
of the reflections excluded from the refinement for the calculation of
Rfree. The initial R factor for the
K230E mutant was 0.366, which dropped to 0.260 (Rfree = 0.321) after refinement at 2.8 Å. The
initial R factor for the E504S mutant was 0.372, which was
reduced to 0.258 (Rfree = 0.302) after
refinement at 2.4 Å. The final models and the calculated |2Fobs
Fcalc| electron
density maps were analyzed using the XFIT program (22).
 |
RESULTS |
Expression and Purification of Wild-type and Mutant
m-Calpains--
Recombinantly expressed wild-type m-calpain and the
mutant enzymes were purified on four successive chromatographic columns to ~90-99% purity based on Coomassie staining of SDS-polyacrylamide electrophoresis gel, depending on the mutant (Fig.
2).

View larger version (59K):
[in this window]
[in a new window]
|
Fig. 2.
SDS-polyacrylamide electrophoresis
gel of m-calpain and mutants. Lane 1,
molecular weight marker; lane 2, wild type; lane
3, E504S; lane 4, K226S; lane 5, K230S;
lane 6, K234S; lane 7, K230E; and lane
8, K234E.
|
|
Effect of Mutations on Ca2+ Requirement and Specific
Activity--
Enzymes were assayed for their ability to hydrolyze
casein at varying concentrations of Ca2+. The calculated
[Ca2+]0.5 served as the basis for the
comparison of the Ca2+ requirement of wild-type and mutant
enzymes in this study. In the conditions employed here, the
[Ca2+]0.5 for wild-type m-calpain was
242 ± 6 µM (Fig. 3),
which is in general agreement with previous reports (1-5). Introducing the mutation E504S in domain III had the most dramatic effect, reducing
the [Ca2+]0.5 to 129 ± 1 µM, corresponding to a 47% reduction in the
Ca2+ requirement compared with wild-type m-calpain. The
effects of mutating each of the three lysine residues (Lys-226,
Lys-230, and Lys-234) individually to serine had varying results.
Mutating Lys-226 or Lys-230 had minor effects on the Ca2+
sensitivity of the enzyme (Fig. 3, Table
I), whereas the mutation of Lys-234
reduced the [Ca2+]0.5 to 183 ± 1 µM (a 24% reduction in the Ca2+
requirement). Further, the mutation of Lys-234 to glutamic acid resulted in a reduction in the [Ca2+]0.5 to
159 ± 3 µM, corresponding to a 34% decrease.
Interestingly, the K230E and K234E mutations decreased the specific
activity of the enzyme to ~16% compared with the wild type, whereas
the K230S and K234S mutations had no significant effect on specific activity.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 3.
Ca2+ titrations of m-calpain
variants. The activity and [Ca2+]0.5
values for m-calpain and the various mutations were measured as
described under "Experimental Procedures." The observed values of
[Ca2+]0.5 were as follows: wild-type
m-calpain ( ), E504S ( ), K226S ( ), K230S ( ), K234S ( ),
K230E ( ),and K234E ( ). The normalized means of duplicate data
points are plotted, and the lines shown were drawn by fitting the data
to the equation y = xn/(kn + xn),
where y is the fraction of maximum activity, k is
[Ca2+]0.5, n is the Hill constant,
and x is [Ca2+]. The calculated values of
[Ca2+]0.5 were 242 ± 6, 129 ± 1, 226 ± 2, 261 ± 14, 183 ± 1, 256 ± 1, and
159 ± 3 µM, respectively.
|
|
X-ray Crystallography--
To ascertain whether mutations had
caused unexpected structural changes that might influence the
Ca2+ sensitivity, we attempted structure determination of
these mutants by x-ray crystallography. We were successful in
crystallizing K226S, K230E, K234S, and E504S mutants in the absence of
Ca2+ in conditions very similar to those for wild-type rat
m-calpain (19). To date, we have solved the structures of the K230E and E504S mutants, from which larger crystals suitable for diffraction were
obtained. The refined structures of these mutants are virtually indistinguishable from wild-type calpain (root mean square
deviation of 0.47 and 0.49 Å on
-carbon atoms for the E504S
mutant and the K230E mutant, respectively), even in the vicinity of the
mutations. Because these two structures were almost identical to the
wild type, we did not proceed with the mutants that only gave rise to
weakly diffracting crystals and assumed that they would have minimal,
if any, conformational changes as well.
 |
DISCUSSION |
The mechanism of calpain activation by Ca2+ is a
fundamental biochemical question that has remained poorly understood. A
particularly interesting feature of these enzymes is that their
in vitro requirements for Ca2+ are orders of
magnitude higher than typical Ca2+ concentrations found in
the cell. This characteristic is not shared by similar members of the
EF-hand family, such as calmodulin, which have in vitro
Ca2+ requirements within the physiological range. Efforts
to convert calpain EF-hand sequences to more canonical forms have been
unsuccessful in reducing the Ca2+ requirement (15),
suggesting that other structural features contribute to calpain's
requirement for Ca2+. The x-ray structure of
Ca2+-free rat m-calpain revealed several features that
appear to maintain the inactive conformation of the enzyme. Of
particular interest is a loop of nine acidic residues
(Glu-392-Asp-400) in domain III, together with Glu-504, which
make several electrostatic contacts with three lysine residues
(Lys-226, Lys-230, Lys-234) on one
-helix in domain II. The
structure of human m-calpain showed an additional interaction between
Glu-504 and Lys-354. It should be noted particularly that Glu-504 would
have the strongest contact with Lys-234. Structures are not yet
available for µ-calpain and calpain 3, but these residues are almost
all conserved in both µ-calpain and calpain 3, so these domain
II-domain III interactions are likely to be present in all three
calpains. It therefore seemed attractive to propose that this set of
salt bridges might affect the ability of domain II to approach domain I
in the process of assembling the active site, leading to the
activation of calpain.
The K234S and E504S mutations both significantly reduced the
Ca2+ requirement without affecting the specific activity of
the enzyme. The reduction caused by the E504S mutation was greater than
that caused by the K234S mutation. This suggests that Glu-504 makes multiple electrostatic contacts and probably reflects the fact that the
E504S mutation abolishes the salt links to Lys-234 and Lys-354. The
K234S mutation abolishes only one of these salt links. The crystal
structure of E504S is virtually identical to that of the wild-type
enzyme in the inactive state, demonstrating that the
reduction in the Ca2+ requirement observed in E504S is not
a result of the disruption of the inactive conformation but must
involve the facilitation of domain movement during Ca2+
activation. Another interesting result was observed with the K234E
mutation, which reduced the Ca2+ requirement of the enzyme
even further than the K234S mutation. This mutation also reduced the
specific activity of the enzyme, illustrating the sensitivity of this
region to changes in the electrostatic potential. This mutant was
expected to exert a repulsive interaction with Glu-504.
Surprisingly, the results of this work showed that the electrostatic
interactions of Lys-226 and Lys-230 with the acidic Glu-392-Asp-400 loop do not appear to be critical to the activation of calpain by
Ca2+ because the K226S and K230S mutations did not affect
either the Ca2+ requirement of the enzyme or its specific
activity. The K230E mutation, which is expected to introduce a strong
repulsion between this position and the acidic loop, did not affect the
Ca2+ requirement of the enzyme. It did, however, greatly
reduce the specific activity of the enzyme, for reasons that we cannot
presently explain. The crystal structure of K230E in the absence of
Ca2+ was identical to that of wild-type m-calpain, showing
that the mutation did not affect the domain II-domain III geometry in
the resting state of the enzyme. Because it has not so far
been possible to crystallize calpain in the presence of
Ca2+, the effects of these mutations on the activated state
of the enzyme cannot be directly observed.
Of the many mutations that have been made in m-calpain, the Lys-234 and
Glu-504 mutations described here are the only examples so far described
that result in a lowering of the Ca2+ requirement. The
question therefore arises of how a loss of salt linkages around Glu-504
can have a major effect whereas alteration at Lys-226 and Lys-230 has
very little effect. The residue Glu-504 is at one end of the
"transducer arm" or linker peptide that connects domain IV to
domain III, and this arm is assumed to communicate Ca2+-induced conformation changes in domain IV to the rest
of the molecule. The evidence therefore suggests that the loss of the salt bridges at Glu-504 makes the movement of domain II toward domain I
"easier," i.e. permits it to occur at a lower
Ca2+ concentration. As can be seen in Fig. 1, Glu-504 is
"strategically" located at the top right end of the salt bridge
series. The movement of domain II in the assembly of the active site,
according to our modeling studies guided by the many well established
thiol protease structures, would be based on a pivotal point in the Glu-504 region. Therefore, given that the Glu-504 and Lys-234 mutations
weaken the interaction of domain II with domain III, we would suggest
that domain II does in fact move apart from domain III during the
activation process. Whether domain III undergoes additional
conformational changes remains unknown in the absence of a
Ca2+-bound structure. Illustrating the importance of this
interaction in the calpain family, the corresponding mutation in
µ-calpain, E515S, also lowers the Ca2+ requirement (data
not shown).
Recalling the resemblance of domain III to C2 domains, it
should also be noted that Glu-504 and the Glu-392-Asp-400 loop are in
positions very similar to the loops in C2 domains that
participate in Ca2+-dependent phospholipid
binding. The Ca2+ requirement of calpain is greatly reduced
in the presence of some phospholipids in vitro, and this is
assumed to reflect membrane binding in vivo. We speculate
therefore that Ca2+ and phospholipid binding to this region
of calpain could disrupt these critical salt links, thereby releasing
some constraints on the movement of domain II and lowering the
Ca2+ requirement of the enzyme. Coordination of
Ca2+ in the acidic loop by the side chains of the acidic
residues could break most or even all of the electrostatic interactions with the basic residues in domain II. With the exception of pivotal Glu-504, perhaps breaking one or two such salt links by point mutation
may not be enough to completely relieve the conformational constraints
to permit the movement of domain II.
Given that a Ca2+-bound structure may not be available in
the foreseeable future, experiments of the type employed here will be
required to more fully understand the mechanism of calpain activation
by Ca2+. In this work, we have for the first time generated
calpain mutants that are responsive to significantly lower
concentrations of Ca2+ for activation. Further, we have
identified a specific structural feature remote from the EF-hand
domains that affects the Ca2+ sensitivity and activation of
calpains. In light of the sequence conservation displayed in this
region, it is clear that these key interdomain interactions are
important factors contributing to the overall Ca2+
requirement of the calpains.