(Received for publication, March 5, 1997, and in revised form, May 8, 1997)
From the Department of Biochemistry, Medical Research
Council Group in Protein Structure and Function, University of
Alberta, Edmonton, Alberta T6G 2H7, Canada and the
§ Department of Biochemistry and Molecular Biology,
University of Texas Medical School, Houston, Texas 77225
The regulation of cardiac muscle contraction must differ from that of skeletal muscles to effect different physiological and contractile properties. Cardiac troponin C (TnC), the key regulator of cardiac muscle contraction, possesses different functional and Ca2+-binding properties compared with skeletal TnC and features a Ca2+-binding site I, which is naturally inactive. The structure of cardiac TnC in the Ca2+-saturated state has been determined by nuclear magnetic resonance spectroscopy. The regulatory domain exists in a "closed" conformation even in the Ca2+-bound (the "on") state, in contrast to all predicted models and differing significantly from the calcium-induced structure observed in skeletal TnC. This structure in the Ca2+-bound state, and its subsequent interaction with troponin I (TnI), are crucial in determining the specific regulatory mechanism for cardiac muscle contraction. Further, it will allow for an understanding of the action of calcium-sensitizing drugs, which bind to cardiac TnC and are known to enhance the ability of cardiac TnC to activate cardiac muscle contraction.
Transient increases in cytosolic Ca2+ levels in the cardiac muscle cell must be recognized by the thin filament to regulate cardiac muscle contraction. This critical function is accomplished by cardiac TnC1 (161 residues), a member of the EF-hand family of Ca2+-binding proteins, which relays the Ca2+ signal via a conformational change to the rest of the troponin-tropomyosin complex, and ultimately signals the activation of the myosin-actin ATPase reaction. Although the sequence of cardiac TnC is 70% identical to that of skeletal TnC, there are significant differences in the first 40 residues, the most crucial being the inactivation of Ca2+-binding site I due to an insertion (Val28) and substitutions of key ligands relative to skeletal TnC (Leu29 and Ala31 in cardiac TnC instead of Asp30 and Asp32 in skeletal TnC) (1). Despite the many functional, binding, and modeling studies performed on cardiac TnC (2), the absence of direct structural data makes the Ca2+-induced conformational change in cardiac TnC unclear. The structures of TnC in the skeletal system, on the other hand, have been solved both in the 2-Ca2+ (3, 4) and 4-Ca2+ states (5), showing TnC to be a dumbbell-shaped molecule with separate N- and C-terminal domains connected by a central linker. Upon Ca2+ binding, the regulatory N-domain of skeletal TnC switches from a "closed" to an "open" conformation, thereby exposing a patch of hydrophobic residues, which is thought to interact with skeletal TnI (6). In this report, we show that, in contrast to predicted models (7-9), the analogous conformational change does not occur in cardiac TnC, and that this is the direct structural consequence of inactivating Ca2+-binding site I. In addition, a structural understanding of cardiac TnC has potential therapeutic value in the understanding of the mechanism of cardiac TnC-binding drugs known as "calcium-sensitizing drugs" (8, 10).
For the purposes of this study, the two Cys residues at positions 35 and 84 of wild type cardiac TnC have been mutated to Ser residues. This prevents the formation of intra- and intermolecular disulfide bonds, which confer Ca2+-independent activity to cardiac TnC when assayed in skeletal muscle myofibrils (11). It has been shown that the conversion of these Cys residues to Ser residues has no effect on the ability of cardiac TnC to recover ATPase activity in TnC-extracted fast skeletal and cardiac myofibrils, and has little effect on Ca2+ binding to site II of cardiac TnC (11). Thus, it is unlikely that the introduction of these two conservative mutations would result in gross conformational changes in the secondary or tertiary structure of cardiac TnC.
For NMR analysis, the protein was uniformly labeled with 13C and/or 15N by expression in Escherichia coli. Triple-resonance NMR experiments were used for assigning the resonances and subsequently to derive distance and dihedral angle restraints. 35 structures were then calculated using the simulated annealing protocol (12). Structural statistics for the 30 lowest energy structures (Table I) show that the N- and C-domains are very well defined separately, with the central linker shown to be flexible by relaxation measurements.2
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To produce high level expression of
chicken cardiac TnC with the mutations C35S and C84S (denoted
cTnC(A-Cys)), the expression plasmid pTnC(A-Cys)PL (11),
which uses the PL promoter, was digested with
NcoI and HindIII, and the small fragment
containing the full amino acid coding region was ligated into the
NcoI/HindIII sites of the plasmid pET-23d
(Novagen), which has a T7 promoter. Plasmids were
maintained, and cTnC(A-Cys) was expressed in the BL21(DE3)pLysS host
strain E. coli after induction with
isopropyl-1-thio--D-galactopyranoside. Isotope
enrichment media consisted of M9 minimal media, in which the
NH4Cl and/or the D-glucose was replaced with
one of the following: 1) 15NH4Cl (1 g/liter)
for uniform 15N labeling; 2)
D-glucose-U-13C6 (2 g/liter) for
uniform 13C labeling; 3) 15NH4Cl (1 g/liter) and D-glucose-U-13C6 (2 g/liter) for uniform 13C and 15N labeling; or
4) D-glucose-U-13C6 (0.5 g/liter)
and D-glucose (1.5 g/liter) for partial 13C
labeling. All isotopes were purchased from Cambridge Isotope Laboratories. After induction, bacteria were collected and lysed, and a
soluble protein fraction was prepared as described previously (13).
This was applied directly to a Macro-Q (Bio-Rad) anion exchange column
and eluted with a 0-500 mM KCl gradient containing 0.5 mM EGTA. Appropriate fractions were pooled, made 3 M in (NH4)2SO4, and
centrifuged to remove precipitates, after which the soluble fraction
was dialyzed against 50 mM MOPS, 0.5 mM
CaCl2 at pH 7.0. The dialyzed sample was then applied to a
semiprep HPLC DEAE 5-PW anion exchange column (Millipore) and eluted
with a 0-500 mM KCl gradient with 0.5 mM
CaCl2. Purified cTnC(A-Cys) was pooled and dialyzed against
10 mM (NH4)HCO3 at pH 8.0, and
lyophilized.
All spectra were collected at
30 °C on a Varian Unity Plus 500-MHz spectrometer or a Varian Unity
Plus 600-MHz spectrometer, both equipped with pulsed field gradient
accessories. The NMR samples contained 2-4 mM cTnC(A-Cys)
in 16 mM CaCl2, 100 mM KCl, 10 mM imidazole at pH 6.7, in either 90% H2O,
10% D2O or 99.99% D2O. The high affinities of
cardiac TnC for calcium (with dissociation constants on the order of
106 M for the N-domain and 10
8
M for the C-domain) ensure that the protein is
Ca2+-saturated at 16 mM CaCl2
(11).
Assignment of the main-chain NH, N, C, and C
resonances were made
based on the three-dimensional experiments HNCACB and CBCA(CO)NH (14).
Three-dimensional 15N-edited total correlation spectroscopy
(TOCSY) (15) and three-dimensional HCCH-TOCSY (16) experiments were
used to assign the chemical shifts for the side-chain atoms. A high
resolution two-dimensional 1H-1H nuclear
Overhauser enhancement spectroscopy (NOESY) experiment and assignments
made by a previous study (17) were used to confirm the assignments for
resonances of aromatic protons. Interproton distances were derived from
three-dimensional 15N- and
15N/13C-edited NOESY experiments (18), both
collected at 50 ms of mixing time. In converting NOE intensities into
distance restraints, the NOE intensities were calibrated for each
residue using its intraresidue dN
(i,
i) and sequential d
N(i
1, i) NOEs as reference intensities (6). In cases where neither
NOE was present, the upper bound distance was given the calibration
factor corresponding to the loosest restraint. In all cases, a 40%
error on NOE intensities was used. Based on the analysis of the crystal structures of skeletal TnC (19), 18 distance restraints to the Ca2+ ion (6 at each of site II, III, and IV) of 2.0-2.8 Å were assigned.
A three-dimensional HNHA experiment (20) was used to derive
3JHNH coupling constants.
restraints
were imposed if the 3JHNH
values were
greater than 8 Hz or less than 5.5 Hz. These restraints were given
different errors depending on the region on the Karplus curve to which
they corresponded, with a minimum error of ±20°.
restraints were
assigned for the (i
1) residue if the
dN
(i, i) to
d
N(i
1, i) NOE intensity
ratio was greater than 1.2 or less than 0.83, which were given loose restraints of -30 ± 110° and 110 ± 110°, respectively
(28). Stereospecific assignments of the methyl protons of valines and leucines (20 out of 21 possible) were made based on the presence of
singlets (pro-S) or doublets (pro-R) in a
two-dimensional 13C-1H HSQC spectrum with the
protein being expressed in 25% [13C]glucose (21).
H
-methylene protons were stereospecifically assigned (32 out of 115 possible) by analyzing 3JH
H
values from
the three-dimensional HACAHB experiment (22),
3JNH
values from the three-dimensional HNHB
experiment (23), and intraresidue NOE intensities.
1
restraints of +60°, 180°, or
60° (±60°) were also given if
all of the above data were consistent with one
1
conformation. All spectra were processed with NMRPipe (24) and
peak-picked with the program PIPP (25).
Fig. 1 shows the solution structures of the N- and
C-terminal domains, with the structural statistics provided in Table
I. The overall solution structure of
Ca2+-saturated cardiac TnC, like the solution structures of
Ca2+-saturated skeletal TnC (5) and calmodulin-target
peptide complex (26), resembles a dumbbell in shape, consisting of two
separate domains connected by a flexible central linker (residues
86-94 in cardiac TnC). However, despite the general structural
similarities to homologous Ca2+-binding proteins, the
regulatory N-domain of Ca2+-saturated cardiac TnC is
significantly more compact than the N-domain of
Ca2+-saturated skeletal TnC (5, 6), exposing approximately
800 Å2 less total accessible surface area (residues 5-84)
than its skeletal counterpart (residues 7-85). In particular, the
B-helix of defunct site I exists in the "closed" conformation,
exhibiting an A-B interhelical angle of 142° (with the A- and
B-helices corresponding to the two helices of the helix-loop-helix
motif in Ca2+-binding proteins; Table II).
The closed conformation is evidenced by 21 NOE connectives observed
between the A- and B-helices (Fig. 2), most of which
would not be observed if the B-helix were in an "open" conformation
as in skeletal TnC (Fig. 3A). A compact regulatory domain is also consistent with a previous
cysteine-reactivity study on wild type cardiac TnC (27).
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The difference in the conformation of the B-helix is reflected most
clearly in the main chain conformation of residue Glu40 in
cardiac TnC (equivalent to Glu41 in skeletal TnC).
Glu41 of skeletal TnC has been proposed to be pivotal in
the mechanism of the coupling between Ca2+ binding and the
Ca2+-induced conformational change in
Ca2+-binding proteins (28, 29). Indeed, the apo form of
skeletal TnC (3) features a kink in the B-helix at Glu41
which straightens out upon Ca2+ binding to sites I and II
(28). In cardiac TnC, however, there exists a kink in the B-helix at
Glu40, even in the Ca2+-bound state
(Ca2+ ions bound at sites II, III, and IV). The non-helical
nature at Glu40 is supported by a
3JHNH value of 7.8 Hz, an absence of an
upfield-shift of its H
resonance (4.37 ppm), and an absence of a
downfield shift in its C
resonance (56.8 ppm), all of which indicate
non-helical conformations (30). On the other hand, both adjacent
residues Lys39 and Leu41 exhibit
3JHNH
values of less than 5.5 Hz, as well as
appropriate shifts in their H
and C
resonances which indicate an
-helical conformation.
The above structural differences between cardiac and skeletal TnC can be explained by what is in fact the most striking functional difference between the two proteins: namely that site I in cardiac TnC is inactive due to an insertion and key substitutions of key ligands. In cardiac TnC, there is no Ca2+ at site I to pull the Glu40 side-chain carboxylate group over to contribute to the enthalpy necessary to overcome the entropic loss associated with exposing buried residues (which occurs in the "opening" of the B- and C-helices relative to helices N, A, and D). Thus, the B-helix remains closed due to favorable packing forces with the A-helix and D-helix (Figs. 1A and 3A; Table II, A-B interhelical angle). On the other hand, with Ca2+ bound at site II, the C-helix is in fact in an open conformation, but does not open up to the extent seen in skeletal TnC (Figs. 1A and 3A; Table II, C-D interhelical angle). These observations demonstrate that the inability of Glu40 to coordinate Ca2+ results in a more compact conformation for the B-helix, and possibly the C-helix, than is observed in skeletal TnC (Fig. 3A); in effect, Ca2+ binding to sites I and II of skeletal TnC locks open the whole regulatory domain, whereas Ca2+ binding to site II of cardiac TnC only partially opens up the regulatory domain. (This discussion assumes that the structure of the apo form of the regulatory domain of cardiac TnC is similar to that of skeletal TnC, as has been recently demonstrated.3) The model of Glu40 acting as a pivot for the N-domain is further supported by a recent structural study of a skeletal TnC mutant in which Glu41 is replaced by Ala41, such that residue 41 can no longer coordinate the Ca2+ ion present at site I (29). In the Ca2+-saturated state of this protein, the single substitution results in a kink at Ala41 and a closed conformation for the B-helix, similar to what is seen in Ca2+-saturated cardiac TnC.
The structure of defunct site I shows that Leu29, which
comes just after the insertion at Val28, forms an extra
half-turn at the end of the A-helix, as evidenced by
dN(i, i + 3) and
d
(i, i + 3) NOE connectives from Ile26 to Leu29. Site I, being
Ca2+-free, is not as well defined as the rest of the
molecule (root mean square deviation of 0.78 Å for backbone atoms of
residues 30-33), and is shown to be more flexible than the rest of the regulatory domain by relaxation measurements.
The structural C-domain of cardiac TnC (Figs. 1B and 3B) is predictably similar to those in skeletal TnC and calmodulin, although the interhelical angles of the two EF-hands in the C-domain indicate that this domain is in fact slightly more compact in cardiac TnC than in its counterparts (10-20° more closed in the E-F and G-H interhelical angles; see Table II). Overall, the backbone atoms of residues 95-157 of cardiac TnC superimpose within 1.9 Å with their equivalent residues (96-158) in the NMR structure of skeletal TnC with Ca2+-saturated N-domain, and 1.3 Å with the same region in the crystal structure of skeletal TnC with apo N-domain.
We have shown for the first time the three-dimensional structure
of Ca2+-saturated cardiac TnC, which reveals an unexpected
compact regulatory domain as a direct consequence of an inactive
Ca2+-binding site I. These results provide a structural
precedent for a Ca2+-binding regulatory protein in which
one of the two sites in the paired set of EF-hands is inactive (for
example, some invertebrate TnCs also have this feature; Ref. 31)). This
unique structural feature sets cardiac TnC apart from other "calcium
sensor" EF-hand Ca2+-binding proteins such as
skeletal TnC and calmodulin, as well as "calcium buffer" EF-hand
proteins such as parvalbumin and calbindin. The compact
regulatory domain is a surprising result because it violates the
general rule with Ca2+-binding proteins that a small
conformational change accompanies Ca2+ binding in buffering
proteins, and that a large conformational change accompanies
Ca2+ binding in regulatory proteins such as cardiac TnC
(32). In particular, it is believed that in general the action of
Ca2+ binding in calcium sensor proteins is to induce an
exposure of a large hydrophobic surface, allowing the protein to
interact with targets to accomplish regulatory functions, whereas the
capture of Ca2+ ions by calcium buffer proteins is
accompanied by only minor conformational changes. Thus, it has long
been believed that the mechanism for the activation of cardiac TnC
involves the exposure of a large hydrophobic patch upon
Ca2+ binding as observed for other calcium sensors. In
fact, cardiac TnC models based on the conformational changes observed
in skeletal TnC have been widely used to interpret the functional,
Ca2+-binding and drug-binding properties of cardiac TnC
(7-9), despite the unique inactive Ca2+-binding site I in
cardiac TnC. The present results show that the hydrophobic exposure in
the Ca2+-saturated regulatory domain of cardiac TnC (Fig.
4B) is dramatically reduced compared with
that of skeletal TnC (Fig. 4A) as well as a previous widely
used model of cardiac TnC (9) (Fig. 4C).
The substantially reduced hydrophobic surface of
Ca2+-saturated cardiac TnC has important implications for
the association of cardiac TnI with cardiac TnC. In particular, given
that in both the cardiac and skeletal systems the
Ca2+-dependent binding of TnI involves the
N-domain of TnC (33), and that residues 5-84 of
Ca2+-saturated cardiac TnC expose less total and
hydrophobic surface area than residues 7-85 of
Ca2+-saturated skeletal TnC (Fig. 4), it is possible that
the mode of interaction between TnI and TnC in cardiac muscle is in
fact different from that in skeletal muscle. A smaller surface of
interaction between 3Ca·cardiac TnC-cardiac TnI as compared with
4Ca·skeletal TnC-skeletal TnI would explain the finding that the free
energy (G) of Ca2+ binding to the TnC-TnI
complex is 4 times smaller in cardiac than it is in skeletal muscle
(34). It may also be that the hydrophobic or electrostatic force
dominates more in one isoform than in the other in the binding of TnI
to TnC. If indeed the interaction between cardiac TnC and cardiac TnI
involves less surface contact than that between skeletal TnC and
skeletal TnI, cardiac TnC would be a more dynamic calcium sensor than
its skeletal counterpart. In fact, a recent study has shown that the
Ca2+ off rate measured for site II in cardiac TnC is about
3-fold faster than observed for the N-terminal signaling domain of
calmodulin or skeletal troponin C.4 On the
other hand, as an alternative to the above proposal, it is possible
that the Ca2+-dependent binding of TnI forces
open the regulatory domain of cardiac TnC, with the end result being
that the cardiac TnI-TnC complex binds in a similar fashion to skeletal
TnI-TnC (here cardiac TnC may adopt a structure similar to that in Fig.
4C). Such a model would be consistent with the finding that
the chemical environment of Met81, which is mostly buried
in this structure (accessible surface area of 14 Å2),
changes upon the binding of cardiac TnI (33). This may also imply that
cardiac TnC opens up to different degrees in response to events of
muscle contraction such as TnI phosphorylation. At present, there is no
compelling evidence to either favor or discount either model for
cardiac TnI-TnC binding.
Cardiac TnC is a potential target in therapy for patients with acute myocardial infarctions and subsequently congestive heart failure, where the diseased myocardium is "desensitized" to increases in cytosolic Ca2+ levels. A novel group of positive inotropic agents known as "calcium sensitizers" (10) is known to increase the affinity of cardiac TnC for Ca2+, possibly by binding to a hydrophobic patch in the N-domain of cardiac TnC (8). The exposed hydrophobic patches in Ca2+-saturated cardiac TnC can now be identified (Fig. 4B). Although several residues (e.g. Phe77, Met81, and Met85) have been implicated in earlier studies as possible binding sites for these drugs (8), the proposed modes of binding must now be re-evaluated since most of these residues lie on the side of the D-helix facing the B-helix, and therefore are more buried by the B-helix than previously suspected (B-D interhelical distance of 12 Å for cardiac TnC versus 18 Å for skeletal TnC). In addition, significant surface topology differences between the cardiac TnC model and the solution structure (Fig. 4) warrants for a re-interpretation of most of the previous drug binding studies performed based on the now-disproved model (8). Thus, the solution structure of cardiac TnC presented here will allow for the accurate modeling of the binding of calcium-sensitizing drugs to cardiac TnC, in addition to revealing the structural basis for the regulation of cardiac versus skeletal muscle contraction.
The atomic coordinates and structure factors (code 1AJ4) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY. Coordinates of the 30 calculated structures for the N-domain (code 2CTN) and the C-domain (code 3CTN) have also been deposited.
We thank G. McQuaid for upkeep of the spectrometers; L. Willard, T. Jellard, and R. Boyko for computer assistance; and L. Kay for generously providing the pulse sequences. We also acknowledge the Protein Engineering Network Center of Excellence for the use of their Varian Unity 600-MHz spectrometer.