Ca2+-induced Conformational Transition in the Inhibitory and Regulatory Regions of Cardiac Troponin I*

Wen-Ji DongDagger, John M. RobinsonDagger, Scott Stagg, Jun Xing, and Herbert C. Cheung§

From the Department of Biochemistry and Molecular Genetics, University of Alabama, Birmingham, Alabama 35294-2041

Received for publication, December 18, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cardiac muscle activation is initiated by the binding of Ca2+ to the single N-domain regulatory site of cardiac muscle troponin C (cTnC). Ca2+ binding causes structural changes between cTnC and two critical regions of cardiac muscle troponin I (cTnI): the regulatory region (cTnI-R, residues 150-165) and the inhibitory region (cTnI-I, residues130-149). These changes are associated with a decreased cTnI affinity for actin and a heightened affinity for cTnC. Using Förster resonance energy transfer, we have measured three intra-cTnI distances in the deactivated (Mg2+-saturated) and Ca2+-activated (Ca2+-saturated) states in reconstituted binary (cTnC-cTnI) and ternary (cTnC-cTnI-cTnT) troponin complexes. Distance A (spanning cTnI-R) was unaltered by Ca2+. Distances B (spanning both cTnI-R and cTnI-I) and C (from a residue flanking cTnI-I to a residue in the center of cTnI-R) exhibited Ca2+-induced increases of >8 Å. These results compliment our previous determination of the distance between residues flanking cTnI-I alone. Together, the data suggest that Ca2+ activation causes residues within cTnI-I to switch from a beta -turn/coil to an extended quasi-alpha -helical conformation as the actin-contacts are broken, whereas cTnI-R remains alpha -helical in both Mg2+- and Ca2+-saturated states. We have used the data to construct a structural model of the cTnI inhibitory and regulatory regions in the Mg2+- and Ca2+-saturated states.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The contractile state of cardiac muscle is regulated by a series of Ca2+ and cross-bridge-dependent interactions among the thin filament proteins, including the subunits of troponin (Tn),1 tropomyosin, and actin. Troponin is composed of the three subunits: a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), which when bound to actin, inhibits myosin ATPase and force generation in relaxed muscle, and a tropomyosin-binding subunit (TnT). Cardiac muscle activation is initiated by the binding of Ca2+ to the single N-domain regulatory site of cTnC. This binding causes structural changes between cTnC and two critical regions of cTnI: the regulatory region (cTnI-R, residues 150-165) and the inhibitory region (cTnI-I, residues 130-149). During activation, cTnI-I dissociates from actin and associates with cTnC (1), and cTnI-R associates with an activation-exposed hydrophobic patch in the N-domain of cTnC (2). These changes, together with movement of tropomyosin on the thin filament (3) and changes in actin structure and dynamics (4), switch on the thin filament, permitting actin-myosin cross-bridge cycling and force development.

Numerous studies have elucidated Ca2+-induced structural changes between TnI and the other thin filament proteins (5, 6). Less is known about Ca2+-induced structural changes within TnI itself. We recently reported that in fully reconstituted cTn, Ca2+ binding to cTnC causes a 9-Å increase in the length of cTnI-I (7). This large extension of the inhibitory region apparently pulls this region away from actin and facilitates movement of the adjacent regulatory region toward the N-domain of cTnC. In the present study, we have extended the previous work by examining the potential for Ca2+-induced conformational changes within the regulatory region and between the regulatory and inhibitory regions. We conclude that internal structural changes in cTnI are confined to the inhibitory region, but the cTnI-I internal changes cause the adjoining regulatory region to be displaced as a rigid body.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sample Preparation-- Wild type recombinant chicken slow skeletal TnC (identical in sequence to mouse cTnC) and an adult rat cTnT mutant, in which both endogenous tryptophan at positions 237 and 288 were converted into phenylalanine, were overexpressed in Escherichia coli BL21(DE3) (Invitrogen) and purified as previously described (7-9). Three constructs of mouse cTnI each containing a single tryptophan and a single cysteine were generated: 129W/160C, cTnI(129W/167C), and cTnI(150W/167C). In these cTnI mutants, the endogenous Trp192 was changed to Phe and the two endogenous Cys81 and Cys98 were mutated to Ser and Ile, respectively. Construction of cTnI mutant clones, protein expression, purification, and characterization of the expressed proteins were as described (10). The single-cysteine cTnI mutants were labeled with 5-(iodoacetamidoethyl)aminonaphthelene-1-sulfonic acid in the presence of 6 M urea (11). The degree of labeling was found to be >0.97 mol of probe/mol of protein. Binary (cTnC-cTnI) and ternary (cTnC-cTnI-cTnT) troponin complexes were reconstituted by incubation at 4 °C for 12 h in the presence of 6 M urea, 30 mM MOPS, pH 7.2, 1 mM dithiothreitol, 5 mM Ca2+ at a ratio of [cTnI]:[cTnC] = 1:3 for the binary complexes and [cTnI]:[cTnT]:[cTnC] = 1:1.2:3 for the ternary complexes. The samples were brought to working buffer conditions (50 mM MOPS, pH 7.2, 1 mM dithiothreitol, 1 mM EGTA, 5 mM MgCl2, 0.15 M KCl) through a series of dialysis steps as described (12). The protein concentrations were determined using the Bradford method. Mutant function was assessed through Ca2+ titrations performed on the binary complexes. The measurements yielded pCa50 values of 6.19 and 6.12 for cTnI mutants cTnI(129W/160C) and cTnI(129W/167C), respectively. These values were similar to those previously reported with wild type cTnI (8).

Fluorescence Measurements-- Steady-state fluorescence measurements were carried out at 20 ± 0.1 °C on an ISS PC1 photon-counting spectrofluorometer, using a 2-nm band pass on both the excitation and emission monochromators (7). The emission spectra were corrected for variation of the detector sensitivity with wavelength and for sample scattering. The spectral measurements were collected using 5 µM protein samples. Ca2+ saturation (2 mM total Ca2+) was achieved by addition from a 0.1 M CaCl2 stock solution. Quantum yields of tryptophan residues were obtained by comparison with known values as described (13).

Time-resolved fluorescence intensity and anisotropy decays were measured at 20 ± 0.1 °C with an IBH 5000 photon-counting lifetime system equipped with a very stable flash lamp operated at 40 kHz in 0.5 atm of hydrogen. FRET measurement requires data collection from donor-only and donor-acceptor samples. For both, fluorescent emission from the donor was collected into 1,024 channels of a multichannel analyzer. Donor-only and donor-acceptor data were obtained under identical experimental conditions. Donor-only decays were analyzed as a sum of exponential terms. Donor-acceptor decays were analyzed as a static Gaussian distribution of distances using the subroutine GAUDIS in the package CFS_LS (cfs.umbi.umd.edu/cfs/software/index.html) (11, 14). The reduced chi squares ratio (chi <UP><SUB><IT>R</IT></SUB><SUP>2</SUP></UP>) was used to judge goodness of fit for the distribution. Anisotropy decays of both donor and acceptor were determined by measuring the emission polarized in the vertical and horizontal directions with vertically polarized excitation. The polarized decay data were fitted to a biexponential function to recover the limiting anisotropy at zero time (13). For all time-resolved FRET measurements, the donor, tryptophan, was selectively excited at 295 nm, and its emission was collected near its 333 nm maximum with a 340-nm interference filter. For all of the samples, the acceptor, when present, was the extrinsic fluorophore AEDANS covalently linked to a single cysteine residue. AEDANS maximum absorbance is near 340 nm. Its emission maximum is near 480 nm. The donor-acceptor overlap integral was determined for each probe pair under each experimental condition. The overlap integrals and the donor-only quantum yields enabled calculation of the Förster distance (Ro) for each experimental condition (Mg2+-saturating, Ca2+-saturating, and denatured).

Structural Modeling-- Both the Mg2+ and the Ca2+ models had a total of 14 rigid pseudoatoms. The models were optimized using 100,000 steps of rigid body Monte Carlo with simulated annealing from 400 to 0 K. The force field consisted of bonds between successive residues, a volume exclusion term, and a term to represent the FRET distances. Bond potential energies were calculated using E<UP><SUB><IT>ij</IT></SUB><SUP><IT>b</IT></SUP></UP> kb(rij - db)2, where kb is the force constant (200 kcal/mol/Å2), rij is the distance between atoms i and j, and db is the optimal bond length (3.79 Å). The potential energy for the volume exclusion term was expressed by E<UP><SUB><IT>ij</IT></SUB><SUP><IT>nb</IT></SUP></UP> knb(rij - dnb)2, if rij < dnb, or E<UP><SUB><IT>ij</IT></SUB><SUP><IT>nb</IT></SUP></UP> = 0, where knb is the nonbonded force constant (50 kcal/mol/Å2) and dnb is the nonbonded contact distance (3.79 Å). Long range distance constraints based on FRET measurements were expressed by the following equation.


E<SUB><UP>fret</UP></SUB>=<FENCE><AR><R><C><UP>k</UP><SUB><UP>fret</UP></SUB>(d−d<SUB><UP>low</UP></SUB>)<SUP>2</SUP></C><C><UP>d</UP><SUB><UP>low</UP></SUB>>d</C></R><R><C><UP>0</UP></C><C><UP>d</UP><SUB><UP>low</UP></SUB>>d>d<SUB><UP>high</UP></SUB></C></R><R><C><UP>k</UP><SUB><UP>fret</UP></SUB>(d−d<SUB><UP>high</UP></SUB>)<SUP>2</SUP></C><C><UP>d>d</UP><SUB><UP>high</UP></SUB></C></R></AR></FENCE> (Eq. 1)
where dlow = dobs - delta  and dhigh = dobs + delta , and kfret was set to 20 kcal/mol/Å2. dobs is the observed FRET distance + 4 Å (to correct for probe-Calpha offset) (see Table III), and delta  was set to 1.5 Å for distances B and C and to 0 for the shorter and more reliably measured distance D. For the Ca2+-saturated distance B, no upper bounds were set (kfret = 0). Mg2+- and Ca2+-saturated models were optimized to near zero energy. The reported models are representative of three different energy-minimized structures.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Fluorescence Properties of cTnI Mutants-- Three intra-cTnI distances were studied (Fig. 1) in the Mg2+- and Ca2+-saturated states: distance A (residues 150-167), distance B (residues 129-167), and distance C (residues 129-160). Distance A spans the cTnI-R. Distances B spans both cTnI-R and cTnI-I. Distance C spans cTnI-I and the N-terminal 10 residues of cTnI-R. Tryptophan at residue 129 or 150 served as energy transfer donors. Cysteines at residues 160 or 167 were selectively labeled with the acceptor probe AEDANS. The steady-state and time-resolved fluorescence properties of the donor tryptophan are summarized in Table I.


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Fig. 1.   Partial sequence of cTnI. cTnI inhibitory (red) and regulatory (blue) regions, including the actin-binding segment (underbar) are shown. FRET pairs were engineered to measure distances: A (residues 150-167), B (residues 129-167), and C (residues 129-160). Distance D (residues 129-152) was measured previously (7).

                              
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Table I
cTnI tryptophan emission properties

Trp129 emission properties were nearly equivalent in the two mutants cTnI(W129/C160) and cTnI(W129/C167) (Table I). Trp129 is sensitive to both cTnT binding and to Ca2+ addition. When denatured, the Trp129 mutants had low quantum yields (0.11), and they decayed biexponentially with mean lifetimes of 3.13-3.14 ns. In working buffer, the decays were triexponential, and the mean lifetimes increased to about 4.2 ns. A small increase in quantum yield was also observed. Ca2+ addition to the binary complexes increased the Trp129 quantum yield by an average of 64% (0.17-0.27 for cTnI(W129,C160) and 0.16-0.27 for cTnI(W129,C167)). There were small increases in the mean lifetimes. Trp129 decayed biexponentially in ternary cTn. In the Mg2+-saturated state, cTnT increased both the quantum yields (0.17-0.22 for cTnI(W129,C160) and 0.16 to 0.23 for cTnI(W129,C167)) and the mean lifetimes (4.20-4.39 ns for cTnI(W129,C160) and 4.18-4.41 ns for cTnI(W129,C167)). These results suggest that cTnT either sterically or allosterically shields Trp129 from the solvent. Ca2+ addition to ternary cTn increased the Trp129 quantum yield to 0.31 and increased its mean lifetime to 4.62 ns in cTnI(W129/C160) and to 4.68 ns in cTnI(W129/C167). The substantial quantum yield increases, and blue spectral shifts suggest that Ca2+ addition causes the transfer of residue 129 from a partially buried to a highly buried conformation. Ca2+-induced environmental changes in cTnI Trp150 are more difficult to interpret. Ca2+ binding to the binary and ternary complexes containing Trp150 produced significant blue shifts in the emission spectra. This change was in the same direction as that observed for Trp129, suggestive of a less exposed environment. However, the binding also induced decreases in the quantum yield and mean lifetime. Subsequent to Ca2+ addition, Trp150 emission is likely partially quenched not by the solvent but by adjacent residues (15).

Ca2+ addition caused AEDANS-labeled cTnI160 in binary cTn to undergo a blue shift of 2.1 nm (484.4-482.3 nm) and experience an 8.6% enhanced peak intensity. As a member of the ternary cTn complex, Ca2+ addition induces a 1.9-nm blue shift and a 13% increase in maximum intensity. In contrast, AEDANS, when attached to Cys167 as a member of the binary troponin complex, undergoes a Ca2+-induced red shift of 1.4 nm (483.3-484.7 nm) and an intensity decrease of 12.3%. Similar features were observed in the ternary complex. In the presence of the dipolar solvent water, probe emission occurs from lower vibrational energy levels within the singlet excited state. Emitted photons contain less energy on average, causing an observable red shift in the emission maximum. These data indicate that Ca2+ addition causes cTnI residue 167 at the C terminus of cTnI-R to migrate from a less to a more polar environment. Ca2+ activation causes residue 160 to become buried.

Steady-state Förster Resonance Energy Transfer-- We have examined FRET using both steady-state and time-resolved methods. The impact of energy transfer between donor Trp129 and acceptor AEDANS attached to Cys160 (distance C) on steady-state emission spectra is shown in Fig. 2. Trp emission is observed as a peak near 330 nm. A portion of the photons absorbed by Trp is nonradiatively transferred to AEDANS. The AEDANS fluorescent emission of the transferred photons (sensitized emission) is observed as a peak near 480 nm. When denatured, donor and acceptor move apart on average, resulting in decreased transfer efficiency (Fig. 2a), because less donor energy is lost to the acceptor. The donor fluorescence increases (toward the level observed in the absence of acceptor), and the sensitized acceptor emission decreases. The addition of Ca2+ elicits an effect similar to denaturation in the binary (Fig. 2b) and ternary (Fig. 2c) complexes. Ca2+ increases the interprobe distance. Distance B underwent Ca2+-induced changes similar to those observed for distance C (data not shown). In contrast to the large Ca2+-induced distance changes observed for distances B and C, distance A was quite insensitive to Ca2+ (Fig. 3). Distance B, which spans both cTnI-I and cTnI-R, encompasses residues in distance A (cTnI-R). The steady-state FRET data implicates cTnI-I as the region undergoing large Ca2+-elicited structural changes. The steady-state spectral data provide an initial indication of substantial Ca2+-induced conformational change in cTnI-I. Time-resolved measurements quantified these initial findings.


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Fig. 2.   Steady-state fluorescence emission spectra of cTnI(129W/160C) labeled with AEDANS used to measure distance C. a, free cTnI in +Mg2+ buffer (solid line) and in +Mg2+ buffer + 4 M GdnHCl (dotted line). b, binary complex (cTnC-cTnI) in +Mg2+ buffer (solid line) and in +Ca2+ buffer (dotted line). c, ternary complex (cTnC-cTnI-cTnT) in +Mg2+ buffer (solid line) and in +Ca2+ buffer (dotted line). Excitation was at 295 nm. +Mg2+ buffer was 50 mM MOPS, pH 7.2, 1 mM dithiothreitol, 1 mM EGTA, 5 mM MgCl2, 0.15 M KCl; +Ca2+ buffer was +Mg2+ buffer with 2 mM CaCl2.


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Fig. 3.   Steady-state fluorescence emission spectra of cTnI(150W/167C) labeled with AEDANS used to measure distance A. a, binary complex (cTnC-cTnI) in +Mg2+ buffer (solid line) and in +Ca2+ buffer (dotted line). b, ternary complex (cTnC-cTnI-cTnT) in +Mg2+ buffer (solid line) and in +Ca2+ buffer (dotted line). The conditions are the same as for Fig. 2.

Time-resolved Förster Resonance Energy Transfer-- Fig. 4 shows the intensity decays of Trp129 used to make the distance C measurement in ternary cTn. Unless otherwise indicated, we shall confine our attention to the most physiologically relevant complex, ternary cTn. In the Mg2+-saturated state, the donor lifetime (Fig. 4, black) is appreciably quenched (Fig. 4, red) through FRET. Upon saturation with Ca2+, the donor lifetime (Fig. 4, blue) is unquenched by the acceptor (Fig. 4, magenta). Ca2+ does not appreciably alter the donor-only lifetime (Fig. 4, black versus blue). Visual inspection of the data indicates a large Ca2+-induced extension of distance C. The recovered distance distribution parameters are reported in Table II. The distribution is parametrized by a mean distance and a full width at half-maximum (half-width). The mean distance C in the Mg2+-saturated ternary complex is 24.5 Å, a distance similar to that observed in free cTnI and in the Mg2+-saturated binary complex. When the donor-acceptor probe mean distance is within the range Ro = 0.5-1.5, the lifetime decay data usually contain sufficient information to resolve the first and second moments of the donor-acceptor distance distribution. Beyond this range it may be possible to recover the mean distance but with less precision. In the Ca2+-saturated case shown, the transfer efficiency is very small, and upon analysis the interprobe distance is observed to be 1.63. The half-width is unattainable. Ca2+ binding causes a 14.7 Å increase in distance C. Similar results were obtained for distance B. Upon Ca2+ saturation, the distance increased from 26.8 Å to 45.7 Å, a change of 18.9 Å. Distance A was not changed by Ca2+ addition, its value remaining near 22.7 Å. The data confirm the observations obtained from steady-state data. Ca2+ activation induces a major structural rearrangement in cTnI-I, whereas the cTnI-R is unaltered.


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Fig. 4.   Representative fluorescence intensity decay curves of Trp129 of cTnI(129W/167C) used to measure distance B. a, free cTnI with unlabeled Cys167 (black) or AEDANS-labeled Cys167 (red). b, ternary complex (cTnC-cTnI-cTnT) with unlabeled Cys167 and Mg2+-saturated (black), unlabeled and Ca2+-saturated (blue), AEDANS-labeled Cys167 and Mg2+-saturated (red), and AEDANS-labeled Cys167 and Ca2+-saturated (green). Donor-only decays nearly superimpose with each other and with the Ca2+-saturated donor-acceptor decay. The conditions are the same as for Fig. 2.

                              
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Table II
cTnI FRET distances

To examine the uncertainty in the mean distance values for distances B and C, one-dimensional chi <UP><SUB>R</SUB><SUP>2</SUP></UP> grid searches were performed by manually varying the distribution mean distance and minimizing around the remaining parameters (Fig. 5) (16, 17). Intersection of the curve with the 68% confidence F statistic (Fig. 5, horizontal line) determines the lower and upper error bounds for the mean distance. The surfaces are not symmetric. In the absence of bound Ca2+, the surfaces are sharp, and the mean distances can be specified to within 1-Å resolution with 68% confidence. In the presence of bound Ca2+, the surfaces are shallow, indicating poor resolution in the recovered mean distance. For both distances B and C, the upper limit on the Ca2+-saturated distance is large (distance B, 9 Å; distance C, 5 Å).


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Fig. 5.   Confidence estimates in the recovered mean distances. A grid search yielded the dependence of the variance ratio (chi <UP><SUB><IT>R</IT></SUB><SUP>2</SUP></UP>/F) on the distance distribution mean distance. The data are from ternary complexes. a, distance C in the Mg2+- and Ca2+-saturated states. b, distance B in the Mg2+- and Ca2+-saturated states. The value of the mean distance (R) was held constant at the value indicated on the horizontal axis, and the half-width (hw) was varied to minimize chi <UP><SUB><IT>R</IT></SUB><SUP>2</SUP></UP>; all other parameters were fixed. The dotted horizontal line shows the 68% confidence level error estimates for the mean distance.

To calculate the Ro, a value of 2/3 was assumed for the orientation factor (kappa 2). This value is based on the assumption that the donor and acceptor probes tumble rapidly and randomly over the course of the measurement (dynamic averaging). One way of testing this assumption is to measure the axial depolarization factors of donor and acceptor probes (18). Time-resolved anisotropy decays were obtained for both donor tryptophan and acceptor AEDANS as members of the protein complexes. The limiting anisotropies of donor and acceptor were determined from the decay profiles. These values were used to calculate the depolarization factors shown in Table II. The depolarization factors of both donor and acceptor in free cTnI and in the binary and ternary complexes were similar. Ca2+ had a negligible effect on the depolarization factors. These results suggest that Ca2+ binding had little effect on the orientational freedom of the probes.

Model of cTnI Regulatory and Inhibitory Regions-- Full atom models of the 39-residue cTnI fragment corresponding to residues 129-167 were built using a peptide builder (Insight 2). The Ca2+-saturated state was modeled as a continuous native alpha -helix. The Mg2+-saturated state was represented as a alpha -helix (residues 1-9)/beta -turn (residues 10-21)/alpha -helix (residues 22-39). Model residues 1-9 correspond to the N terminus of the inhibitory region (TnI-I), believed to form a stable alpha -helical coiled-coil with TnT (19, 20). Residues 10-21, corresponding to the C terminus of cTnI-I, associate with the actin filament in the Mg2+-saturated state. Model residues 22-39 encompass the regulatory region (TnI-R) that our present data (distance A) suggest is alpha -helical. The FRET distances obtained in this and our previous study (7) were used to set long range constraints. The starting models did not meet the FRET-derived distance criteria. Reduced representation models were constructed using the computer program YAMMP (21), by representing amino acid residues as alpha -carbon-centered pseudoatoms. The models were further reduced by treating the alpha -helical segments 1-9 and 22-39 as rigid units. The remaining residues (residues 10-21) were treated as rigid bodies forming a random coil. Distances B, C, and D were used to impose long range energy constraints. Representative energy-minimized Mg2+- and Ca2+-saturated models are shown in Fig. 6. The two states have been superimposed along the regulatory region. Table III shows the agreement between the FRET-derived and model-based distances.


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Fig. 6.   Reduced atom models of the TnI-I and TnI-R regions in the Mg2+- and Ca2+-saturated states. Stereo views of the Mg2+- (blue) and Ca2+-saturated (red) states, which are superimposed along the regulatory region. The N-terminal portion of TnI-I (white) is represented as an alpha -helix. TnI-R (green) is alpha -helical in both Mg2+ and Ca2+ states (distance A). Distances between the Calpha of residue 129 (white sphere) and residues 152, 160, and 167 (green spheres) were used to impose long range energy constraints on the models. The addition of Ca2+ causes the C terminus of TnI-I to undergo a beta -turn/coil (blue) to quasi-alpha -helix (red) transition.

                              
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Table III
Comparison of model and observed FRET distances


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Ca2+-dependent interaction between TnC and TnI is the key triggering step in striated muscle contraction. Despite years of intense investigation in many laboratories (22-27), the molecular details of the Ca2+-switch mechanism in cardiac troponin remain incompletely understood. To elucidate the switching mechanism, it is necessary to understand intersubunit interactions within the troponin complex as well as the conformational transitions that occur within the individual troponin subunits. We have previously reported a large Ca2+-induced extension of cTnI-I in reconstituted binary (cTnC-cTnI) and ternary (cTnC-cTnI-cTnT) complexes. We proposed that the Ca2+-induced extension of cTnI-I accommodates the movement of the adjacent cTnI-R toward the Ca2+-exposed hydrophobic patch on N-cTnC (8, 28). TnI-I release from actin (26) is apparently required for its extension. The switching mechanism therefore involves movement in TnI-I, TnI-R, and N-TnC. These structural arrangements are coupled with the transition of tropomyosin from the blocked (B) to the Ca2+-induced (C) states (29).

The present study has focused on Ca2+-induced changes in cTnI-R itself and the relative movements between cTnI and cTnI-R. In particular, we were interested in whether Ca2+-induced cTnI conformational changes were confined to cTnI-I or extended into the adjacent cTnI-R. Three engineered FRET pairs were used to measure three different intra-cTnI distances under Mg2+- and Ca2+-saturating conditions: distance A (residues 150-167) spanned only cTnI-R, distance B (residues 129-167) spanned both cTnI-R and cTnI-I, and distance C (residues 129-160) spanned cTnI-I and the N-terminal portion of cTnI-R extending approximately to its midpoint. Distance A was unchanged by Ca2+ addition. The FRET measured mean interprobe distances (Mg2+, 22.7 Å; Ca2+, 22.6 A) were within 4 Å of the Calpha -Calpha distance of a 18-residue native alpha -helix (26.4 Å). The ~4-Å distance difference can be attributed to donor and acceptor probe-Calpha offset distances. NMR studies on the Ca2+-saturated complexes formed between N-cTnC and the cTnI-R peptide indicate that the C terminus of the peptide (residues 150-157) is unstructured (28). Our results, based on full-length cTnI, indicate that cTnI-R is alpha -helical throughout. This was observed in both cTnC-cTnI and cTnC-cTnI-cTnT complexes under Mg2+- and Ca2+-saturating conditions. The secondary structure of cTnI-R under Mg2+ saturation has not been previously reported.

TnI-R Is Unbound in the Mg2+-saturated State-- An NMR study of the Ca2+-saturated cTnC-TnI-R peptide complex suggests that the central region of TnI-R binds to the N-TnC hydrophobic patch, whereas the C terminus of TnI-R extends beyond the patch in a solvent-exposed position (28). The spectral properties of AEDANS-labeled cTnI residues 160 and 167, indicating that residue 160 is buried and that residue 167 is solvent-exposed under saturating Ca2+, are compatible with this model. The spectral and quantum yield properties of AEDANS-labeled cTnI160 in the Mg2+-saturated state are similar to the solvent-exposed AEDANS-labeled Cys167 in the Ca2+-saturated state. These results suggest that the central portion of cTnI-R is unbound and solvent-exposed in the Mg2+-saturated state.

Relative Movement of the cTnI-I and cTnI-R Regions-- We have previously reported the Ca2+-induced distance changes between 24 residues (129-152) spanning cTnI-I (distance D). The Ca2+-saturated distance D, when crudely corrected from probe-Calpha offset by adding 4 Å to the FRET distance, (32.5 A), closely matches that of a 24-residue alpha -helix (33.7 A). The present measurements extend this work by retaining the donor position (Trp129) and placing the acceptor at residues 160 (distance C) and 167 (distance B). The large Ca2+-induced changes in distances B (18.9 Å) and C (14.7 Å) corroborate our earlier results. We have constructed a reduced representation molecular model of cTnI-I/cTnI-R in the Mg2+- and Ca2+-saturated states (Fig. 6) consistent with distances A-D (Fig. 1 and Table III). According to the model, cTnI-I/cTnI-R exists as an extended nearly continuous alpha -helix in the Ca2+-saturated state. The cTnI-R portion of the helix (residues 150-166) is supported by contacts with N-cTnC (28). Meanwhile, the N-terminal portion of cTnI-I associates with the C-domain of cTnC. This arrangement requires that the C-terminal portion of cTnI-I, which is accessible to solvent (19), bridges the N- and C-terminal cTnC domains, running roughly anti-parallel to the cTnC linker region. In the Mg2+-saturated state, cTnI-R remains alpha -helical. The C-terminal portion of cTnI-I (residues 138-149) have been previously modeled as a beta -turn (30) and presently modeled as a loop. This C-TnI-I sequence has been identified as the minimum length peptide capable of inhibiting actin-myosin ATPase (31, 32). The N-terminal portion of cTnI-I (residues130-137) likely remains associated with cTnC in the Mg2+-saturated state. We propose that Ca2+ activation causes an internal beta -turn/loop to alpha -helix transition in the actin-binding portion of the TnI inhibitory region. The extension in TnI-I caused by this secondary structure transition allows the TnI inhibitory region to span the TnC linker region, which is required for the adjacent TnI regulatory region to access sites on N-TnC.

Movement of TnI-I and TnI-R Relative to the Actin Filament-- For ease of visualization in Fig. 6, we have aligned the Mg2+- and Ca2+-saturated models along cTnI-R, perhaps giving the impression that cTnI-R hardly moves with respect to the actin surface. This is not the case. In the model, the transition of cTnI-I from a beta -turn/coil to bent alpha -helix is associated with a 19.7 Å increase in the intra cTnI-I/cTnI-R separation (distance B). In the intact thin filament, sTnI133 (cTnI167) is observed to move by 23.6 Å relative to actin filament,2 and this movement is predominantly in the plane of the actin surface. N-cTnI-I is believed to remain fixed to cTnT/C-cTnC (19, 20), and the proximity of the C- and N-cTnC domains is essentially unaltered by Ca2+ addition (33). Assuming a relatively fixed position of N-TnC relative to the actin filament (34), the 19.7 Å increase in distance B can be attributed to the translation of cTnI-R into the N-cTnC hydrophobic pocket. Narita et al. (34) have proposed that Ca2+ activation induces a ~50-Å TnC C-domain repositioning on the surface of actin, which would also reposition N-cTnI-I on the actin surface.

In summary, we have shown a large Ca2+-induced extended conformation of the contiguous inhibitory and regulatory regions of cTnI in cTn. This conformational transition arises from an extension of the inhibitory region and an orientational change of the regulatory region away from the inhibitory region. The regulatory region does not experience a Ca2+-induced increase in its end-to-end distance. The present results strengthen our previous proposal that the inhibitory region in the cTnC-cTnI interface provides the main Ca2+-induced switching mechanism between relaxation and activation. The transition of the inhibitory region of cTnI from a helix-loop-helix motif to a more extended conformation enables TnI-I to span distance between the N- and C-cTnC domains. This switching event places TnI-R near the N-domain of cTnC where it can bind to the activation-exposed hydrophobic patch.

    ACKNOWLEDGEMENT

We thank Dr. Takeda for discussing Tn Calpha -Calpha distances prior publication.

    FOOTNOTES

* This work was supported in part by Grant HL52508 from the National Institutes of Health (to H. C. C.) and by Grant 0330170N from the American Heart Association (to W. J. D.) and by the University of Alabama at Birmingham M.D./Ph.D. program (to J. M. R.).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.

Dagger These authors contributed equally to this work.

§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Genetics, University of Alabama, Rm. 490, MCLM, 1530 3rd Ave. S., Birmingham, AL 35294-0005. Tel.: 205-934-2485; Fax: 205-975-4621; E-mail: hccheung@uab.edu.

Published, JBC Papers in Press, January 2, 2003, DOI 10.1074/jbc.M212886200

2 J. M. Robinson, W. J. Dong, and H. C. Cheung, unpublished result.

    ABBREVIATIONS

The abbreviations used are: Tn, troponin; TnC, troponin C; N-TnC, N-domain of troponin C; TnI, troponin I; TnI-I, troponin I inhibitory region; TnI-R, troponin I regulatory region; TnT, troponin T; c, cardiac muscle; cTnI(129W/160C), mutant cTnI(C81S/C98I/L129W/L160C/W192F); cTnI(129W/167C), mutant cTnI(C81S/C98I/L129W/S167C/W192F); cTnI(150W/167C), mutant cTnI(C81S/C98I/I150W/S167C/W192F); FRET, Förster resonance energy transfer; MOPS, 3-(N-mopholino)propanesulfonic acid; GdnHCl, guanidine hydrogen chloride; AEDANS, aminonaphthelene-1-sulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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