In the presence of inhibitory peptide at relatively lower concentrations the myosin peptides and a troponin I peptide C-terminal to the inhibitory region, rcTnI161–181, all dissociate fr
Trang 1The inhibitory region of troponin-I alters the ability of F-actin
to interact with different segments of myosin
Valerie B Patchell1, Clare E Gallon2, Matthew A Hodgkin3, Abdellatif Fattoum4, S Victor Perry1
and Barry A Levine1,2
1
Department of Physiology, School of Medicine and2School of Biosciences, University of Birmingham, Birmingham, UK;3School
of Biological Sciences, University of Warwick, Warwick, UK;4CRBM, CNRS, INSERM U249, F-34090 Montpellier, France
Peptides corresponding to the N-terminus of skeletal
myosin light chain 1 (rsMLC1 1–37) and the short loop of
human cardiac b-myosin (hcM398–414) have been shown
to interact with skeletal F-actin by NMR and fluorescence
measurements Skeletal tropomyosin strengthens the
binding of the myosin peptides to actin but does not
interact with the peptides The binding of peptides
cor-responding to the inhibitory region of cardiac troponin I
(e.g hcTnI128–153) to F-actin to form a 1 : 1 molar
complex is also strengthened in the presence of
tropomyo-sin In the presence of inhibitory peptide at relatively
lower concentrations the myosin peptides and a troponin I
peptide C-terminal to the inhibitory region, rcTnI161–181,
all dissociate from F-actin Structural and fluorescence
evidence indicate that the troponin I inhibitory region and
the myosin peptides do not bind in an identical manner to F-actin It is concluded that the binding of the inhibitory region of troponin I to F-actin produces a conformational change in the actin monomer with the result that inter-action at different locations of F-actin is impeded These observations are interpreted to indicate that a major conformational change occurs in actin on binding to troponin I that is fundamental to the regulatory process in muscle The data are discussed in the context of tropo-myosin’s ability to stabilize the actin filament and facilitate the transmission of the conformational change to actin monomers not in direct contact with troponin I
Keywords: Cardiac troponin I, tropomyosin, myosin pep-tides, actin, conformational change
The property of troponin I (TnI) of being able to inhibit the
MgATPase of actomyosin in a manner that can be
neutralized by the calcium-binding protein troponin C in
the presence of calcium ions suggests that TnI occupies a
key position in the regulation of contraction in striated
muscle In the absence of tropomyosin and the other
components of the troponin complex, TnI inhibits the
MgATPase of actomyosin maximally when there is
approximately one molecule of TnI per actin monomer
[1,2] This implies that TnI prevents the interaction of actin
with the myosin head that leads to the activation of the
MgATPase In the presence of tropomyosin, the inhibitory
influence of TnI is much increased and the maximum effect
is obtained when the stoichiometry approaches one
mole-cule of TnI to seven actin monomers [1–5] When troponin
C and troponin T are absent this inhibition is calcium
insensitive [6] but otherwise corresponds to the off state in
intact muscle
The region of rabbit fast skeletal TnI represented by
residues 96–116, known as the inhibitory peptide (IP),
possesses properties that are very similar to the intact molecule in that it binds to troponin C, and in the presence of tropomyosin the inhibition of the MgATPase
of actomyosin by the peptide is markedly increased [7] The inhibitory peptide in the presence of tropomyosin is about 50% as effective as the intact TnI molecule when assayed under similar conditions Only about half of the residues of IP, as originally isolated, appear to be essential for this property because a synthetic duodecapeptide comprising residues 104–115 (short IP) has equivalent inhibitory activity [8] Recent evidence suggests that additional regions of TnI, C-terminal to the IP, may be required to obtain inhibitory activity equal to the intact molecule [9,10]
The mechanism of action of TnI on the regulation of the contractile process is not as yet understood (see [11] for a review) Despite the inhibitory properties of TnI the current view is that tropomyosin regulates the actomyosin ATPase in situ by a steric mechanism [12–14] and it has been suggested that the role of TnI is to induce the binding of tropomyosin to actin [3] Nevertheless the ability of TnI to bind to actin must be of special significance, as by blocking the binding site it could prevent the interaction with myosin that leads to the activation of the MgATPase Alternatively binding could involve an allosteric mechanism whereby a conformational change is induced in the actin monomer that results in regions elsewhere on the molecule no longer being able to interact with myosin to activate the MgATPase Any proposed mechanism must explain the ability of tropo-myosin to extend the inhibitory activity of the troponin I molecule from one to seven actin monomers
Correspondence to S V Perry, Department of Physiology, School of
Medicine, University of Birmingham, Birmingham B15 2TT.
Fax: + 44 121414 6919, Tel.: + 44 121414 6930,
E-mail: S.V.Perry@bham.ac.uk
Abbreviations: IP, inhibitory peptide; TnI, troponin I; ATPase,
adenosinetriphosphatase; MLC1, myosin light chain 1; IAEDANS,
5-((((2-iodoacetyl)amino)ethyl)amino)napthalene-1-sulfonic acid;
SPR, surface plasmon resonance
(Received 14 June 2002, revised 19 August 2002,
accepted 5 September 2002)
Trang 2The nature of the interaction of the myosin head with
actin is still a matter for discussion but it is clearly complex
and may involve more than one actin monomer (for review
see [15]) The major contacts of myosin with actin appear to
involve several regions of the myosin heavy chain It is
considered that there is a large primary contact site on the
surface of actin flanked on three sides by additional contacts
involving myosin surface loops [16] One of these loops,
Pro402–Lys415, is modelled as interacting with actin near
residues 331–332 [16] at the junction of subdomains 1 and 3
of actin and appears to be important for normal muscle
activity Deletion of this loop region resulted in the loss of
strong binding of myosin to actin [17] while a single amino
acid residue change, ArgfiGln, in this loop region of the
b-chain of human cardiac myosin is associated with familial
hypertrophic cardiomyopathy [18,19] and has been reported
to result in altered kinetic properties of the myosin
subfragment 1 ATPase [20]
Although there is no doubt that tropomyosin moves on
contraction it is difficult, in view of the somewhat limited
knowledge of the nature of the actin–myosin interaction, to
decide on the role of actin in the activation process X-ray
analysis provides some evidence for movement of the actin
domains during contraction [21] and it is likely that in model
systems using mutant proteins the movement of
tropo-myosin observed in the presence of tropo-myosin and troponin is a
consequence of conformational changes in actin [22,23] The
binding of ligands at discrete and specific binding sites on
actin during the contractile cycle would be expected to
induce conformational changes that influence its interaction
with myosin Cross linking studies with the zero length
carbodiimidate reagent specific for lysine–carboxylate
con-tacts suggest that one such ligand, TnI, binds close to the
region represented by residues 1–12 of actin [24] Proton
MR studies have indicated that IP interacts with residues
1–7and 24–25 of the N-terminal region of actin [25] These
observations and the fact that only about half of the
residues of the IP are required for inhibitory activity suggest
that the interaction of only a small region of TnI with the
N-terminal region of actin is the minimum requirement to
prevent activation of the myosin MgATPase
To throw light on the role of TnI and its relation to that of
tropomyosin in the regulatory process we have studied how
the actin-binding properties of peptides representing regions
of the myosin molecule are affected by the interaction of
actin with peptides incorporating the inhibitory domain of
TnI The myosin peptides are displaced from F-actin by the
IP but not by tropomyosin; indeed their binding is strengthened in the presence of the latter protein Evidence
is provided for the induction of conformational changes in
at least two regions of the actin molecule on binding the inhibitory domain of TnI to a third independent site These observations have important significance for understanding the role of TnI in the regulation of contraction in striated muscle
Some aspects of this work have been briefly described in abstract form [26]
M A T E R I A L S A N D M E T H O D S
Peptides The N-terminal region of the myosin light chain (MLC1), residues 1–37, was prepared as described by Henry et al [27] The peptides encompassing the inhibitory region of human cardiac TnI, hcTnI128–153, dansylated hcTnI128–
153 (T128 replaced by a dansyl group), hcTnI136–148 and the other peptides used in this study (Table 1) were synthesized by Alta Bioscience (Birmingham University) using Fmoc polyamide chemistry and purified as described previously [28] The peptide comprising residues 398–414 of human cardiac b-myosin was synthesized by Syntem (Montpellier) and purified as reported previously [29] The composition and purity of all peptides was confirmed by NMR and mass spectral analysis
Muscle proteins Freeze dried actin prepared by the method of Spudich and Watt [30] was dissolved in 5 mM triethanolamine/HCl,
pH 8.0, 0.2 mMCaCl2, 0.2 mMATP, 0.2 mMdithiothreitol, and dialysed for 3 h against the same buffer until fully depolymerized It was then centrifuged at 30 000 g for
30 min and the concentration of the G-actin in the supernatant determined by measuring absorbance at
290 nm using an absorption coefficient of 0.63 mgÆ
mL)1Æcm)1 The G-actin was polymerized by making the solution 2 mM with respect to MgCl2 and 50 mM with respect to KCl The F-actin was then dialysed overnight against several changes of 5 mMsodium phosphate buffer,
pH 7.0, in H2O or [2H]2O F-actin–tropomyosin complex was made by adding G-actin prepared as described above to
Table 1 Peptides used in this study Unless otherwise indicated the N-terminus is acetylated and the C-terminus is in the amide form The N-terminus of the long MLC1 peptide is trimethylalanine The trimethylalanine of wild type MLC 1–13 was replaced by N-acetyl alanine The HA peptide corresponds to the well characterized immunodominant epitope of influenza haemagglutinin, residues 306–318 TnI numbering based on N-acyl terminus as occurs in vertebrate proteins and not methionine as occurs in recombinant TnI.
Trang 3a stock solution of 1 mgÆmL)1rabbit skeletal tropomyosin
in 50 mMTris/HCl, pH 7.6, 100 mM KCl, to give a final
concentration of 2.5 mgÆmL)1 actin, 0.5 mgÆmL)1
tropo-myosin, i.e a molar ratio of actin : tropomyosin of
approximately 7: 1 The complex was dialysed into several
changes of 5 mM phosphate buffer, pH 7.0, in H2O or
[2H]2O Complex formation and the absence of free protein
was confirmed by comparison of the electrophoretic
patterns of the free proteins by electrophoresis on 10%
nondenaturing polyacrylamide gels run in 10% (v/v)
glycerol, 25 mMTris)80 mMglycine, pH 8.3
Actin labelled at Cys374 with IAEDANS was prepared
according to the method of Miki et al [31] F-actin
(2 mgÆmL)1) in 5 mMtriethanolamine/HCl, pH 8, 0.2 mM
ATP, 0.2 mMCaCl2(Buffer A) to which 50 mMKCl and
2 mMMgCl2had been added was incubated with 10-fold
molar excess of IAEDANS for 2 h at room temperature
The reaction was terminated by the addition of dithiothreitol
to 2 mM The actin was then centrifuged at 100 000 g for 1 h
and the pellet resuspended in Buffer A This was dialysed
extensively against Buffer A to remove excess IAEDANS
The concentration of the resulting G-actin was determined
using an absorption coefficient of 0.63 mgÆmL)1Æcm)1 at
290 nm A correction for the IAEDANS contribution at
290 nm was made using absorbance at 290 nm¼
0.21· absorbance at 336 nm, for bound IAEDANS
The concentration of IAEDANS was determined using
the absorption coefficient of 6100M )1Æcm)1at 336 nm The
extent of labelling was normally 0.8–0.9 molÆmole)1
G-actin The labelled G-actin was polymerized by making
the solution 50 mMwith respect to KCl, 2 mMwith respect
to MgCl2, and stored frozen in aliquots
Fluorescence measurements
All fluorescence emission spectra were obtained using a
Perkin-Elmer LS50B fluorescence spectrometer interfaced
to a computer The excitation wavelength for tryptophan
was 280 nm and the IAEDANS probe was excited at
340 nm The fluorescence emission spectra of the dansylated
TnI peptide was recorded between 400 and 550 nm after
excitation of the dansyl group at 340 nm Emission
fluorescence intensity values were corrected for the
corres-ponding solvent emission fluorescence values and the
dilution effects (< 5%) resulting from the titration carried
out The dissociation constants (Kd) for the complexes
formed were calculated by using a nonlinear regression
procedure fitting the fluorescence data obtained in three
separate titrations in each case to a 1 : 1 binding curve
in combination with the use of a reciprocal linear plot
(Fo)Fmin)/(Fo)F) vs (concentration of added peptide))1
For all calculations it was assumed that the fractional
change in fluorescence was directly proportional to the
fraction of the complexes formed The accuracy of the Kd
values was gauged from curve fit obtained, the associated R2
value (> 0.95) and the requirement that iterative fit of the
linear representation of the experimental data extrapolated
to an intercept value of 1
Surface plasmon resonance
Direct binding of the TnI inhibitory peptide to actin was
investigated using surface plasmon resonance (SPR)
analysis to evaluate the association and dissociation rate constants, Ka and Kd respectively, for the binding of the peptide to immobilized F-actin using a BIAcore 3000 system F-actin or BSA was covalently linked to carboxy-methyldextran surfaces using standard amine coupling One surface was derivatized in the absence of protein Following immobilization the chip surfaces were capped with ethanolamine and subject to surface equilibration (BIApplications Handbook, 1993) Non-specific binding was monitored using the control BSA and underivatized flow cells Sensorgrams were obtained using different immobilization densities and the binding of the TnI inhibitory peptide was assessed at various flow rates (5–30 lLÆmin)1) and over a range of concentrations (1–50 lM) Sensorgrams were analysed using BIAEVALUA TION 3 software taking account of the small amount of nonspecific binding of the TnI peptide to the control surfaces The association and dissociation rate constants were obtained from these sensorgrams by fitting the experimental data to a model obeying 1 : 1 complex formation and the Langmuir binding isotherm The apparent equilibrium constant (dissociation constant Kd) was calculated as Kd/Ka There was no significant change
in the Kd derived for the peptide concentrations in the range 1–10 lM Curve fitting of the dissociation phase for each concentration was also separately carried out as for
an AB complex dissociation
NMR studies The NMR spectral assignment of peptide resonances was carried out using standard TOCSY and NOESY proce-dures Spectra were obtained at 500 MHz on a Bruker spectrometer at a sample temperature of 285K Titration
of the peptides with F-actin was carried out by addition of aliquots of F-actin (10 mgÆmL)1) or F-actin–tropomyosin (5 mgÆmL)1 F-actin) Titration of the inhibitory peptide with F-actin or F-actin–tropomyosin was also carried out
by the addition of small aliquots (1–5 lL) of a stock solution of the peptide to 0.5 mL of solution containing F-actin at a concentration of 2.5–4.0 mgÆmL)1 The broad signals of the spectrum of F-actin obtained at these concentrations contributed relatively little to the spectra of the peptides in the presence of actin Two-pulse spin-echo spectra (1024 transients) were obtained using a (180-t-90-t) sequence with a delay time, t¼ 60 ms, and an overall interpulse delay of 3 s to enable complete magnetization recovery Signal amplitude in these experiments is modu-lated by the corresponding coupling constant and relax-ation time of each resonance and is a very sensitive indicator of the effect of binding As observed in previous studies of actin binding [25,28] interaction results in marked reduction of the bound peptide ligand resonances consistent with the high molecular weight and slow tumbling of the complex Both direct signal linewidth and the signal intensity in the two-pulse spin-echo spectra were used therefore to evaluate the perturbation resulting from interaction with actin The spectral changes were also visualized by difference spectra taken at each stage of the titration Quantification of the binding stoichiometry and affinity of complex formation was confirmed by equilibrium fluorescence measurements and by surface plasmon resonance studies
Trang 4R E S U L T S
The interaction of the different peptides with F-actin was
assayed using a variety of biophysical techniques to
characterize their interaction affinity, to determine the
nature of the residues involved and the extent of any
competition between the peptides on binding A diagnostic
test for the binding of a peptide ligand to F-actin or
tropomyosin is the observation of significant broadening of
the ligand resonances detected in the NMR spectrum The
flexibility in a peptide ligand is manifest in the narrow line
widths of the peptide NMR resonances since linewidth is a
monotonic function of the effective correlation time [32]
Upon complex formation the bound groups of the peptide
ligand would experience the longer rotational correlation
time of the macromolecular assembly and a decrease in
segmental mobility Interaction is therefore apparent from
changes in the linewidth of the peptide resonances since
complex formation results in an increased relaxation rate
due to the longer correlation time of the protein and the
motional constraints imposed by binding of the peptide to
the protein surface Spectral linewidth increases on addition
of F-actin to inhibitory regions of troponin I and caldesmon
have been reported elsewhere [25,33] Resonance line width
changes can also originate, however, from any increase in
solution viscosity that significantly alters the rotational
diffusion of the ligand As F-actin solutions have significant
viscosity we therefore first studied the effect of an increase in
solution viscosity on the linewidth characteristics of the IP
Minimal spectral effects were observed for the hcTnI128–
153 peptide over a concentration range of 0–500 lMin 10%
(v/v) deuterated glycerol (MSD Isotopes) These
observa-tions indicated that viscosity effects on resonance and
linewidth in the peptide spectrum were not significant
Evidence for the absence of viscosity effects on peptide
resonance and linewidth as a result the presence of actin
were obtained by comparing the spectrum of a control
peptide, the HA peptide (Table 1), in the presence and
absence of F-actin (Fig 1) The absence of detectable
alterations in the spectrum of the peptide indicated that any
changes in viscosity due to the presence of F-actin have
negligible effects on the rotational diffusion in solution and
hence linewidth of the peptide resonances These results
(Fig 1) also served as control data indicating that there was
no nonspecific HA peptide interaction with F-actin
Inspection of the spectrum of the HA peptide in the
presence of F-actin also indicates that although interaction
did not occur, there is a detectable contribution to signal
intensity deriving from F-actin at the relatively high
concentrations of the protein used in this control experiment
(Fig 1) The broad signals of the spectrum of F-actin did
not, however, mask the resonances of other peptides used in
this study due to the lower protein concentrations required
to induce spectral broadening Figure 1 shows that specific
resonance broadening occurred during titration of the
rcTnI161–181 peptide with F-actin indicating complex
formation characterized by fast exchange between the free
and actin-bound states of the peptide Most markedly
altered by interaction with F-actin are the sidechain signals
of Arg168, His170, Gln173 and the composite methyl group
resonance while the sidechain resonance deriving from the
five lysine residues of the peptide remained relatively
unperturbed Correlation with the peptide sequence
(Table 1) indicates that residues 165–174 represent that part of the peptide rcTnI161–181 whose molecular motions are most restricted by binding to the surface of F-actin The possibility of nonspecific binding of the TnI peptides
to protein was examined using BSA As shown in Fig 2 the presence of an equimolar concentration of the protein did not give rise to any significant changes in the spectrum of peptide hcTnI128–147indicating the absence of nonspecific interaction with the protein Clearly resolved in the spectra
is the composite signal of the five Arg dCH2groups of the inhibitory peptide ( 3.2 p.p.m., Fig 2) that, as in the case
of rcTnI161–181 peptide (Fig 1), can be used to monitor the effect of F-actin on the spectrum of the peptide The arginine residues of the IP are located in the central portion
of the region associated with inhibitory activity
Interaction of the cardiac inhibitory peptide region with F-actin
To investigate the interaction between actin and the inhibitory region of TnI, we monitored the NMR spectral
Fig 1 Proton magnetic resonance spectra demonstrating that the presence of F-actin does not result in broadening of signals of peptide in the absence of complex formation whilst interaction with F-actin results
in specific spectral changes Spectra determined in 5 m M sodium phosphate buffer, pH 7.4, T ¼ 285K (A) HA306–318 peptide,
200 l M , (B) HA306–318 peptide, 200 l M , in the presence of F-actin,
200 l M The spectral region between 1.2 and 1.4 p.p.m under these conditions is shown on an expanded scale as inset The fine structure for the HA306–318 peptide resonances is retained indicating lack of interaction with F-actin and the absence of broadening due to non-specific viscosity effects over the actin concentration range studied (0–8 mgÆmL)1) Peak at 1.34 p.p.m in inset B is due to actin (C) rcTnI161–181 peptide, 200 l M (D) rcTnI161–181 peptide, 200 l M , in the presence of F-actin, 35 l M Specific resonance broadening occurs during titration of the peptide rcTnI161–181 with increasing concen-trations of F-actin indicating complex formation characterized by fast exchange between the free and actin-bound states of the peptide population The residues whose signals are most markedly affected by interaction (e.g Arg168, His170 and Gln173) indicate the region of the peptide whose molecular motions are most restricted by binding to the surface of F-actin.
Trang 5changes during titration with F-actin of two peptides,
hcTnI128–153 and hcTnI136–147, corresponding to
over-lapping regions of human cardiac TnI (Table 1) Peptide
hcTnI128–153 comprises the inhibitory region of TnI with
the additional six residue C-terminal sequence, ISADAM,
which is present in all of the mammalian isoforms of TnI
sequenced so far and may be of functional significance The
smaller peptide, hcTnI136–147represents the region
cor-responding to the minimal inhibitory sequence of TnI [8]
Addition of F-actin to the human cardiac inhibitory
peptide in molar excess produced a marked reduction in
resonance intensity of the side chain groups of the free
peptide, indicating that interaction had occurred (Fig 3)
The progressive variation in linewidth and intensity for the
peptide signals occurred in the absence of any chemical shift
change as is characteristic of relatively rapid exchange on
the relaxation time scale [32,34] The marked reduction of
the peptide ligand resonance intensity upon addition of
F-actin is consistent with the high molecular weight and
slow tumbling of the complex formed Similar results were
reported earlier [25] for the binding to F-actin by the
inhibitory peptide from rabbit fast skeletal muscle TnI
(residues 96–116) that differs from the homologous human
cardiac peptide by four conservative replacements Since
almost all the resonances of the peptide hcTnI128–153 were
affected in the presence of F-actin (Fig 3) the extent of the
spectral changes suggests that the entire length of the
peptide is constrained by attachment to the actin filament
The kinetics of the interaction of the TnI inhibitory
region with actin were characterized using surface plasmon
resonance to monitor binding to immobilized F-actin The
sensorgrams obtained recorded the association and
disso-ciation phases of the interaction (Fig 4A) Analysis of the
dissociation phase for hcTnI128–153 peptide concentrations
in the range 1–10 lMgave an off rate constant of103Æs)1 consistent with the NMR observation of fast exchange on the relaxation time scale The equilibrium constant for the interaction was obtained by fitting the sensorgram data to a model employing 1 : 1 complex formation The value of the dissociation constant derived, 3 lM(Table 2) was consistent with an analysis of the dependence of the equilibrium plateau signal on the concentration of the TnI inhibitory peptide
Tropomyosin enhances the affinity of the TnI inhibitory peptide for F-actin
Titrations of F-actin with peptide hcTnI128–153 were carried out with the molar ratio peptide : actin varied over the range of 1 : 1 to 6 : 1 in the presence and absence of tropomyosin As shown in Fig 4B, a steady change was observed in the signal corresponding to the side chains of arginine as the molar ratio of peptide to actin was increased The broad resonance linewidth of the bound peptide reduced to that of the free peptide as the ratio of the bound
to free peptide decreased during titration with the peptide
In the presence of tropomyosin the continuous reversion of the signal lineshape to that of the free peptide is found to saturate at close to a 1 : 1 molar ratio In the presence of tropomyosin, saturation occurred at a lower peptide : actin ratio than was the case in the absence of tropomyosin (Fig 4B) The rate of the exchange process between actin-bound and free peptide is therefore altered by the presence
Fig 2 Proton magnetic resonance spectra demonstrating the absence of
non-specific interaction of the TnIinhibitory peptide with BSA Spectra
determined in 5 m M sodium phosphate buffer, pH 7.4, T ¼ 285K (A)
peptide hcTnI128–147, 120 l M (B) BSA, 120 l M (C) 120 l M peptide
hcTnI128–147, in the presence of 120 l M BSA This spectrum is
indistinguishable from the algebraic sum of the individual spectra
(A + B) indicating lack of nonspecific interaction with BSA Signals
deriving from the hcTnI128–147are labelled.
Fig 3 Interaction of the human cardiac TnI inhibitory peptide with F-actin illustrated by proton magnetic resonance spectroscopy to show the residues involved in complex formation Spectra determined in 5 m M
sodium phosphate buffer, pH 7.2, T ¼ 293K (A) peptide hcTnI128–
153, 200 l M , (B) hcTnI128–153, 200 l M , in the presence of F-actin,
18 l M (C) hcTnI128–153, 200 l M , in the presence of F-actin, 50 l M (D) difference spectrum, A–C, highlighting the residues whose side-chain signals are perturbed by binding to F-actin Signals of the hcTnI128–153 are labelled Complex formation characterized by rel-atively fast exchange between the free and actin-bound states of the peptide population is indicated by the resonance broadening that occurs during titration with increasing concentrations of F-actin Note that the lack of spectral change for the signal originating from the buffer (*) confirms the absence of nonspecific viscosity effects.
Trang 6of tropomyosin The observation that in the presence of
tropomyosin the signal linewidth returned to that of the free
peptide at a 1 : 1 ratio and altered more dramatically at low
peptide : actin ratios indicates a retarded exchange process
and tighter binding of hcTnI128–153 to actin–tropomyosin
In this intermediate exchange range, the rate of the exchange
process also contributes to the relaxation process and
resonance linewidths are expected on this exchange
time-scale to revert to those of the free peptide in a manner
dependent upon the exchange off-rate constant [34–36] The
NMR data are therefore consistent with a 1 : 1 complex
formation between the IP and actin–tropomyosin and an
increase in affinity for actin resulting from the presence of
tropomyosin
To supplement the binding data obtained from the NMR
investigations fluorescence studies using dansylated
hcTnI128–153 were undertaken to evaluate binding
stoi-chiometry and affinity The intrinsic fluorescence emission
of actin tryptophan residues was not significantly altered by
the presence of the IP whereas titration of the dansylated
peptide with F-actin or F-actin–tropomyosin resulted in
enhancement of the emission intensity of the dansyl group
(Fig 5) The titration data gave excellent fits to a 1 : 1
binding curve and provided direct evidence of a significant
increase in affinity in the presence of tropomyosin (Fig 5,
Table 2) Dansyl emission was unaltered in the presence of tropomyosin alone while competition with unlabelled IP reversed the enhancement seen in the presence of F-actin or F-actin–tropomyosin in a manner consistent with the derived affinity of the IP (Table 2) These data confirmed that the IP formed a 1 : 1 complex with F-actin the affinity
of which is enhanced by tropomyosin
Interaction of the myosin light chain N-terminal peptide with actin and reversal by the TnI inhibitory peptide The effect of the IP on the interaction with F-actin of the myosin light chain peptides, MLC1 1–37and MLC1 1–13 was studied in view of the evidence that this region, localized
to the head of the myosin molecule, can bind to the C-terminal of actin [15,37,38] The MLC1 peptides were found to bind to F-actin both in the absence and presence of tropomyosin with the interaction resulting in the reduction
of the NMR resonance intensity for the majority of the peptide sidechain signals (Fig 6) Tropomyosin alone did not affect the MLC1 peptide spectra nor did it result in the dissociation of the MLC1 peptides from F-actin On the contrary it increased their affinity The progressive changes observed with increasing concentrations of F-actin reflected complex formation in fast exchange and indicated the
Fig 4 Interaction of the TnI inhibitory peptide with F-actin determined by surface plasmon resonance (25 mm Hepes, pH 7.4, 150 m M NaCl) and by proton magnetic resonance spectroscopy (5 mm sodium phosphate buffer pH 7.2) (A) Sensorgrams showing the kinetics of binding of the human cardiac TnI128–153 inhibitory peptide to immobilized F-actin at the peptide concentrations indicated The fit of these data to 1 : 1 complex formation yielded a dissociation constant of 3 ± 2 l M for the F-actin complex (Table 2) (B) The cardiac TnI inhibitory peptide forms a complex with F-actin whose affinity is enhanced by tropomyosin as shown by the influence of tropomyosin on the change in resonance line width of the composite signal of the dCH 2 groups of the arginine residue side chains of the inhibitory peptide as a function of the peptide : actin molar ratio j, F-actin-tropomyosin (molar ratio 7: 1), m, F-actin The concentration of actin was in each case was 40 l M with < 5% dilution during titration with the inhibitory peptide up to a concentration of 160 l M , pH 7.2 Saturation of the linewidth change at lower molar ratios of actin–tropomyosin compared to actin alone is indicative of the enhanced affinity of F-actin for the peptide in the presence of tropomyosin The dotted lines show that in the presence of tropomyosin the return to the linewidth of the free peptide occurred at approximately a 1 : 1 ratio of peptide : F-actin–tropomyosin indicative of 1 : 1 complex formation.
Table 2 Dissociation constants for the different peptide complexes with F-actin determined from fluorescence and surface plasmon resonance meas-urements The K d values quoted were derived from the nonlinear regression fit (GraphPad Prism) of the fluorescence data to a 1 : 1 binding curve for peptide–actin complex formation The corresponding standard errors are quoted The K d obtained for the myosin loop peptide, hcM398–414, upon interaction with F-actin is consistent with the value previously reported using a peptide labelled at Cys400 [29] The K d quoted for the unlabelled TnI inhibitory peptide was obtained by curve fitting of the dissociation phase of the SPR data to derive the off-rate constant and an on-rate of 5 · 10 8
M )1 Æs with curve fitting of the association phase.
F-actin ( M )6 ) F-actin–tropomyosin ( M )6 )
Trang 7involvement of the N-terminal residues of MLC1 in actin
binding both in the absence and presence of tropomyosin
(Fig 6i)
Addition of hcTnI128–153 at a much lower relative con-centration than either MLC1 peptide brought about disso-ciation of the latter from F-actin and F-actin–tropomyosin
Fig 5 The TnIinhibitory peptide forms a 1 : 1 complex with F-actin whose affinity is enhanced by tropomyosin as indicated by fluorescence emission spectra The experimental conditions were 5 m M phosphate buffer, pH 7.2, T ¼ 293K The relative fluorescence intensity is shown in arbitrary units Excitation was at 340 nm and the spectra were recorded from 420 to 600 nm (A) Fluorescence emission spectra of dansylated TnI inhibitory peptide complexed with F-actin-tropomyosin Titration of the dansylated TnI inhibitory peptide with F-actin or F-actin–tropomyosin (molar ratio
of actin : tropomyosin of 7: 1) resulted in enhancement of the fluorescence emission intensity of the dansyl group Shown are fluorescence emission spectra of 10 l M dansylated hcTnI128–153 in the presence of increasing concentrations of F-actin–tropomyosin (2, 5, 20 and 60 l M actin,
in traces 2–5, respectively) Competition by 2 l M unlabelled hcTnI128–153 in the presence of 60 l M actin led to a reduction in emission enhancement, trace 6, consistent with the dissociation constant (Table 2) derived from curve fitting of the data to 1 : 1 complex formation with F-actin–tropomyosin (B) Fluorescence changes observed upon titration of 10 l M dansylated hcTnI128–153 with F-actin (j), or F-actin–tropo-myosin (m) (molar ratio of actin : tropoF-actin–tropo-myosin of 7: 1) The binding curves shown are the nonlinear regression fits obtained (R 2
> 0.97 ) for
1 : 1 complex formation using data obtained in three separate titrations in each case.
Fig 6 The interaction of the N-terminal region of MLC1 with F-actin is weakened by the binding of the TnIinhibitory region Spectra determined in
5 m M sodium phosphate buffer, pH 7.2, T ¼ 293K (i) Proton magnetic resonance spectra of the MLC1 1–13 peptide during titration with F-actin and upon subsequent addition of hcTnI136–147 (A) MLC1 1–13 peptide, 200 l M , (B) MLC1 1–13 peptide, 200 l M , in the presence of F-actin,
60 l M (C) As for (B) but in the presence of 55 l M hcTnI136–147 (D) MLC1 1–13 peptide as in (A), but spectrum acquired by the use of a two-pulse spin-echo sequence (E) MLC1 1–13 peptide in the presence of F-actin as in (B) but spectrum acquired by the use of a two-two-pulse spin-echo sequence Spectral accumulation in this way is sensitive to even small changes in signal linewidth resulting in readily detectable changes in intensity The linebroadening of the MLC1 peptide signals resulting from interaction with F-actin is markedly diminished by the presence of hcTnI136–147 (ii) Spectra of MLC1 1–37during titration with F-actin and upon subsequent addition of hcTnI128–153 (A) 200 l M peptide MLC1 1–37, in the presence of 25 l M F-actin (B) As for A and upon addition of 10 l M cardiac inhibitory peptide, hcTnI128–153 (C) Difference spectrum, B-A, showing the sidechain groups of MLC1 1–37whose resonances displayed actin-dependent broadening that is reversed by the presence of the inhibitory peptide The increase in signal intensity of the proton NMR spectra of the MLC1 peptide indicates that its interaction with F-actin is abolished in the presence of the inhibitory peptide.
Trang 8This was clearly indicated by the reversal of the
actin-associated spectral changes for resonances unique to the
MLC1 peptide, e.g the trimethylalanine signal (Fig 6ii)
Taken together these results suggested that, while
tropo-myosin on its own did not hinder the binding of MLC1 to
actin, the dissociation of the MLC1 1–37by the IP binding
to F-actin or F-actin–tropomyosin may have resulted from
a conformational change in subdomain 1 of actin rather
than as a consequence of competition for binding at
identical or overlapping sites The possibility that the IP
produced its effect by inducing a conformational change in
actin was explored further by studying its influence on the
binding of the loop peptide hcM398–414 This region of the
myosin molecule is believed to dock at the junction between
subdomain 1 and 3 of actin [16,39] whereas the LC1 peptide
binds close to the C terminus of actin
Interaction of the myosin loop peptide, residues
398–414, with F-actin occurs at a region
that does not overlap with the binding site
for the TnI inhibitory peptide
Characterization of the interaction of cardiac b-myosin
residues 398–414 with F-actin was carried out so as to
explore any influence of tropomyosin on the inhibitory
region of TnI The binding affinity of the myosin loop
peptide hcM398–414 to F-actin and F-actin–tropomyosin
was initially determined from the changes in intrinsic
tryptophan fluorescence of F-actin observed upon titration
with the peptide The dissociation constant for the F-actin
complex formed was calculated using a nonlinear regression
procedure in each case to fit the data to a 1 : 1 binding curve
(Fig 7A) The Kd value obtained in the presence of
tropomyosin was 18 ± 4 lM The affinity of the complex
with the peptide was higher than that found for F-actin
alone (Table 2) indicating that tropomyosin enhanced the
binding of hcM398-414 to F-actin
Since the loop region of the myosin head, comprising
residues 398–414, is believed to interact near the C-terminus
of actin, F-actin labelled with 1,5 IAEDANS at Cys374 was
titrated with increasing concentrations of the hcM398-414
peptide This was undertaken in order to determine an
alternative value for the binding affinity using as a readout the spectral properties of a probe located on subdomain 1 of actin in the vicinity of the presumed binding site Addition
of hcM398–414 led to quenching of IAEDANS emission with an overall intensity reduction of some 3% at saturation (Fig 7B) Comparable quenching effects were observed in the presence of tropomyosin (1 : 7, tropomyosin : actin) while the titration data were consistent with 1 : 1 complex formation as judged by the goodness of fit of the data to a
1 : 1 binding curve The derived Kdvalues were similar to those obtained by monitoring the actin–tryptophan fluor-escence changes on the addition of the hcM398–414 peptide (Table 2)
Titration of IAEDANS-labelled F-actin with the inhi-bitory peptide, hcTnI128–153 was also carried out in the absence and presence of tropomyosin Under both condi-tions the inhibitory peptide led to enhancement of the IAEDANS emission ( 16% enhancement at saturation, Fig 7B) with a shift of the fluorescence emission maximum from 475–470 nm These titration data were consistent with
1 : 1 complex formation and yielded Kdvalues similar to those obtained using unlabelled F-actin (Table 2) The observations that binding of the TnI inhibitory region led to fluorescence enhancement and a blue-shift of the emission maximum are consistent with the IAEDANS label on Cys374 experiencing a less polar environment upon complex formation This contrasts with the change in environment of the label upon interaction of actin with the hcM398–414 myosin loop peptide The markedly different response of the IAEDANS label to the binding of the two peptides provides direct experimental evidence that the myosin loop and the TnI inhibitory peptides bind on different sites on F-actin Resolution of the nature of the residues of the hcM398–414 myosin loop peptide involved in interaction with F-actin was achieved by monitoring the NMR spectral changes resulting from complex formation in the presence and absence of tropomyosin As was the case with the other peptides used in this study that bound to F-actin and F-actin–tropomyosin, peptide hcM398–414 did not interact with tropomyosin alone under the conditions described Titration of peptide hcM398–414 with F-actin resulted in marked spectral broadening of the readily identifiable sidechain resonances
Fig 7 The binding of hcM398–414 and hcTnI128–153 to F-actin as monitored by intrinsic (A) and extrinsic (B) fluorescence emission changes Experimental conditions were 5 m M sodium phosphate buffer, pH 7.4, T ¼ 293K (A) Intrinsic tryptophan fluorescence emission spectra of the F-actin–tropomyosin complex during titration with hcM398–414 The inset shows the decrease in fluorescence emission observed as a function of increasing hcM398–414 concentration (0–50 · 10)6M ) The curve shown is the fit of the data to 1 : 1 complex formation at an F-actin concen-tration of 5 · 10)6M (B) Variation of the IAEDANS emission upon titration of Cys374-labelled F-actin with hcM398–414 (m) or hcTnI128–153 (j), at a concentration of F-actin equal to 5 · 10)6M The curves shown are the nonlinear regression fits to 1 : 1 complex formation The derived dissociation constants are reported in Table 2.
Trang 9of His401, Arg403, Asn408, Tyr410 and Thr412 that
indicated complex formation with F-actin (Fig 8) Less
notably perturbed is the sidechain signal of Val411 The
nature of the residues affected was unchanged upon
interac-tion with F-actin–tropomyosin while the increased
broaden-ing effects observed at low peptide : actin in the presence of
tropomyosin are consistent with an enhanced affinity
result-ing from a decrease in peptide dissociation kinetics
The binding of the TnI inhibitory region simultaneously
displaces peptides bound at nonoverlapping sites
on actin
Competition experiments were carried out to monitor the
ability of different peptides to simultaneously bind to
F-actin The peptide derived from TnI, rcTnI161–181,
bound to F-actin in the presence or absence of tropomyosin
without displacing the myosin loop peptide (Fig 9i) This
TnI peptide represents a region C-terminal to the IP of TnI
and has been proposed as an additional actin-binding site
[9,10] Binding of the rcTnI161–181 peptide was judged
from the spectral broadening of its clearly distinguishable
sidechain signals (e.g His170, c.f Fig 1) that occurred
without any concurrent changes in the resonances unique to
the hcM398–414 myosin loop peptide (His401 and Tyr410,
c.f Fig 8) Competition from the myosin loop peptide with
peptide rcTnI161–181 for interaction with actin would have
resulted in its displacement and the consequent appearance
of signals broadened as a consequence of interaction with
F-actin These results indicated that the myosin loop peptide
and rcTnI161–181 are bound simultaneously at different
sites on actin as might be expected from the differences in
the composition of the two peptides
Titration of the hcTnI128–153 inhibitory peptide into this system up to a concentration equimolar to that of F-actin resulted in the dissociation of both hcM398–414 and rcTnI161–181 from actin whether in the absence or presence
of tropomyosin (Fig 9i) This dissociation was also induced
by the shorter peptides encompassing the TnI inhibitory region, hcTnI128–147and hcTnI136–147although consistent with its lower actin affinity (Table 2), higher hcTnI136–147peptide : actin ratios were required to achieve dissociation of hcM398–414 and rcTnI161–181 Their simultaneous displacement was readily seen from the reappearance of the signals unique to these peptides that had been broadened by their interaction with F-actin in the absence of the inhibitory peptide (Fig 9i) At the same time signals unique to the IP broadened in the manner described above (Figs 3 and 4) Since the TnI inhibitory peptide forms
a 1 : 1 complex with F-actin these observations indicate that the association of the TnI inhibitory region with F-actin antagonized the binding of the myosin loop peptide and rcTnI161–181 to their individual binding sites on actin These effects cannot be ascribed to site competition and simple steric displacement
Since the binding of the TnI inhibitory peptide appeared
to induce a conformational change in the actin molecule that altered the ability of F-actin to interact with different segments of myosin we went on to monitor the binding of hcM398–414 at an actin : Tm : TnI peptide ratio of 7: 1 : 1 over a range of myosin peptide concentrations Figure 9ii presents data obtained using a myosin peptide concentration of 50 lM The presence of the TnI inhibitory peptide (0.55 lM) led to a decrease in the amount of myosin peptide bound to F-actin–tropomyosin (with hcM398–414
at 100-fold excess over TnI peptide) This is seen from the
Fig 8 Proton MR spectral changes upon titration of hcM398–414 with F-actin identifying the residues involved in complex formation Spectra determined in 5 m M sodium phosphate buffer, pH 7.2, T ¼ 293K (i) (A) Peptide hcM398–414 (200 l M ) (B) In the presence F-actin, 28 l M (C) Difference spectrum, A-B, highlighting the residues whose sidechain signals are perturbed by binding to F-actin (ii) as in (i) but spectra acquired by the use of a two-pulse spin-echo sequence Spectral accumulation in this way distinguishes signals on the basis of their J-coupling patterns and highlights even small changes in signal linewidth resulting in readily detectable changes in intensity Signals of hcM398–414 are labelled Complex formation characterized by relatively fast exchange between the free and actin-bound states of the peptide population is indicated by the resonance broadening that occurs during titration with increasing concentrations of F-actin The unique sidechain resonances of His401, Arg403, Tyr410, Val411 and Thr412 display marked perturbation upon complex formation.
Trang 10change in the myosin peptide signals, for example, Arg403,
Val411 and Thr412 that, as highlighted by difference
spectroscopy (Fig 9ii), revert towards those of the free
peptide in the presence of hcTnI128–147at an
actin : Tm : TnI peptide ratio of 7: 1 : 1 These
observa-tions reinforce the suggestion that conformational changes
which occur when one molecule of troponin I interacts with
the actin monomer are transmitted to other actin monomers
in the filament not associated with TnI
D I S C U S S I O N
The TnI inhibitory region is an early example of a growing
family of short peptide sequences capable of emulating the
ability of the parent proteins to interact with their
physio-logical targets The biophysio-logical activity characteristic of the
whole molecule is held to derive from the retention of
specific protein–protein recognition by such isolated
pep-tides and their resulting ability to inhibit receptor/effector
interactions Examples of such intervention range from the
inhibition of the replication of simian virus 40 DNA by the
Proliferating Cell Nuclear Antigen-binding peptide of
p21WAF1 [40] to the myosin loop peptide, hcM398–414,
used in this study In keeping with its apparent contribution
to the actomyosin interface [16,17,20] the latter peptide
inhibited actin-activated MgATPase activity [29] while the
short TnI inhibitory peptide, some 6% of the parent
molecule, preserves both the inhibitory and the tropomyosin
accentuation effects characteristic of troponin-I
The NMR data clearly indicate that peptides corres-ponding to the N-terminus of myosin LC1 interact speci-fically with F-actin in the absence of inhibitory peptide derived from TnI This conclusion is consistent with the results of earlier NMR investigations [38,39] and cross linking studies [37,41] that the N-terminal region of skeletal LC1 is one of the sites involved in the interaction of myosin with actin From these studies and electron microscopy of C-terminally labelled actin [42] it can be concluded that the N-terminus of MLC1 binds close to the C-terminus of actin The N-terminal region APKK (residues 1–4) of MLC1 appears to be particularly important since modification of these residues by recombinant DNA technology results in changes in the kinetics of the actomyosin MgATPase [41] Other residues at the N-terminus of MLC1 are involved in binding and have indeed been shown to be important for the activity of cardiac myosin A peptide corresponding to residues 5–14 of human ventricular MLC1 increased the contractility of intact and skinned human heart fibres [43] and a similar peptide added to rat cardiac myofibrils induced a supramaximal increase in the MgATPase activity
at submaximal calcium levels [44]
The NMR and fluorescence studies both indicate inter-action of F-actin with another region of myosin, the loop peptide, hcM398–414 The interaction appears to occur at a region on F-actin that is different from that involved in binding the TnI inhibitory peptide as shown by the distinctive response of the IAEDANS probe to each peptide This is consistent with the earlier observations that
Fig 9 (i) The TnIinhibitory region displaces both peptide hcM398–414 and peptide rcTnI161–181 that interact concurrently with F-actin at distinct binding locations The aromatic sidechain NMR resonances are shown since these provide unique reporter signals for each of the peptides Spectra determined in 5 m M phosphate buffer, pH 7.2, T ¼ 293K (A) Myosin loop peptide (hcM398–414), 108 l M (B) hcM398–414, 108 l M in the presence of 74 l M F-actin The signals of His401 and Tyr410 of the myosin loop peptide are markedly broadened by complex formation (C) As for (B), but upon the addition of 186 l M rcTnI161–181 Binding of this TnI peptide is indicated by broadening of its His171 resonances No displacement of the myosin loop peptide has occurred since its signals remain broad The two peptides are therefore concurrently bound to F-actin (D) As for (C) but upon titration with inhibitory peptide hcTnI128–153 (here 102 l M ) The reappearance of the myosin loop peptide signals and those of rcTnI161–181 indicates their simultaneous displacement by the inhibitory peptide The signals unique to the inhibitory peptide, Phe132 and
138, are broad indicating that the peptide is bound to F-actin (ii) The binding of hcM398–414 to F-actin–tropomyosin is altered by the presence of hcTnI128–153 at an actin : Tm : nI inhibitory peptide ratio of 7: 1 : 1 This is detected by the increased spectral contribution of free hcM398–414 signals clearly identified by difference spectroscopy The two-pulse spin-echo spectra shown (c.f Fig 8ii) were obtained in 5 m M phosphate buffer,
pH 7.2, T ¼ 293K (A) Myosin loop peptide (hcM398–414), 50 l M , (B) as for (A) but in the presence of F-actin–tropomyosin (3.7 l M F-actin) (C)
As for (A) but in the presence of an actin : Tm : TnI inhibitory peptide ratio of 7: 1 : 1 (0.55 l M hcTnI128–153) (D) Difference spectrum (C-B) showing the reappearance of the myosin loop peptide signals.