The Cardiomyopathy Mutation, R146G Troponin I, Stabilizes the

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The cardiomyopathy mutation, R146G TnI, stabilizes the intermediate "C" state of regulated actin at high and low free Ca2+ conditions. Dylan James Johnson, Mohit C. Mathur, Tomoyoshi Kobayashi, and Joseph Michael Chalovich Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b01359 • Publication Date (Web): 25 Jul 2016 Downloaded from http://pubs.acs.org on July 28, 2016

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Biochemistry

1

1

The cardiomyopathy mutation, R146G TnI,

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stabilizes the intermediate "C" state of regulated actin

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at high and low free Ca2+ conditions. ᴪ

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Dylan Johnson†,Ʃ, Mohit C. Mathur†, Tomoyoshi Kobayashi‡,

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and Joseph M. Chalovich†*

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ᴪ

This work was funded by NIH grant number AR44505 to J.M.C.

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†

Department of Biochemistry and Molecular Biology, Brody School of Medicine,

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East Carolina University, Greenville, North Carolina; ƩDepartment of Chemistry, East

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Carolina University, Greenville, North Carolina and ‡Department of Physiology and

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Biophysics and Center for Cardiovascular Research, College of Medicine, University of

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Illinois, Chicago, Illinois

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*Correspondence: [email protected]

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1 ACS Paragon Plus Environment

Biochemistry

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Abbreviations and Textual Footnotes

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EGTA, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid; MOPS, 3-(N-

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Morpholino]-propanesulfonic acid; regulated actin, actin-tropomyosin-troponin; S1, myosin

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subfragment 1; TnT, troponin T; TnI, troponin I; TnC, troponin C.

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Biochemistry

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Abstract

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The R146G mutation of troponin I is associated with hypertrophic

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cardiomyopathy in humans. Earlier data pointed to stabilization of the intermediate, C

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state, of actin-tropomyosin-troponin by this mutant. Because cardiac disorders appear

32

to be linked to changes in regulated actin distributions we determined the extent to

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which the R146G TnI mutant alters the distribution of states at low and high Ca2+

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concentrations.

35

We show, from measurements of the kcat for actin-activated ATPase activity at

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saturating Ca2+, that R146G TnI reduced the population of the active, M, state to 25% of

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the wild type level. Together with acrylodan tropomyosin fluorescence measurements of

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the B state it appeared that the C state was populated at about 91% of the total for

39

R146G TnI containing actin filaments. The C state was also more heavily populated at

40

low Ca2+. Acrylodan tropomyosin fluorescence changes showed a large diminution in

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the inactive state relative to the wild type value without a comparable increase in the

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active state. Furthermore, the rate of binding of rigor S1 to pyrene labeled actin

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filaments containing R146G TnI was faster than to wild type filaments at low free Ca2+

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concentrations. These results indicate that the inhibitory region of TnI affects both the

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B/C and M/C equilibria of actin-tropomyosin-troponin. The observation that a mutation in

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the inhibitory region affects the M/C equilibrium may point to a novel regulatory

47

interaction.

48 49 50


Biochemistry

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Introduction Muscle contraction results from a cyclic interaction between myosin and actin

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that is driven by the hydrolysis of ATP. The actin-binding complex of tropomyosin and

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the three troponin subunits regulates the rate of ATP hydrolysis by myosin in cardiac

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and skeletal muscles. Activation of contraction occurs when Ca2+ binds to troponin C

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(TnC) and exposes a hydrophobic patch to which troponin I (TnI) can bind 1,2. Because

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TnT links TnI to tropomyosin (reviewed 3) this change results in movement of

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tropomyosin across the actin helix 4,5,6. The regulatory complex was originally thought to be a binary switch with active,

59 60

(now called M), and inactive (now called B) states. Data from several laboratories show

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that there are 3 structural states of regulated actin 7,8,9,10,11,12 that are commonly called

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the B, C and M states. The C state or Ca2+-state predominates at saturating Ca2+ and it

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is in equilibrium with the B and M states. The B state is heavily populated at low Ca2+.

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States B and C are both inefficient in stimulating myosin S1 ATPase activity 13. The M

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state accelerates myosin ATPase activity; it is stabilized by rigor type myosin binding to

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actin.

67

Mutations in troponin, as well as normal adaptive responses, may change the

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distribution of these regulatory states at both low and high Ca2+ concentrations.

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Phosphorylation of either TnI 14 or TnT 15 changes both the Ca2+ sensitivity of ATPase

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activity and minimal and maximal levels of activity at low and high Ca2+, respectively.

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Mutations of TnI that simulate protein kinase C phosphorylation stabilize the inactive B

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state of regulated actin 13. Deletion of the COOH-terminal 14 amino acids of cardiac

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TnT, as occurs in one form of hypertrophic cardiomyopathy, stabilize the active M state


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Biochemistry

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16 17

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to full activation with high affinity myosin S1 binding 17, equilibrium binding of myosin S1

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to regulated actin, binding kinetics and acrylodan tropomyosin fluorescence changes 16.

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. Evidence for these state changes is based on ATPase measurements normalized

An intriguing class of troponin mutants causes too little activation at saturating

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Ca2+ and too much activation at low Ca2+ concentrations. An example of this type of

79

mutant is an Arg to Gly substitution within the unstructured of cardiac TnI (R146G

80

mouse sequence or R145G human sequence). Myofibrils 18 or trabeculae 19 containing

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R145G TnI produce increased force, relative to wild type troponin, at low Ca2+ but

82

decreased force in activating conditions. Skinned papillary fibers from transgenic mice

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expressing R145G TnI have decreased force at saturating Ca2+ 20. The sliding speed of

84

isolated thin actin filaments containing R145G TnI is greater than wild type at low Ca2+

85

but lower than wild type at high Ca2+ 21. ATPase rates at low Ca2+ conditions are

86

elevated (22

87

type values (22 23 24 25 26). ATPase rates, measured relative to those with actin filaments

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stabilized in the M state with N-ethylmaleimide modified S1 (NEM-S1)16, are consistent

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with an increase in the intermediate C state for R146G TnI containing filaments 24.

90

23 24 25 26 27

) while the rates at saturating Ca2+ are low compared with wild

The results of studies with different mutants indicate that alterations in the

91

distribution of actin-tropomyosin-troponin states results in cardiac dysfunction. We

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currently cannot predict how a mutation in any region of troponin will affect these

93

equilibria. One would not have predicted, even from detailed studies of the inhibitory

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peptide 28 that the R146G TnI mutation would stabilize the intermediate C state. By

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investigating the effect of mutations in all of the troponin subunits on the distribution of

96

actin states it may be possible to predict the effect of mutations and, more importantly,


Biochemistry

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6 97

to predict how to restore normal function. We have taken several approaches to map

98

the effects of the R146G TnI mutation on actin state distributions at both high and low

99

Ca2+ concentrations.

100

We now show, that at saturating Ca2+, actin filaments containing the R146G

101

mutant occupied the active, M, state with only 25-30% of the frequency of wild type

102

regulated actin. However, there was no corresponding increase in the inactive, B, state.

103

We also probed the behavior of actin filaments containing the R146G TnI mutant at low

104

Ca2+. Rigor S1 was able to bind more quickly to mutant regulated actin filaments.

105

Furthermore, acrylodan tropomyosin fluorescence changes following rapid S1

106

detachment showed a diminished population of the inactive state. The decreased

107

population of the inactive, B, state was greater than can be accounted for by an

108

increase in the active, M, state. Thus the R146G TnI mutation associated with cardiac

109

dysfunction stabilized the intermediate, C, state relative to wild type actin filaments at

110

both saturating Ca2+ and at very low Ca2+ concentrations. The ability of a mutation

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within the inhibitory region of TnI to alter the stability of the C state relative to the M

112

state is particularly intriguing.

113 114 115 116 117 118 119


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Biochemistry

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Experimental Procedures

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Proteins. Myosin and actin were isolated from rabbit back muscle 29 The

122

catalytic fragment of myosin, S1, was prepared by digestion of myosin with

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chymotrypsin (Worthington Biochemical, Freehold, NJ) 30. The A1 isoform of S1 was

124

prepared by ion exchange chromatography 30. Pyrene labeled actin was prepared by

125

reacting actin with N-(1-pyrenyl) iodoacetamide 31. Bovine cardiac tropomyosin was

126

isolated by a standard method 32. Acrylodan labeled cardiac muscle tropomyosin was

127

prepared by our modification 33 of another procedure 34. Mouse cardiac TnC and TnI in

128

pET3d and TnT in psBET were expressed and purified as described earlier 25. Protein

129

concentrations were measured by absorbance (280-340 nm). The extinction coefficients

130

(ε0.1%) used were actin, 1.15; myosin-S1, 0.75; tropomyosin, 0.23; and troponin, 0.37.

131

ATPase rates. Actin-activated ATPase rates were measured by following the

132

liberation of 32Pi from γ32P-labeled ATP 35. The conditions are given in the figure

133

legends. Rates were measured as a function of the actin concentration and values of KM

134

and kcat were determined by fitting the Michaelis-Menten equation to the data using

135

Sigma Plot (Systat Software, Inc., San Jose, CA).

136

Rapid Kinetics. Rapid kinetics measurements were made using an SX20 model

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double mixing stopped flow apparatus (Applied Photophysics Ltd, Leatherhead, Surrey,

138

United Kingdom) equipped with a constant-temperature circulating water bath.

139

Excitation wavelengths were set with a monochrometer using slit widths of 0.5 or 1 mm.

140

Emission wavelengths were set with filters. Excitation for acrylodan tropomyosin

141

fluorescence was at 391 nm and emission was monitored with a long-pass filter with a

142

370 nm cut on, a midpoint of 400 nm and a plateau at 475 nm. Excitation for pyrene


Biochemistry

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8 143

actin fluorescence was measured with excitation at 365 nm with emission measured

144

through a 384 nm cut on long-pass filter. Light scattering measurements were made by

145

increasing the excitation light to 600 nm with the same emission filters.

146

The fraction of actin in the inactive, B, state at low Ca2+ was estimated by the

147

amplitude of acrylodan tropomyosin fluorescence following the rapid dissociation of S1

148

from regulated actin 33,36,17. Acrylodan was used in preference to pyrene because it is

149

smaller, less likely to form excimers, produces a larger signal and is useful over a wider

150

temperature range 33. Solutions containing S1 and actin were mixed in Teflon beakers

151

and allowed to incubate for 5 minutes at the temperature of the assay prior to mixing

152

with ATP in stopped-flow studies.

153

We also estimated the inactive state from the ratio of rates of S1 binding to

154

pyrene labeled actin at low Ca2+ to that at saturating Ca2+ as described by others 8. The

155

equilibrium constants describing the distribution of states are KB = [intermediate C

156

state]/[inactive B state] and KT = [active M state]/[intermediate C state]. In cases where

157

KT is small compared to KB, the value of KB is 1/[(kCa2+/kEGTA) -1] 37.

158

The normal mixture of S1 isoforms was compared with the A1 isoform of S1 in

159

terms of binding to actin by light scattering. We observed the same kinetics for rapid

160

light scattering measurements of S1 and A1-S1 binding to wild type actin-tropomyosin-

161

troponin (not shown). In our hands, unfractionated S1 is more stable than A1-S1 and

162

less prone to stabilize the active M state so the unfractionated S1 was used in the

163

studies that follow.

164 165


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Biochemistry

9 166 167

Results Occupancies of regulated actin states at saturating Ca2+. We reported earlier

168

that regulated actin containing the R146G mutant of TnI had 25% of the wild type

169

ATPase activity at saturating Ca2+ and a low actin concentration. We now report the

170

actin-tropomyosin-troponin concentration dependencies of ATPase rates for filaments

171

containing wild type and R146G TnI over the actin concentration range of 6.7-120 µM.

172

The actin:tropomyosin:troponin proportion was 7:3:3 and the S1 concentration was 0.1

173

µM. Figure 1 shows that differences were observed in ATPase activities of wild type and

174

mutant actin filaments throughout that range of concentrations.

175

Fitting the Michaelis-Menten equation to the data of Figure 1 showed that the

176

concentrations of regulated actin required to reach 50% of the maximum velocities were

177

117 µM for both wild type and R146G TnI containing actin filaments. The lower ATPase

178

activity for mutant containing actin filaments was due to reduction of the kcat from 23 s-1

179

to 6 s-1. That is, in the limit of full binding of actin filaments to myosin S1, those filaments

180

containing the R146G TnI mutant were less competent in stimulating ATP hydrolysis.

181

Furthermore, at saturating Ca2+, actin filaments containing the R146G mutant occupy

182

the active, M, state with 26% of the frequency as wild type actin filaments.

183

We estimated earlier that, at saturating Ca2+, wild type actin filaments occupied

184

the active state approximately 33% of the time 24. That is, the combined occupancy of

185

the B and C states was 67%. Because of the decrease in occupancy of the active M

186

state for the R146G mutant, the B and C states must be occupied 91% of the time. It is

187

likely the occupancy of the C state was near 0.91 because the inactive B state is

188

sparsely occupied at high Ca2+.


Biochemistry

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10 189

It is possible to observe the presence of the B state by using an acrylodan probe

190

on tropomyosin 33. In order to insure that acrylodan-tropomyosin functioned normally we

191

measured the actin-activated ATPase activity of myosin S1 at 10 µM actin, 2.1 µM

192

troponin and 2.1 µM tropomyosin under the conditions of Figure 1. Acrylodan labeling of

193

tropomyosin reduced the ATPase activity to 72% and 56% of the control values at high

194

and low Ca2+, respectively. The acrylodan labeled had 19-fold regulation of ATPase

195

activity compared with 15-fold for regulated actin containing unlabeled tropomyosin.

196

Rapid changes of acrylodan-tropomyosin fluorescence were used to determine if

197

the R146G TnI mutant increased the occupancy of the B state at saturating Ca2+. The

198

basis of the assay is that regulated actin can be maintained in the active state, even at

199

low Ca2+, by high levels of bound S1. Upon rapid addition of ATP, the S1-ATP

200

dissociates leaving regulated actin to relax from the active state through the

201

intermediate, C, state and then to the inactive, B, state if conditions permit 7,33.

202

Acrylodan tropomyosin fluorescence is high for actin-tropomyosin-troponin in the active

203

and inactive states but low in the intermediate state 33.

204

Figure 2 shows that following dissociation of S1-ATP in saturating Ca2+ there was

205

a rapid decrease in fluorescence for actin filaments containing either wild type or R146G

206

troponin. In this condition of saturating Ca2+ the regulated actin filaments passed from

207

the active state to a mixture of states dominated by the intermediate state. Longer

208

traces showed no evidence of transition to the inactive state at saturating Ca2+ for either

209

wild type or R146G TnI containing actin filaments. Because the B state was unoccupied,

210

the mole fraction of regulated actin in the C state was increased from about 0.67 for wild


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Biochemistry

11 211

type to 0.91 for R146G containing filaments (Table I). That represents a decrease in the

212

equilibrium constant between the C and M states (KT) from 0.49 to 0.1.

213

Occupancies of regulated actin states at low Ca2+. Our earlier study, showed

214

that actin filaments containing R146G TnI exhibited an increase in the active M state in

215

low Ca2+ conditions. By normalizing the ATPase rates to the minimum level of activity

216

(S45E TnI, 0.017/s) 13 and an average value of the maximum NEM-S1 activated activity

217

(7.8/s) the mole fractions of the active state in wild type and mutant filaments was

218

calculated to be 0.014 and 0.028, respectively. In order to calculate the change in the

219

intermediate C state distribution, it is necessary to have a measure of the inactive B

220

state in each case.

221

Changes in acrylodan tropomyosin fluorescence, following rapid S1 detachment

222

from regulated actin, were measured at low Ca2+ for wild type and for the R146G TnI

223

mutant (Figure 3). For both wild type (curve 1) and R146G TnI containing actin filaments

224

(curve 2) there was a rapid fluorescence decrease as regulated actin shifted from the

225

active M state to the C state (Figure 3A). Subsequently, both types of actin filaments

226

produced an increase in fluorescence as the occupancy of the B state increased.

227

However there was a marked depression of the amplitude of the signal for actin

228

filaments containing the R146G TnI mutation. The magnitude of the amplitude for the

229

R146G TnI case was too small to accurately measure.

230

Reducing KCl to 90 mM (Figure 3B) or 30 mM (Figure 3C) increased the

231

fluorescence amplitudes for both wild type and R146G TnI containing filaments. Figure

232

3D shows the dependencies of reaction amplitudes on the square root of the ionic

233

strength (see the Debye-Hückel law 38 39). The ratio of amplitudes of R146G TnI to wild


Biochemistry

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12 234

type was 0.27 at 30 mM KCl. The solid line in Figure 3D shows the actual measured

235

amplitudes while the dashed line shows a constant 0.27 ratio of amplitudes. In light of

236

errors in measuring small signals for R146G TnI at higher salt conditions, we assumed

237

that R146G TnI decreased the fraction of the B state to 0.27 of that of wild type

238

filaments throughout the range of conditions studied. Because the fraction of M state is

239

negligible in each case, there must have been a large increase in the C state for the

240

R146G mutant. From the conservation of mass the fractional increase in the C state due

241

to the mutation is equal to (1-0.27* BWT)/(1-BWT) where BWT is the fraction of wild type

242

actin filaments in the B state. A reasonable value for the wild type fraction of state B is

243

0.62 (Table I). That would give a 2-fold increase in the C state due to the mutation.

244

Pyrene actin fluorescence measurements report isomerization of the low affinity

245

S1-actin complex to a higher affinity complex that occurs only in the absence of ATP. By

246

using pyrene-labeled actin to monitor S1 binding it is possible to estimate the fraction of

247

actin in the inactive state by comparing the rate of binding in Ca2+ relative to that at low

248

Ca2+ 8,37. This is most readily done with actin in excess over the S1.

249

Figure 4 shows pyrene-actin fluorescence changes for S1 binding to both wild

250

type and R146G containing actin filaments at both saturating Ca2+ and in the presence

251

of EGTA. The single exponential phases of the curves were analyzed as shown by the

252

fitted lines. At long time intervals we noted a slow increase in fluorescence especially

253

with the R146G filaments. The curves were unchanged when the single S1 isoform A1-

254

S1 was used. We did note that the inclusion of ADP in the assay did eliminate that

255

fluorescence increase and did minimize the initial mixing artifact.


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Biochemistry

13 256

The rate of binding of S1 to R146G TnI containing actin filaments, in Ca2+, was

257

similar for wild type and R146G TnI containing actin filaments (Figure 4A and Table I).

258

This is expected if the inactive state is unpopulated in the presence of Ca2+.

259

Binding of S1 to regulated actin was slower in EGTA than at saturating Ca2+ for

260

both wild type and R146G TnI containing actin filaments. However, troponin with R146G

261

TnI did not inhibit the rate of S1 binding as much as did wild type troponin. That is,

262

R146G TnI containing actin filaments did not favor the inactive state to the same extent

263

as did wild type filaments (Figure 4B and Table I). The results, listed in Table I, show a

264

1.9-fold increase in the fraction of the intermediate state for the R146G TnI mutant with

265

respect to wild type at low Ca2+. Table I also lists the result of a study done at a higher

266

concentration (1.6x) of troponin. That study gave similar 2.1-fold increase in the

267

intermediate state with the inclusion of R146G TnI.

268


Biochemistry

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14 269

Discussion

270

The results show that R146G TnI containing actin filaments increased the

271

population of the C state in both EGTA and saturating Ca2+. That resulted is an elevated

272

ATPase activity at low Ca2+ because of an increase in the active state M from the

273

equilibrium KT (see Fig. 5). In contrast, the activity of the mutant system was less than

274

that of wild type at saturating Ca2+ because the M state population decreased at the

275

expense of the inactive C state. The net result is a loss of regulation.

276

The R146G TnI mutation is another example of an alteration in troponin that

277

changes the distribution of regulated actin states and results in cardiac dysfunction. This

278

mutation is unusual in that it stabilizes the intermediate C state. This means that when

279

trying to manage cardiomyopathies it is necessary to restore the normal distribution

280

among all regulated actin states. The drug Bepridil that was used to increase Ca2+

281

sensitivity actually appeared to stabilize the C state 40 which is an unfavorable outcome.

282

The reported differences in distributions from wild type to R146G TnI containing

283

actin filaments are more reliable than the actual reported distribution of each type of

284

actin filament. Different assays were needed for measurements of each of the two

285

equilibrium constants state and those assays have different limitations. The active M

286

state is readily defined by ATPase activities. However, those assays had to be done at

287

somewhat lower ionic strength than used for the measurement of the B state.

288

Measurements of the B state were done using pyrene labeled actin or acrylodan labeled

289

tropomyosin, These labels are likely to alter the distributions somewhat. We showed

290

that acrylodan labeling of tropomyosin reduced the ATPase rates in both Ca2+ and

291

EGTA indicating a reduction in the M state but the extent of regulation was maintained.


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Biochemistry

15 292

The acrylodan and pyrene assays were used to calculate the B state occupancy only at

293

low Ca2+ where the M state was sparsely occupied. Therefore, an error in the

294

occupancy of the M state would have little effect on the calculated distributions. Acrylodan tropomyosin fluorescence undergoes changes in going both from M to

295 296

C and from C to B 16, 33, 17. The limitation of this method is that a standard is needed to

297

define the intensity equivalent to 100% occupancy of the B state. No such standard is

298

known so values of the B state are given relative to wild type levels. The kinetics of

299

binding of S1 to regulated pyrene-actin have more often been used to define the B state

300

7 37 8

301

binding at saturating Ca2+ where the B state is assumed to be negligible. A further

302

assumption is that S1 binds with identical rates to the C and M states. The acrylodan

303

and pyrene methods predict similar but not identical occupancies of the C state at low

304

Ca2+ (0.8 for acrylodan tropomyosin measurements and 0.69-0.78 for pyrene actin

305

measurements). However, both methods predict that the R146G TnI mutation produces

306

a 2-fold increase in the occupancy of the C state relative to wild type containing actin

307

filaments (Table I).

308

,

, . Calculation of the fraction of the B state at low Ca2+ is made relative to rate of

The intensity of the acrylodan tropomyosin fluorescence change corresponding

309

to the formation of the B state decreased with increasing ionic strength (Figure 3D).

310

That salt dependence probably results from a change in quenching of the probe or an

311

increase in the strength of interaction of troponin to actin-tropomyosin. Thus, the ratio of

312

the signal intensities of the mutant to wild type filaments was approximately constant

313

over the ionic strengths explored. Furthermore, earlier studies suggested that the B


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

16 314

state is actually stabilized by high ionic strength 37,41 and the M state is destabilized by

315

high salt 42 so it is unlikely that the B state decreased at high salt concentrations.

316

This work and our earlier study with R146G TnI mutant 24 support the notion that

317

there are three structurally different states of regulated actin as proposed by others

318

7,8,9,10,11,12

319

acrylodan tropomyosin fluorescence followed by an increase shown in Figure 3.

320

However, the B and C states are degenerate with respect to activation of ATPase

321

activity. The Hill model of regulation, proposed some years ago, made the simplifying

322

assumption that that the states corresponding to the B and C states could be treated as

323

a single inactive state 43,44. That assumption was correct. Because alterations in the

324

distributions of the three structural states leads to cardiac dysfunction it is necessary

325

now to distinguish between the B and C states. Figure 5 shows a modification of the Hill

326

model to explicitly show the B and C states.

327

. In the case of a pure two state model there would not be a decrease in

While we are using the popular B, C and M notation to describe the states of

328

regulated actin we note that the term blocked (B) can lead to misunderstanding of the

329

functional consequences of tropomyosin movement. Binding of rigor S1 to regulated

330

actin is cooperative at low Ca2+ and the affinity increases with the occupancy of S1 on

331

actin 45. Furthermore, the kinetics of rigor S1 and S1-ADP binding are Ca2+-sensitive 7, 8

332

,46. However, myosin, HMM and S1 can bind to actin in the B state during steady-state

333

ATP hydrolysis 47 35 41 48 49 50 51 52. That is why Figure 5 shows binding of S1-ADP-Pi to

334

the B state.

335 336

When myosin S1 binds to regulated actin in the absence of both nucleotide and Ca2+, as shown in Figure 4, the binding probably proceeds stepwise from state B to C to


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Biochemistry

17 337

M, as shown in Figure 5. When S1 is rapidly dissociated with ATP, the reverse stepwise

338

process occurs (Figure 3). However, there is no apparent reason that binding of S1

339

cannot occur directly to the C or M states. This is not shown in Figure 5 because of the

340

complexity of the resulting figure.

341

We showed earlier that there was little effect of Ca2+ on the KM for actin activated

342

ATPase activity 35,53. In those studies the B state was heavily occupied at low Ca2+ but

343

at high Ca2+ there was a mixture of states with > 50% of the actin being in the C state.

344

The results of Figure 1 show that in going from the wild type mixture of states at

345

saturating Ca2+ to about 91% occupancy of the C state, with the R146G mutant, there is

346

no change in the KM. Taken together, these results indicate that the apparent KM for

347

actin is the same for the B and C states. Figure 1 shows further than when the fraction

348

of actin in the C state was increased at the expense of the M state, the kcat was

349

reduced. This supports the view that both the B and C states are inactive toward

350

stimulating myosin ATPase activity.

351

The R146G mutation occurs within the 336 of TnI. The peptide (Gly-Lys-Phe-Lys-

352

Arg-Pro-Pro-Leu-Arg-Arg-Val-Arg) mimics the inhibitory region of fast skeletal muscle

353

and replicates some activities of intact TnI 54. This peptide gave 43% of the inhibition of

354

the actin-tropomyosin stimulated ATPase activity of myosin S1 seen

355

with intact TnI

356

the inhibitory activity of the peptide to 13%. That mutation also tripled the

357

concentration of peptide required to reach 50% of the maximum effect. That skeletal

358

peptide differs from the inhibitory region of cardiac TnI only in that the second Pro is

359

replaced with a Thr in cardiac muscle. That substitution appears to be important in

28

. Substitution of the Arg equivalent to R146 (in bold) with Gly reduced


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Page 18 of 34

18 55

360

length dependent changes in activation of cardiac muscle

361

amino acid residues within the TnI inhibitory region with Gly-Ala raised the ATPase

362

activity in relaxing conditions by 2.1-5.8 fold

363

56

. Replacement of 4-10

.

The region of TnI containing R146 is normally bound to actin in relaxing

364

conditions. That interaction is thought to hold tropomyosin in the B position on actin

365

where the tropomyosin overlaps the binding site of some myosin complexes including

366

nucleotide-free myosin or myosin-ADP. A mutation within this region of TnI might be

367

expected to destabilize the B state at low Ca2+ and lead to an increase in the

368

intermediate C state. It is more surprising that, at high Ca2+, the R146G mutation

369

destabilizes the M state to increase the C state. The inhibitory region of TnI may be

370

involved in forming both the B and M states of regulated actin.

371

Such a link between the B and M states is not unreasonable. The COOH terminal

372

region of TnT is required for the inactive B state at low Ca2+ but it tends to destabilize

373

the active M state at saturating Ca2+

374

B state and the active M state. Substitution of larger regions of the TnI inhibitory region

375

with Gly-Ala led to the conclusion that, the inhibitory region promotes the inactive B

376

state and destabilizes the active M state

377

17

. Residue R146 in TnI promotes both the inactive

56

.

These observations suggest that TnI and TnT cooperate in forming the different

378

states of regulated actin. That cooperation may be the basis for the ability of a

379

mutation in one troponin subunit to rescue the effects of a mutation in the other

380

subunit. The TnT R278C mutation (within the COOH terminus of TnT near the IT arm)

381

rescues the reduction of the actin filament sliding speed caused by the R145G mutation


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Biochemistry

19 21

382

at saturating Ca2+ and worsens the inhibitory activity at low Ca2+

383

∆14 TnT mutation on activation, but not inhibition, can be rescued by a

384

phosphomimetic mutation on TnI

385

some cases, to restore normal function to troponin by altering the tertiary structure of a

386

troponin subunit distant from the mutation.

17

. The effects of the

. These long-range effects may make it possible, in

387


Biochemistry

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20 388

Tables

389 390

Table I. Estimated distributions of states of actin-tropomyosin-troponin MATPasea

CAcrylodana,b

BAcrylodana

wild type Ca2+

0.33c

≈ 0.67

≈0

-

-

R146G Ca2+

0.09d

≈ 0.91

≈0

-

-

wild type EGTA

0.014c

R146G EGTA

0.028c

0.80

0.17f

R146G EGTAg

CPyrenea,b

BPyrenea

0.37e

0.62e

0.69e

0.28e

0.78e

0.19e

391

a

392

estimating the fractions of the active, M, intermediate, C, and inactive, B, states.

393

b

394

(CAcrylodan) or pyrene (Cpyrene) measurements.

395

c

396

d

397

e

398

the case where KT is small 37; KB = 1/(kCa2+/kEGTA) -1). KB = [C]/[B] at equilibrium.

399

f

400

occupancy of the B state obtained from pyrene actin measurements.

401

g

The subscript (ATPase, acrylodan, pyrene) identifies the method used for

C = 1 - (M + B) calculated using estimates of state B from either acrylodan

From an earlier study 24. From Figure 1. From conservation of mass and the estimated value of KB from the equation for

From ratio of acrylodan fluorescence changes in Figure 3 and the wild type

From a separate study with troponin increased from 0.86 to 1.42 µM.

402 403 404


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Biochemistry

21 405

Figure Legends

406

Figure 1. S1 ATPase rates as a function of the actin-tropomyosin-troponin

407

concentration at saturating Ca2+. Actin filaments contained either wild type (circles) or

408

R146G TnI (squares). Dashed lines show 95% confidence intervals. Values of the kcat

409

were altered (wild type = 23/s, R146G = 6/s), but the apparent values of KM values were

410

117 µM in both cases. Inset: double reciprocal showing the highest actin concentrations.

411

The dashed line defines 1/[S] = zero. Each tic on the abscissa is 0.02/µM and each tic

412

mark on the ordinate is 0.5 sec. Conditions: 25oC, pH 7, 1 mM ATP, 10 mM MOPS, 3

413

mM MgCl2, 33 mM NaCl, 1 mM dithiothreitol, 1 mM EGTA or 0.5 mM CaCl2 with 0.1 µM

414

S1, and actin:tropomyosin:troponin = 7:3:2.5.

415 416

Figure 2. The rate of transition from the active, M, state to the intermediate, C,

417

state in the presence of Ca2+ at high ionic strength. Acrylodan-tropomyosin fluorescence

418

changes occurred after rapid S1-ATP dissociation. The main figure shows wild type

419

over a long time interval. The inset compares the wild type (curve 1) with R146G TnI

420

containing actin filaments (curve 2). Apparent rates: wild type = 354/s, R146G = 218/s.

421

Conditions: 2 µM actin, 0.86 µM troponin, 0.86 µM acrylodan labeled tropomyosin, 2 µM

422

S1 in 20 mM MOPS buffer pH 7, 4 mM MgCl2, 152 mM KCl, 0.5 mM CaCl2 and 1 mM

423

dithiothreitol was rapidly mixed with 2 mM ATP in 20 mM MOPS buffer pH 7, 4 mM

424

MgCl2, 152 mM KCl, 0.5 mM CaCl2 and 1 mM dithiothreitol at 10°C.

425 426 427

Figure 3. The rate of transition from the active M state to the C state to the inactive B state at low Ca2+. Curve 1, wild type actin filaments; Curve 2, actin filaments


Biochemistry

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22 428

with R146G TnI. A. At the same conditions as Fig. 2 (152 mM KCl but with EGTA and

429

no added Ca2+) the apparent rates of the first phase were 449/s and 401/s for wild type

430

and R146G, respectively. The apparent rates for the slow phase were wild type = 15/s,

431

R146G = 59/s. B. At 90 mM KCl the apparent rates for the slow phase were: wild type =

432

13/s, R146G = 27/s. C. At 30 mM KCl the apparent rates for the slow phase were: wild

433

type = 13/s, R146G = 15/s. D. Fluorescence amplitudes for regulated actin containing

434

wild type (solid circles) and R146G TnI (open circles) as a function of the square root of

435

the ionic strength. The solid lines are best fits to the data. The dashed line shows a

436

constant ratio of mutant to wild type amplitudes of 0.27. Conditions: 2 µM actin, 0.86 µM

437

troponin, 0.86 µM tropomyosin, 2 µM S1 in 20 mM MOPS buffer pH 7, 4 mM MgCl2, 2

438

mM EGTA and 1 mM dithiothreitol was rapidly mixed with 2 mM ATP in 20 mM MOPS

439

buffer pH 7, 4 mM MgCl2, 2 mM EGTA and 1 mM dithiothreitol at 10°C.

440 441 442

Figure 4. Rate of binding of rigor S1 to pyrene labeled actin filaments containing

443

tropomyosin and troponin at saturating Ca2+ (A) or at very low Ca2+ (B). Curve 1, wild

444

type actin filaments; Curve 2, actin filaments with R146G TnI. Apparent rates with

445

standard deviations in the presence of Ca2+: wild type = 0.38 +/- 0.004/s, R146G = 0.37

446

+/- 0.007/s. Apparent rates at low Ca2+: wild type = 0.14 +/- 0.003/s, R146G = 0.27 +/-

447

0.007/s. Conditions: 2 µM phalloidin stabilized pyrene actin, 0.86 µM tropomyosin 0.86

448

µM troponin in a buffer containing 152 mM KCl, 2 mM EGTA, 20 mM MOPS buffer pH

449

7, 4 mM MgCl2, 1 mM dithiothreitol was rapidly mixed with 0.4 µM myosin S1 in 152 mM

450

KCl, 2 mM EGTA, 20 mM MOPS buffer pH 7, 4 mM MgCl2, 1 mM dithiothreitol at 10°C. 22 ACS Paragon Plus Environment

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Biochemistry

23 451 452

Figure 5. Possible pathway of actin-activated ATP hydrolysis by subfragment 1 of

453

myosin (S1). Only the phosphate release step is shown for simplicity. Actin-

454

tropomyosin-troponin exists in 3 states, B, C and M described by equilibrium constants

455

KB and KT in the absence of bound S1. The values of KB and KT depend on Ca2+ and on

456

mutations of troponin. Direct binding of S1 intermediates to the C and M actin states is

457

not shown for clarity although it was shown in the Hill model 43 57,

458


Biochemistry

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24 459

Figures

460

Figure 1.

461 462 463 464 465 466 467


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Biochemistry

25 468

Figure 2. 469 470 471 472 473 474 475 476 477

478 479 480 481


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

26 482

Figure 3.

483 484 485 486 487 488 489 490 491 26 ACS Paragon Plus Environment

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Biochemistry

27 492

Figure 4.

493 494


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

28 495

Figure 5.

496 497

498


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Biochemistry

29 499

Graphic for the Table of Contents

500

The R146G TnI mutation has an attenuated inactive state at low calcium

501

concentrations.

502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522

References

523


Biochemistry

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30 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574

[1] Herzberg, O., Moult, J., and James, M. N. (1986) A model for the Ca2+-induced conformational transition of troponin C. A trigger for muscle contraction, J Biol.Chem. 261, 2638-2644. [2] Gagn‚, S. M., Tsuda, S., Li, M. X., Smillie, L. B., and Sykes, B. D. (1995) Structures of the troponin C regulatory domains in the apo and calcium-saturated states, Nature Structural Biology 2, 784-789. [3] Chalovich, J. M. (1992) Actin mediated regulation of muscle contraction, Pharmacol.Ther. 55, 95-148. [4] Huxley, H. E. (1972) Structural changes in the actin and myosin containing filaments during contraction, Cold Spring Harbor Symposia on Quantitative Biology 37, 361-376. [5] Haselgrove, J. C. (1972) X-ray evidence for a conformational change in the actin containing filaments of vertebrate striated muscle, Cold Spring Harbor Symposia on Quantitative Biology 37, 341-352. [6] Parry, D. A. D., and Squire, J. M. (1973) Structural role of tropomyosin in muscle regulation: analysis of the X-ray diffraction patterns from relaxed and contracting muscles, Journal of Molecular Biology 75, 33-55. [7] Trybus, K. M., and Taylor, E. W. (1980) Kinetic studies of the cooperative binding of subfragment 1 to regulated actin, Proceedings of the National Academy of Sciences of the United States of America 77, 7209-7213. [8] McKillop, D. F. A., and Geeves, M. A. (1993) Regulation of the interaction between actin and myosin subfragment 1: Evidence for three states of the thin filament, Biophys.J. 65, 693701. [9] Pirani, A., Xu, C., Hatch, V., Craig, R., Tobacman, L. S., and Lehman, W. (2005) Single particle analysis of relaxed and activated muscle thin filaments, J Mol Biol 346, 761-772. [10] Lehman, W., Rosol, M., Tobacman, L. S., and Craig, R. (2001) Troponin organization on relaxed and activated thin filaments revealed by electron microscopy and threedimensional reconstruction, Journal of Molecular Biology 307, 739-744. [11] Poole, K. J. V., Lorenz, M., Evans, G., Rosenbaum, G., Pirani, A., Craig, R., Tobacman, L. S., Lehman, W., and Holmes, K. C. (2006) A comparison of muscle thin filament models obtained from electron microscopy reconstructions and low-angle X-ray fibre diagrams from non-overlap muscle, Journal of Structural Biology 155, 273-284. [12] Kimura, C., Maeda, K., Hai, H., and Miki, M. (2002) Ca 2+ - and S1-induced movement of troponin T on mutant thin filaments reconstituted with functionally deficient mutant tropomyosin, Journal of Biochemistry 132, 345-352. [13] Mathur, M. C., Kobayashi, T., and Chalovich, J. M. (2008) Negative Charges at Protein Kinase C Sites of Troponin I Stabilize the Inactive State of Actin, Biophysical Journal 94, 542-549. [14] Layland, J., Solaro, R. J., and Shah, A. M. (2005) Regulation of cardiac contractile function by troponin I phosphorylation, Cardiovascular Research 66, 12-21. [15] Risnik, V. V., Vorotnikov, A. V., and Gusev, N. B. (1987) Phosphorylation of troponin T by Ca-phospholipid-dependent protein kinase, Biomed.Biochim.Acta 46, S444-S444. [16] Gafurov, B., Fredricksen, S., Cai, A., Brenner, B., Chase, P. B., and Chalovich, J. M. (2004) The Delta14 mutation of human cardiac troponin T enhances ATPase activity and alters the cooperative binding of S1-ADP to regulated actin, Biochemistry 43, 15276-15285. [17] Franklin, A. J., Baxley, T., Kobayashi, T., and Chalovich, J. M. (2012) The C-Terminus of Troponin T Is Essential for Maintaining the Inactive State of Regulated Actin, Biophysical Journal 102, 2536-2544. [18] Kruger, M., Zittrich, S., Redwood, C., Blaudeck, N., James, J., Robbins, J., Pfitzer, G., and Stehle, R. (2005) Effects of the mutation R145G in human cardiac troponin I on the kinetics of the contraction-relaxation cycle in isolated cardiac myofibrils, J Physiol 564, 347-357. 30 ACS Paragon Plus Environment

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[19] Burton, D., Abdulrazzak, H., Knott, A., Elliott, K., Redwood, C., Watkins, H., Marston, S., and Ashley, C. (2002) Two mutations in troponin I that cause hypertrophic cardiomyopathy have contrasting effects on cardiac muscle contractility, Biochemical Journal 362, 443-451. [20] Wen, Y., Pinto, J. R., Gomes, A. V., Xu, Y., Wang, Y., Wang, Y., Potter, J. D., and Kerrick, W. G. (2008) Functional consequences of the human cardiac troponin I hypertrophic cardiomyopathy mutation R145G in transgenic mice, The Journal of biological chemistry 283, 20484-20494. [21] Brunet, N. M., Chase, P. B., Mihajlovic, G., and Schoffstall, B. (2014) Ca(2+)-regulatory function of the inhibitory peptide region of cardiac troponin I is aided by the C-terminus of cardiac troponin T: Effects of familial hypertrophic cardiomyopathy mutations cTnI R145G and cTnT R278C, alone and in combination, on filament sliding, Arch Biochem Biophys 552-553, 11-20. [22] Takahashi-Yanaga, F., Morimoto, S., Harada, K., Minakami, R., Shiraishi, F., Ohta, M., Lu, U. W., Sasaguri, T., and Ohtsuki, I. (2001) Functional consequences of the mutations in human cardiac troponin I gene found in familial hypertrophic cardiomyopathy, Journal of Molecular and Cellular Cardiology 33, 2095-2107. [23] Lang, R., Gomes, A. V., Zhao, J. J., Housmans, P. R., Miller, T., and Potter, J. D. (2002) Functional analysis of a troponin I (R145G) mutation associated with familial hypertrophic cardiomyopathy, Journal of Biological Chemistry 277, 11670-11678. [24] Mathur, M. C., Kobayashi, T., and Chalovich, J. M. (2009) Some cardiomyopathy causing troponin I mutations stabilize a functional intermediate actin state, Biophysical Journal 96, 2237-2244. [25] Kobayashi, T., and Solaro, R. J. (2006) Increased Ca2+ Affinity of Cardiac Thin Filaments Reconstituted with Cardiomyopathy-related Mutant Cardiac Troponin I, Journal of Biological Chemistry 281, 13471-13477. [26] Kobayashi, T., Patrick, S. E., and Kobayashi, M. (2009) Ala scanning of the inhibitory region of cardiac troponin I, The Journal of biological chemistry 284, 20052-20060. [27] Elliott, K., Watkins, H., and Redwood, C. S. (2000) Altered regulatory properties of human cardiac troponin I mutants that cause hypertrophic cardiomyopathy, Journal of Biological Chemistry 275, 22069-22074. [28] VanEyk, J. E., and Hodges, R. S. (1988) The biological importance of each amino acid residue of the troponin Iinhibitory sequence 104-115 in the interaction with troponin C andtropomyosin-actin, Journal of Biological Chemistry 263, 1726-1726. [29] Kielley, W. W., and Harrington, W. F. (1960) A model for the myosin molecule, Biochim.Biophys.Acta 41, 401-421. [30] Weeds, A. G., and Taylor, R. S. (1975) Separation of subfragment-1 isozymes from rabbit skeletal muscle myosin, Nature 257, 54-56. [31] Kouyama, T., and Mihashi, K. (1981) Fluorimetry study of N-(1-pyrenyl)iodoacetamidelabeled F-actin: local structural change of actin protomer both on polymerization and on binding of heavy meromyosin, European Journal of Biochemistry 114, 33-38. [32] Smillie, L. B. (1982) Preparation and identification of alpha- and beta-tropomyosins, In Methods in Enzymology (Frederiksen, D. W., and Cunningham, L. W., Eds.), pp 234241, Academic Press, New York. [33] Borrego-Diaz, E., and Chalovich, J. M. (2010) Kinetics of regulated actin transitions measured by probes on tropomyosin, Biophys.J 98, 2601-2609. [34] Lehrer, S. S., and Ishii, Y. (1988) Fluorescence properties of acrylodan-labeled tropomyosin and tropomyosin-actin: Evidence for myosin subfragment 1 induced changes in geometry between tropomyosin and actin, Biochemistry 27, 5899-5906.


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The R146G TnI mutation has an attenuated inactive state in the absence of calcium. 50x44mm (300 x 300 DPI)

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The Cardiomyopathy Mutation, R146G Troponin I, Stabilizes the

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