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The C Terminus of Cardiac Troponin I Stabilizes the Ca2+-Activated State of Tropomyosin on Actin Filaments

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The C Terminus of Cardiac Troponin I Stabilizes the Ca2+-Activated State of Tropomyosin on Actin Filaments
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  -L. Albert Wang, William Lehman and D. Brian FosterAgnieszka Galinska, Victoria Hatch, Roger Craig, Anne M. Murphy, Jennifer E. Van Eyk, C. Tropomyosin on Actin Filaments-Activated State of 2+The C Terminus of Cardiac Troponin I Stabilizes the Ca Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 2009 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Circulation Research doi: 10.1161/CIRCRESAHA.109.2100472010;106:705-711; srcinally published online December 24, 2009; Circ Res. http://circres.ahajournals.org/content/106/4/705 World Wide Web at: The online version of this article, along with updated information and services, is located on the  http://circres.ahajournals.org/content/suppl/2009/12/23/CIRCRESAHA.109.210047.DC1.html Data Supplement (unedited) at:  http://circres.ahajournals.org//subscriptions/ is online at: Circulation Research Information about subscribing to Subscriptions:  http://www.lww.com/reprints Information about reprints can be found online at: Reprints:  document. Permissions and Rights Question and Answer about this process is available in thelocated, click Request Permissions in the middle column of the Web page under Services. Further informationEditorial Office. Once the online version of the published article for which permission is being requested is can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Circulation Research in Requests for permissions to reproduce figures, tables, or portions of articles srcinally published Permissions:  at WELCH MED LIBR-JHU-MEYER SERIA on March 13, 2013http://circres.ahajournals.org/ Downloaded from  The C Terminus of Cardiac Troponin I Stabilizes theCa 2  -Activated State of Tropomyosin on Actin Filaments Agnieszka Galin´ska,* Victoria Hatch, Roger Craig, Anne M. Murphy, Jennifer E. Van Eyk,C.-L. Albert Wang, William Lehman,* D. Brian Foster* Rationale: Ca 2  control of troponin–tropomyosin position on actin regulates cardiac muscle contraction. Theinhibitory subunit of troponin, cardiac troponin (cTn)I is primarily responsible for maintaining a tropomyosinconformation that prevents crossbridge cycling. Despite extensive characterization of cTnI, the precise role of itsC-terminal domain (residues 193 to 210) is unclear. Mutations within this region are associated with restrictivecardiomyopathy, and C-terminal deletion of cTnI, in some species, has been associated with myocardial stunning.Objective: We sought to investigate the effect of a cTnI deletion–removal of 17 amino acids from the C terminus–on the structure of troponin-regulated tropomyosin bound to actin.Methods and Results: A truncated form of human cTnI (cTnI 1–192 ) was expressed and reconstituted with troponinC and troponin T to form a mutant troponin. Using electron microscopy and 3D image reconstruction, we showthat the mutant troponin perturbs the positional equilibrium dynamics of tropomyosin in the presence of Ca 2  .Specifically, it biases tropomyosin position toward an “enhanced C-state” that exposes more of the myosin-binding site on actin than found with wild-type troponin.Conclusions: In addition to its well-established role of promoting the so-called “blocked-state” or “B-state,” cTnIparticipates in proper stabilization of tropomyosin in the “Ca 2  -activated state” or “C-state.” The last 17 aminoacids perform this stabilizing role. The data are consistent with a “fly-casting” model in which the mobile Cterminus of cTnI ensures proper conformational switching of troponin–tropomyosin. Loss of actin-sensingfunction within this domain, by pathological proteolysis or cardiomyopathic mutation, may be sufficient toperturb tropomyosin conformation. ( Circ Res . 2010;106:705-711.)Key Words:  troponin    thin filament    myocardial stunning    cardiomyopathy L esions of the myofilament proteins are a common causeof inherited and acquired forms of heart disease. 1,2 Suchdefects in the thin filament protein, cardiac troponin (cTn)I, havebeen implicated in both hypertrophic and restrictive cardiomy-opathy, as well myocardial stunning. For example, mutationswithin the C-terminal domain of the cTnI gene that cause aminoacid substitutions R192H, G203S, and K206Q lead to hypertro-phic and/or restrictive cardiomyopathy, 3,4 whereas removal of the C-terminal 17 amino acids from cTnI by Ca 2  -dependentproteolysis has been implicated in models of myocardial stun-ning, 5–9 a condition that arises from brief ischemia that substan-tially depresses contractile function without causing celldeath. 2,10 In fact, expression of truncated cTnI 1–193 , at levels  20% relative to endogenous cTnI, is sufficient to substantiallycompromise systolic and diastolic function 11 in mice. Moreover,C-terminal degradation of cTnI has been observed in patientsundergoing coronary artery bypass graft surgery. 12 Given therole of this domain of cTnI in genetic and acquired heart disease,efforts are underway to fully understand its function.TnI is 1 of the 3 subunits that comprise the troponin complexthat regulates muscle contraction by controlling the position of tropomyosin on actin filaments in response to Ca 2  . 13,14 Knownas the inhibitory subunit of troponin, it prevents myosin bindingto actin, in diastole, by maintaining tropomyosin over the outeredge of actin filaments. In systole, Ca 2  binds to the Ca 2  -receptor subunit of troponin, TnC, which causes a conforma-tional change that promotes its interaction with cTnI andcoincident release of TnI inhibitor regions from actin. In turn,this causes the average position of tropomyosin to shift acrossthe face of actin and thereby exposes myosin-binding sites thatare then accessible for myosin to begin crossbridge cycling. 15 The domains of TnI, which contain multiple binding sites forTnC, TnT, actin, and tropomyosin, have been extensivelycharacterized, 16 and their organization has been clarified by the Original received September 28, 2009; revision received December 14, 2009; accepted December 15, 2009.From the Department of Physiology & Biophysics (A.G., V.H., W.L., D.B.F.), Boston University School of Medicine, Mass; Department of CellBiology (R.C.), University of Massachusetts Medical School, Worcester; Departments of Pediatrics (A.M.M.) and Medicine (J.E.V.E., D.B.F.), Divisionof Cardiology; and Departments of Biological Chemistry and Biomedical Engineering (J.E.V.E.), Johns Hopkins University School of Medicine,Baltimore, Md; and Boston Biomedical Research Institute (C.-L.A.W., D.B.F.), Watertown, Mass.*These authors contributed equally to this work.Correspondence to D. Brian Foster, PhD, Department of Medicine, Division of Cardiology, Johns Hopkins School of Medicine, 720 Rutland Ave,Baltimore, MD 21205; E-mail dbrianfoster@jhmi.edu or to William Lehman, PhD, Department of Physiology & Biophysics, Boston University Schoolof Medicine, 72 E Concord St, Boston, MA 02118; E-mail wlehman@bu.edu© 2010 American Heart Association, Inc. Circulation Research  is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.109.210047 705 at WELCH MED LIBR-JHU-MEYER SERIA on March 13, 2013http://circres.ahajournals.org/ Downloaded from  low- and high-Ca 2  crystal structures of cardiac and skeletalmuscle troponin. 17,18 However, only an incomplete picture of theregulatory switching of tropomyosin on actin can be garneredfrom the troponin structures, because the C terminus of TnI isunresolved, owing to its high flexibility. 19 Given the pathologi-cal significance of lesions with the C terminus of cTnI, wesought to determine how truncation of TnI affects the primefunctionoftroponin,namely,itsabilitytomodulatetropomyosinpositiononactinfilaments.Wediscussnewlyacquiredstructuraldata in the context of recent biochemical and biophysical studiesof cTnI 1–192  and a newly proposed model of cTnI function. 20 Methods Protein Preparation F-Actin and bovine cardiac tropomyosin were purified by standardmethods. 21 Methods describing the expression, purification, andreconstitution of troponin subunits are described in the Online DataSupplement, available at http://circres.ahajournals.org. Electron Microscopy Thin filaments were reconstituted by mixing actin, tropomyosin, andwild-type or mutant troponin in a ratio of 7:2:2 (F-actin: 10 to20   mol/L) in both low- and high-calcium buffers (low Ca 2  :5 mmol/L PIPES/5 mmol/L sodium phosphate buffer [pH 7.1],100 mmol/L NaCl, 3 mmol/L MgCl 2 , 0.2 mmol/L EGTA, 1 mmol/LNaN 3 , 1 mmol/L DTT; high-Ca 2  : same buffer supplemented with2 mmol/L CaCl 2 ). Uranyl acetate staining is described in the OnlineData Supplement. Electron microscopy was carried out on a PhilipsCM120 transmission electron microscope using low-dose methods(12 e   /Å), the details of which are described elsewhere. 22–24 Three-Dimensional Image Reconstruction FromElectron Micrographs Electron micrographs were digitized and analyzed by 2 distinct yetcomplementary methods of image reconstruction. 22,23 First, datawere analyzed by helical reconstruction, a Fourier-space filtering andaveraging method, using the Brandeis Helical Package essentially asdetailed in. 23 Given the subtle, yet statistically and biologicallysignificant, changes that we observed in thin filament structure, theresults were cross-validated by further analysis of micrographs froman independent protein preparation using the real-space single-particle averaging method of Egelman, 25 as described by Pirani etal. 22 (For a comparison of results obtained from both reconstructionmethods, see Figure III in the Online Data Supplement athttp://circres.ahajournals.org.) Results Electron Microscopy and Three-DimensionalReconstruction of Wild-Type andcTnI 1–192 -Containing Thin Filaments Thin filaments were formed from F-actin, cardiac tropomy-osin and troponin complexes under conditions known tosaturate the filaments with regulatory proteins. 22–24 “Wild-type” and mutant troponin complexes, reconstituted fromsubunits expressed in  Escherichia coli , were used for com-parison. The mutant troponin contained a truncated form of cTnI (cTnI 1–192 ) but included otherwise normal troponinsubunits, human cTnC and cTnT. Filaments were negativelystained in uranyl acetate and recorded by low-dose electronmicroscopy. 23 Electron microscopy (EM) of the thin fila-ments showed characteristic double-helical arrays of actinmonomers, tropomyosin strands, and troponin densities re-peating with a 40 nm periodicity (Figure 1). EM images of reconstituted filaments prepared from separately expressedand purified proteins were analyzed independently by the firstand last authors; the raw images and 3D reconstructionsgenerated from the 2 data sets were indistinguishable fromeach other and thus combined for analysis here. Filamentswere studied by both helical reconstruction of relatively longfilament stretches (  200 to 400 nm) 26 and by single-particlemethods on filament segments (  40 nm) 25 ; results from the 2methods were completely consistent and reproducible.Reconstructions of thin filaments showed actin subunits anddensities that were attributable to tropomyosin (Figure 2). Thelongitudinally continuous tropomyosin strands were well de-fined in both control filaments containing “wild-type” troponin– Non-standard Abbreviations and Acronyms B-state  blocked state of the thin filament C-state  Ca 2  -induced closed state of the thin filament cTn  cardiac troponin cTnI 1–192  truncated cTnI lacking 17 amino acids at the C terminus EM  electron microscopy M-state  myosin-induced fully active open state of the thin filament PIPES  piperazine- N  , N   -bis(ethanesulfonic acid) Tn  troponin Figure 1.  Electron micrographs of reconstituted thin filaments.a, Bare actin filaments. b, Actin filaments reconstituted withwild-type troponin and tropomyosin in Ca 2  -free buffer. c, Actinfilaments reconstituted with wild-type troponin and tropomyosinin the presence of Ca 2  . d, Actin filaments reconstituted withmutant troponin and tropomyosin in Ca 2  -free buffer. e, Actinfilaments reconstituted with mutant troponin and tropomyosin inthe presence of Ca 2  . Arrowheads point to the globular headsof the troponin complex. The white arrows highlight tropomyosinstrands. Scale bar  50 nm. 706 Circulation Research  March 5, 2010  at WELCH MED LIBR-JHU-MEYER SERIA on March 13, 2013http://circres.ahajournals.org/ Downloaded from  tropomyosin and in filaments containing the mutant cTnI 1–192 .Inspection of the reconstructions showed that the mutation didnot interfere with the ability of tropomyosin to undergo aCa 2  -induced shift from the outer domain (A o ) to the innerdomain (A i ) of actin; thus, the impact of both the wild-typetroponin and mutant troponin on directed tropomyosin move-ment is normal in both sets of filaments. In fact, in high-Ca 2  conditions, tropomyosin localized further onto A i  in filamentscontaining mutant cTnI (Figures 2c and 3c) than it did infilaments with the wild-type TnI (Figures 2b and 3b). Thus,whereas the direction of the tropomyosin movement was thesame in both samples, the magnitude of the movement wasgreater in the mutant (superimposed in Figure 2d and Figure 3d).As a consequence, in high Ca 2  , less lingering density touchedA o  in the mutant filaments than in the wild-type. In contrast, noobvious differences in tropomyosin position on F-actin werefound for the low Ca 2  data (Figures 2g and 3g). At Hight Ca 2  , the Effect of cTnI 1–192  IsStatistically Significant Helical projection, ie, projection of densities down the helicalaxis of F-actin and tropomyosin, provides a means of definingthe average position of tropomyosin relative to actin in recon-structions. Comparison of helical projections confirmed thattropomyosin is localized differently in wild-type and mutant thinfilaments, but again such a distinction was only detected for thehigh Ca 2  -treated sample. The distinction was subtle but be-came obvious following difference density analysis that isolated Figure 2.  Mutant troponin affects the average posi-tion of tropomyosin on actin. Three-dimensionalreconstructions of filaments are depicted in longitu-dinal view. b through d show 3D structures of fila-ments incubated in Ca 2  , whereas e through gdepict reconstructions of Ca 2  -free filaments. Thebare actin filament is depicted in a. Subdomains 1and 2 comprise the outer domain of actin (A  o  ),whereas the inner domain (A  i  ) consists of subdo-mains 3 and 4. b depicts actin–tropomyosin withwild-type troponin in Ca 2  . c depicts of mutant-controlled tropomyosin on actin in Ca 2  . In d, thepositions of tropomyosin from b and c are superim-posed. e and f show the average position of tropo-myosin conferred by wild-type and mutant troponin,respectively, in the absence of Ca 2  . The results ofe and f are superimposed in g. All structures aresuperimposed in h. Figure 3.  Cross-sections of wild-type– and mutant-controlled thin filaments. Images correspond to those described in Figure 2. bthrough d, Structures in Ca 2  . e through g, Structures in EGTA. b, Wild-type plus Ca 2  . c, Mutant plus Ca 2  . b and c are superim-posed in d. e, Wild-type in EGTA. f, Mutant in EGTA. e and f are superimposed in g. All structures are superimposed in g. Galin´ska et al C Terminus of cTnI Stabilizes the C-State  707   at WELCH MED LIBR-JHU-MEYER SERIA on March 13, 2013http://circres.ahajournals.org/ Downloaded from  the respective tropomyosin densities from actin. Here, maps of F-actin (no tropomyosin) were simply subtracted from those of thin filaments. The resulting tropomyosin densities then weresuperimposed on reference maps of bare F-actin and compared(Figure 4e). In the presence of Ca 2  , tropomyosin controlled bymutant troponin was shifted azimuthally by about 9° more thanit was by wild-type troponin (Figure 4e). Point by point analysisof the maps using Student  t   test methodology 27,28 showed thatthis difference in tropomyosin position was statistically signifi-cant at 95% confidence levels. (Also see Online Figure IV,which demonstrates further that the distinctions noted are statis-tically significant.) Because the average position of tropomyosinin the mutant is further from the low Ca 2  , blocking state than itisincontrolfilaments,wecallitthe“enhancedC-state.”Differencesin tropomyosin positions for low-Ca 2  filaments were not obviousor statistically significant. Tropomyosin Equilibrium Position on ThinFilaments Is Altered by cTnI 1–192 Tropomyosin is thought to oscillate laterally over a narrowregion of the flat surface of actin 22,29 ; however, in the presenceoftroponin,itsequilibriumbalancebecomesmorebiasedtowardspecific regulatory positions on actin, 24,30,31 namely, those of thelow-Ca 2  B-state or the high-Ca 2  C-state. The results abovesuggest that the mutant caused a rebalancing between positionalstates or possible development of a new equilibrium position fortropomyosin. Cross-correlation tools 32,33 comparing the experi-mental data to thin filament models with different tropomyosinlocations were used to sort and classify short filament segmentsinto positional categories. An analysis of high Ca 2  filamentsindicated that  3.5 times more mutant filament data fitted betterto the “enhanced C-state” than to the wild-type C-state position,whereas the reverse was true for wild-type data, where more of the data belonged to the C-state category (Table). Discussion Control of Tropomyosin Conformation by TnI 1–192 The C-terminal half of cTnI harbors 3 well-characterizeddomains: (1) an actin-binding region that inhibits actomyosinATPase activity (inhibitory peptide; residues 128 to 147); (2)a region that binds to the N-terminal domain of troponin C inthe presence of Ca 2  (switch peptide; residues 148 to 163);and (3) a second actin-binding site (residues 168 to 188). Thefunction of the remaining C-terminal residues (residues 189to 210) is largely unknown. In the absence of Ca 2  , thishighly flexible domain 19 adopts a more defined structure as itbinds to actin. Image reconstruction of thin filaments satu-rated with the C-terminal half of TnI show that the inhibitoryregion binds to actin at its N terminus (subdomain 1).Residues downstream of the inhibitory peptide span the cleftof the long-pitch helical actin strands, much like the smoothmuscle inhibitory protein caldesmon, 34 and drape over sub-domains 3 and 4 of the adjacent actin, where they abuttropomyosin and stabilize it in the blocked state (B-state). 31 The human cTnI 1–192  construct, like the form that recapitu-lates the phenotype of myocardial stunning in mice, 11 lacks thelast 17 amino acids. Previous biochemical studies 35 showed thatcTnI 1–192 , alone, bound both actin and actin-tropomyosin withthe same affinity as full length cTnI. Yet when cTnI 1–192  wasreconstituted into troponin, the complex could not fully inhibitATPase activity in the absence of Ca 2  . This suggested thateither cTnI could not maintain tropomyosin in a fully competentB-state or that equilibrium dynamics between the B- andC-states of the thin filament of might be altered. As shown inFigures 2g and 3g, mutant troponin caused no statisticallydiscernible difference in the average position of tropomyosin inthe presence of EGTA. Thus, residues 193 to 210 of cTnI,downstream of its major actin-binding regions, are not requiredto generate the B-state position of tropomyosin.In the presence of Ca 2  , the mutant troponin displays highermaximal Ca 2  - activated actin–tropomyosin–S1 ATPase thandoes the wild-type troponin. 35,36 Similar observations of bothhigher basal and Ca 2  -activated ATPase activity 35 were noted instudies of the murine variant of restrictive cardiomyopathy Figure 4.  Helical projections illustrate the impactof mutant troponin on tropomyosin position. a, Actin–tropomyosin–troponin filaments in Ca 2  -free solution. Tropomyosin sits on the outerdomain of actin. b, In Ca 2  , tropomyosin adoptsan average position over the inner domain ofactin. c, Mutant troponin containing cTnI 1–192  isalso responsive to Ca 2  and tropomyosin againadopts and average position over the innerdomain of actin. d, Superimposing results fromb and c shows that tropomyosin, controlled bymutant troponin, has shifted azimuthally toadopt an average position further from the outeractin domain by about 9°. e, To better visualizethe image densities arising from the tropomyosinstrands, the image density of bare actin fila-ments was subtracted from b and c. The densi-ties of tropomyosin controlled by wild-type (lightgreen) or mutant troponin (dark green) were superimposed over the helical projection of bare actin (blue). The difference in thecentroid positions of tropomyosin density were statistically significant at   95%. Table. Distribution of Filament Segments Sorting to DifferentRegulatory States Sample B-State (%) C-State (%)EnhancedC-State (%)Ca 2  -treated filaments withwild-type troponin29 43 28Ca 2  -treated filaments withmutant troponin26 17 57 708 Circulation Research  March 5, 2010  at WELCH MED LIBR-JHU-MEYER SERIA on March 13, 2013http://circres.ahajournals.org/ Downloaded from
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