Mini-Thin Filaments Regulated by Troponin–Tropomyosin

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Mini-thin filaments regulated by troponin–tropomyosin

Huiyu Gong*, Victoria Hatch†, Laith Ali‡, William Lehman†, Roger Craig§, and Larry S. Tobacman‡¶

*Department of Internal Medicine, University of Iowa, Iowa City, IA 52242; †Department of Physiology and Biophysics, Boston University, Boston, MA 02118; §Department of Cell Biology, University of Massachusetts, Worcester, MA 01655; and ‡Departments of Medicine and Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL 60612

Edited by Edward D. Korn, National Institutes of Health, Bethesda, MD, and approved December 9, 2004 (received for review September 29, 2004) Striated muscle thin filaments contain hundreds of actin monomers normal-length thin filaments. They also would make possible and scores of troponins and tropomyosins. To study the coopera- approaches to thin-filament structural analysis. We report here tive mechanism of thin filaments, ‘‘mini-thin filaments’’ were the design and purification of mini-thin filaments with the generated by isolating particles nearly matching the minimal intended composition and compare their function to the function structural repeat of thin filaments: a double helix of actin subunits of conventional-length thin filaments. with each strand approximately seven actins long and spanned by Ca2ϩ regulates muscle contraction in the heart and in skeletal a troponin–tropomyosin complex. One end of the particles was muscle by binding to specific site(s) in the NH2 domain of the capped by a gelsolin (segment 1–3)–TnT fusion protein (substitut- troponin subunit, TnC. Significantly, Ca2ϩ activates tension very ing for normal TnT), and the other end was capped by tropomodu- cooperatively (3, 4) even in cardiac muscle, in which each TnC lin. EM showed that the particles were 46 ؎ 9 nm long, with a ϩ has only one regulatory Ca2 site (5). This observation implies knob-like mass attributable to gelsolin at one end. Average actin, that multiple troponins on each thin filament bind Ca2ϩ inter- tropomyosin, and gelsolin–troponin composition indicated one dependently in contracting muscle. Because mini-thin filaments troponin–tropomyosin attached to each strand of the two- stranded actin filament. The minifilaments thus nearly represent have only two troponins, one on each actin strand, they should single regulatory units of thin filaments. The myosin S1 MgATPase provide an opportunity to investigate this cooperative mecha- rate stimulated by the minifilaments was Ca2؉-sensitive, indicating nism. As shown in the results below, thin-filament cooperativity that single regulatory length particles are sufficient for regulation. is complex. Some aspects of cooperative regulation were dis- Ca2؉ bound cooperatively to cardiac TnC in conventional thin rupted in the particles, but other aspects were retained. The filaments but noncooperatively to cardiac TnC in minifilaments in findings suggest which processes depend on end-to-end contacts the absence of myosin. This suggests that thin filament Ca2؉- between multiple contiguous units in the thin filament. More binding cooperativity reflects indirect troponin–troponin interac- generally, the results provide perspectives on the regulation of tions along the long axis of conventional filaments, which do not muscle contraction and on the function of the thin filament as a .occur in minifilaments. Despite noncooperative Ca2؉ binding to large protein assembly -minifilaments in the absence of myosin, Ca2؉ cooperatively acti vated the myosin S1-particle ATPase rate. Two-stranded single Materials and Methods regulatory units therefore may be sufficient for myosin-mediated Design of a Gelsolin–TnT Fusion Protein. By using standard tech- ؉ Ca2 -binding cooperativity. Functional mini-thin filaments are well niques, the cDNA encoding residues Met-1–Tyr-418 of human suited for biochemical and structural analysis of thin-filament gelsolin [a gift from D. Kwiatokowski, Harvard Medical School, regulation. Boston (6)] was inserted into the NcoI site of pET3d and succeeded in frame by bovine cardiac TnT cDNA (7) (minus the ͉ ͉ actin cooperativity muscle initiating ATG of TnT). Gelsolin contains an Ϸ26-residue linker connecting domain 3 to domain 4 (8). In the construct, this linker olecular motors produce force and movement by interact- plus the structurally variable N-terminal part of domain 4 form Ming with large filamentous protein assemblies such as the the connection between gelsolin domains 1–3 and the TnT N actin-based thin filaments along which myosin motors translo- terminus. Coding regions were deemed to be without errors by cate thick filaments or organelles. In vertebrate and many automated DNA sequencing. invertebrate striated muscles, thin filaments also contain tropomyosin and troponin, which regulate actin–myosin interactions Protein Purification. The gelsolin–TnT fusion protein was ex- and thus muscle contraction. The present work describes the pressed in BL21 (DE3). After washing twice with 50 mM isolation and characterization of ‘‘mini-thin filaments,’’ which Tris⅐HCl (pH 8.5)͞2 M urea͞1 mM EDTA, inclusion bodies were approximate single regulatory units of the thin filament and dissolved in 50 mM diethanolamine (pH 8.9)͞8 M urea͞1mM represent an approach for investigating these large assemblies EDTA͞10 mM DTT and applied to a Q-Sepharose FastFlow and their regulation. Striated muscle thin filaments typically are Ϸ1 ␮m long, column. Protein elution used a 0–0.8 M NaCl gradient in column buffer [25 mM diethanolamine (pH 8.9)͞5 M urea͞1mM comprise several hundred actin monomers, and contain one ͞ tropomyosin and one troponin for every seven actins. The EDTA 1 mM DTT]. Chicken E tropomodulin was purified by actin͞tropomyosin–troponin ratio is determined by the near using a pGEX-KG expression plasmid obtained as a gift from V. correspondence between the 38.5-nm span of seven actin mono- Fowler (The Scripps Research Institute, La Jolla, CA) (9). mers along each long-pitch helical strand of the actin filament (1) Tropomodulin was purified further by FPLC (Resource Q and the Ϸ40-nm length of the tropomyosin coiled coil (2). We column). Bovine cardiac troponin, troponin subunits, and tro- reasoned that short thin filaments Ϸ40 nm in length might be pomyosin and rabbit fast skeletal muscle myosin S1 and actin assembled by placing proteins that cap actin filament ends at were purified as described (10). opposite ends of tropomyosin. Because actin filaments are two-stranded helices, such particles would contain 14 actins, two tropomyosins, and two troponins if precisely constructed. These This paper was submitted directly (Track II) to the PNAS office. minifilaments would allow investigation of Ca2ϩ-, myosin-, and Abbreviation: IAANS, 2-(4Ј-(iodoacetamido)anilino)naphthalene-6-sulfonic acid. troponin–tropomyosin-induced cooperativity independently of ¶To whom correspondence should be addressed. E-mail: [email protected]. tropomyosin–tropomyosin end-to-end linkage that occurs on © 2005 by The National Academy of Sciences of the USA

656–661 ͉ PNAS ͉ January 18, 2005 ͉ vol. 102 ͉ no. 3 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0407225102 Downloaded by guest on October 1, 2021 Preparation of Cardiac TnC Labeled on Cys-84 with 2-(4؅-(Iodoacet- ␮M phalloidin). Free Ca2ϩ concentrations were controlled by amido)anilino)naphthalene-6-sulfonic Acid (IAANS). Recombinant addition of CaCl2 in varying amounts (15). The MgATPase rate cardiac TnC (C35S) (a gift from M. Regnier, University of of myosin S1 alone (0.032 sϪ1) was subtracted. Neither thin Washington, Seattle) was expressed in BL21 (DE3) cells. The filaments nor minifilaments had detectable MgATPase rates crude lysate was directly applied to a Q-Sepharose FastFlow under these conditions in the absence of myosin S1. The program column, and eluting TnC was resolved further on an FPLC SCIENTIST (MicroMath, Salt Lake City) was used for nonlinear Resource Q column. The purified protein was dialyzed into 0.1 least-squares fitting MgATPase rate vs. Ca2ϩ data to the Hill mM DTT͞0.2 M KCl͞30 mM Mops (pH 7.0)͞2 mM EGTA, equation. Curve fitting used averaged data for each Ca2ϩ, followed by dialysis without DTT. The TnC was adjusted to a weighted according to variance of three to five measurements. concentration of 1–2 mg͞ml, and 6 M urea plus a 4-fold excess However, similar fits resulted when all measurements were fit as of IAANS were added to effect labeling of Cys-84 at 4°C one data set. overnight. After quenching with DTT and either dialysis or G-25 ATPase measurements were made in the presence of low actin desalting, final protein concentration was determined by protein concentrations, resulting in rates very far below the Vmax of assay with unlabeled TnC as a standard. Labeling stoichiometry myosin S1. The low rates resulted from two interrelated causes: was 0.88 mol of fluorophore͞mol of protein, as determined by relatively low concentrations of the particles were available, and ⌭ ϭ Ϫ1⅐ Ϫ1 IAANS 325 24,900 M cm . S1-particle binding was inhibited by the presence of KCl required during particle preparation (particle isolation required 100 mM Reconstitution and Purification of a Gelsolin–Troponin Complex. Gel- KCL, of which 40 mM KCl remained under ATPase conditions). solin–TnT and bovine cardiac muscle TnI and TnC (or IAANS- In control experiments, three minifilament preparations were labeled TnC for experiments so indicated) were mixed under examined by electron microscopy in the presence of ATP and denaturing conditions in 1:1:1 ratios in the presence of 2 M myosin S1 under conditions identical to those of the ATPase ͞ ͞ ⅐ ͞ urea 1 M KCl 20 mM Tris HCl (pH 7.8) 1 mM DTT. Protein experiments. No long filaments were observed, and no effect of molarities were calculated by absorbance (11). Mixtures were myosin S1 on the particles was detected. Thus, myosin S1 did not dialyzed in consecutive solutions containing 1, 0.7, 0.5, 0.3, 0.2, ⅐ ͞ induce filament elongation in the presence of ATP, and ATPase and 0.06 M KCl plus 10 mM Tris HCl (pH 7.8) 1mMDTT. results reported below were due to the interactions between Samples were clarified by centrifugation, applied to an FPLC myosin S1 and short thin-filament particles. Resource Q column equilibrated with the same buffer, and eluted with a 0–0.8 M NaCl gradient. Fluorescence Measurement. Mini-thin filaments labeled with ␮ IAANS TnC were prepared as described for unlabeled TnC and Assembly and Isolation of Mini-Thin Filaments. First, 15 M G-actin, examined at an actin concentration of 1 ␮M. Fluorescence 3 ␮M tropomyosin, 3 ␮M tropomodulin, 3 ␮M gelsolin– measurements were performed on 1.8-ml samples by using a troponin, and 15 ␮M phalloidin were mixed in the presence of FluoroMax-3 spectrofluorometer. Calcium titrations were per- 10 mM Mops (pH 7.0)͞0.2 mM DTT͞0.2 mM ATP, and then 2 formed by sequential addition of CaCl . Free calcium concen- mM MgCl and 100 mM KCl were added. After incubation at 2 2 trations were calculated as described (16). Conditions were 25°C room temperature for 30 min, the 3- to 3.5-ml solution was and 20 mM Mops (pH 7.0)͞1mMDTT͞3 mM MgCl ͞0.1 M chromatographed over Sephacryl HR S-500 equilibrated in 10 2 KCl͞0.2 mM ATP͞1 mM EGTA. Conventional thin filaments mM Mops (pH 7.0)͞2 mM MgCl2͞0.2 mM DTT͞0.2 mM ATP and 50 mM or 100 mM KCl. Fractions containing mini-thin also were studied by reconstitution of purified proteins to final concentrations of 1.4 ␮M F-actin, 0.5 ␮M tropomyosin, 0.2 ␮M filaments with concentrations at least 50% of the peak concen- ␮ tration were pooled and characterized. Of Ϸ2 mg of initial actin, IAANS-troponin, and 1.4 M phalloidin, incubated together for Ϸ1 mg was recovered as minifilaments. To determine protein 1 h at room temperature. These concentrations were designed to ͞ maximize saturation of actin with troponin–tropomyosin so that ratios, SDS PAGE of the particles was compared quantitatively 2ϩ to SDS͞PAGE standard curves of actin (42 kDa), tropomyosin cooperativity could be examined. Ca increased the fluores- (dimer, 66 kDa), and TnI (23.5 kDa) as described (12). (The cence intensity of these thin filaments by 36%, similar to the 39% actin standard curve also included tropomodulin at a 1:14 ratio increase for other samples, which were designed to maximize the ␮ relative to actin.) Results indicated a 7:(0.97 Ϯ 0.11):(0.97 Ϯ fraction of troponin that was bound to the thin filament (5 M ␮ ␮ 0.10) ratio of actin͞tropomyosin͞gelsolin–troponin T. F-actin, 1.8 M tropomyosin, and 0.2 M IAANS-troponin). Fluorescence intensities were corrected for progressive dilution Ϸ 2ϩ Electron Micrographs and Image Analysis. Solutions containing ( 3% maximum). Ca decreased the fluorescence intensity of mini-thin filaments were applied to holey carbon grids or holey IAANS-troponin in the absence of actin by 2% (data not shown), carbon coated with an additional carbon layer and negatively similar to previous findings (17). SCIENTIST was used for non- stained with 1% uranyl acetate (13). Unchromatographed sam- linear least-squares fitting to the Hill equation or to a nonco- ples were viewed and recorded with a Hitachi (Tokyo) H-7000 operative binding isotherm by using pooled data from multiple transmission electron microscope. Sephacryl HR S-500 chro- fluorescence titrations. matographed particles were examined under low-dose conditions (Ϸ12 electron͞Å2) in a Philips (Eindhoven, The Nether- Results lands) CM120 electron microscope. Magnifications were Strategy for the Creation of Mini-Thin Filaments. Gelsolin caps the calibrated to 2% accuracy by using a diffraction grating with ‘‘barbed’’ (‘‘plus’’) end of the two-stranded actin filament, pre- 2,160 lines per mm, and unselected filaments were measured venting actin monomer addition and dissociation. The N- particle by particle by using the program IMAGE J. terminal three domains of gelsolin bind to the end of one actin strand, and domains 4–6 bind to the end of the other strand (8, Myosin S1 ATPase Assay. Myosin S1 ATPase activity was deter- 25). We therefore reasoned that short F-actin particles could be mined as described in ref. 14. Conditions were: 25°C, 1 ␮M produced by using troponin to position gelsolin domains 1–3 on myosin S1͞5 mM imidazole (pH 7.1)͞3.5 mM MgCl2͞40 mM each of the two strands, at seven-actin intervals along thin KCl͞1mMDTT͞1mMATP͞0.5 mM dibromo-1,2-bis(2- filaments. More specifically, gelsolin domains 1–3, fused to the aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid, and either TnT N terminus, might cap thin filaments near the site of normal mini-thin filaments (0.9 ␮M actin) or conventional thin filaments end-to-end overlap of adjacent tropomyosins in long thin fila- BIOPHYSICS (0.9 ␮M F-actin, 0.4 ␮M tropomyosin, 0.4 ␮M troponin, and 0.2 ments. The other, ‘‘pointed’’ (‘‘minus’’) end of the filaments then

Gong et al. PNAS ͉ January 18, 2005 ͉ vol. 102 ͉ no. 3 ͉ 657 Downloaded by guest on October 1, 2021 Fig. 1. SDS͞PAGE of troponin and gelsolin–troponin fusion protein. Lane 1, bovine cardiac troponin prepared by reconstitution from isolated subunits TnT, TnI, and TnC followed by ion-exchange chromatography; lanes 2–3 (duplicates), similar to lane 1 except the reconstituted complex contained the gelsolin–TnT fusion protein instead of TnT; lane 4, native bovine cardiac troponin; Stnd, molecular weight standards. Results are from a 14% acryl- amide gel. Fig. 2. Electron microscopy of mini-thin filaments. Conditions were as follows: 5 mM Mops, pH 7.0͞2 mM MgCl2͞100 mM KCl͞0.2 mM ATP͞0.2 mM DTT. Proteins were incubated in the following concentrations: 5 ␮M G-actin, could be capped with tropomodulin, which interacts with the 1.2 ␮M tropomyosin, 1 ␮M gelsolin–troponin, 1 ␮M tropomodulin, and 5 ␮M tropomyosin N terminus (18). phalloidin. The samples were diluted 5-fold immediately before application to We considered that this approach might succeed for the grids. (A) Tropomyosin was omitted. (B) Tropomodulin was omitted. (C) All components were present. (D) Higher magnification of minifilaments pre- following reasons: The troponin tail domain, comprised of pared with all components as in C and then chromatographed by using N-terminal 170–200 amino acids of TnT, attaches to tropomy- Sephacryl S-500 (see Materials and Methods). osin near the C terminus of the latter (4, 19, 20); hence, an appropriately engineered gelsolin–TnT construct would be po- sitioned to make it feasible, in principle, to cap F-actin just Electron Microscopy of Mini-Thin Filaments. After size-exclusion beyond the end of troponin–tropomyosin. In addition, gelsolin chromatography (Fig. 3 Lower) mini-thin-filament structure was fragments comprising domains 1–3 cap filaments regardless of examined by negative-stain electron microscopy. Minifilament Ca2ϩ concentration, presumably with one fragment linked to the width was relatively constant and was indistinguishable from barbed end of each actin strand (21, 22). Finally, TnC and TnI normal F-actin–tropomyosin width. However, particle lengths attach to TnT by means of the C-terminal region of the latter (Fig. 4) were much shorter than those of normal thin filaments, Ϯ ϭ (24), indicating that attachment of a fusion protein to the TnT with a mean of 45.7 8.8 nm (n 583), which corresponds to N terminus is unlikely to interfere with formation of a complex F-actin filaments approximately seven to eight monomers long, between TnT and the two other troponin subunits. with capping proteins on both ends. The average particle size Before attempting to generate mini-thin filaments, cardiac therefore approximated the seven-actin span of a single troponin–tropomyosin complex, as intended. Virtually all the particles TnC, cardiac TnI, and the cardiac TnT–gelsolin fusion protein Ͻ were combined to form a ternary, gelsolin–troponin complex, (97%) were 80 nm and therefore could not accommodate two end-to-end tropomyosin lengths. Nevertheless, the length distri- which was purified by FPLC. SDS͞PAGE indicated successful bution (Fig. 4) was broad, indicating that the particles were not complex formation (Fig. 1). Mini-thin filaments then were grown uniform. from monomeric G-actin polymerized by addition of KCl and Except for their short length, minifilaments appeared similar MgCl in the presence of tropomyosin, gelsolin–troponin, and 2 to reconstituted F-actin or native thin filaments (Fig. 2D). A tropomodulin. Phalloidin was included to help stabilize the knob-like mass, however, was observed on one end of many minifilaments. Electron microscopy showed that under these ϽϽ particles (arrows). This feature cannot be troponin, because the conditions only short filaments (mostly 100 nm) were formed globular, core domain of troponin binds closer to the middle (Fig. 2C). Similar results were obtained in the presence or than to the end of tropomyosin (19, 24) and thus would not be 2ϩ absence of Ca , i.e., with the addition of either CaCl2 or EGTA expected to contribute to density at the end of the particles. (data not shown). Tropomyosin, tropomodulin, and gelsolin– Instead, the extra density primarily must represent the gelsolin troponin were each required for minifilament formation. Omis- portions of gelsolin–troponins, one on each strand, each with a sion of tropomyosin (Fig. 2A) or tropomodulin (Fig. 2B) yielded mass (Ϸ45 kDa) comparable to that of an actin monomer. In relatively long filaments (several hundred nanometers), still structural models of gelsolin binding to F-actin, domains 1–3 shorter than pure F-actin filaments but not useful for the present protrude from the filament (8, 25). In contrast, models of experiments. Omission of gelsolin–troponin yielded filaments tropomodulin binding (26) do not suggest a large protrusion of with normal lengths, little different in appearance from F-actin– this type. tropomyosin filaments (data not shown). Multiple attempts to form particles by severing F-actin with gelsolin–troponin rather SDS͞PAGE of Mini-Thin Filaments. After size-exclusion chromatog- than limiting growth from G-actin were unsuccessful and yielded raphy to remove any free tropomodulin, gelsolin–troponin, mixtures of completely depolymerized actin and relatively long tropomyosin, and monomeric actin, the composition of the filaments. minifilaments was examined by SDS͞PAGE (Fig. 3). The par-

658 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0407225102 Gong et al. Downloaded by guest on October 1, 2021 Fig. 5. Mini-thin filaments activate myosin S1 ATPase activity. Addition of mini-thin filaments to myosin S1 increased ATPase activity in a Ca2ϩ- dependent manner, with a 2.6-fold overall activation by Ca2ϩ (filled circles), which indicates that short filaments, only one regulatory unit long, were sufficient for regulation. Each point corresponds to an average of three to five measurements. The results indicate a cooperative transition (solid line) with 2ϩ 6 Ϫ1 nH ϭ 2.4 Ϯ 0.5 and Ca Kapp ϭ 1.34 Ϯ 0.14 ϫ 10 M . Adjusted data for conventional thin filaments are shown also (open circles), after normalization and scaling to the same maximum and minimum rates (see Results).

that of actin, and the two proteins closely comigrate on SDS͞ Fig. 3. Isolation of mini-thin filaments using size-exclusion chromatography. PAGE (data not shown). To test for the presence of tropomodu- HR S500 chromatography (Lower) and gradient gel SDS͞PAGE (Upper)of mini-thin filaments are shown. The minifilament peak is indicated on the lin on minifilaments, the actin band (including any proteins that chromatogram, performed with a column volume of Ϸ90 ml. Other peaks comigrate with actin) was excised from the gel and digested with were variable in size from preparation to preparation and contained low trypsin, and the proteolytic fragments were analyzed by protein concentrations. OD is in arbitrary units. Lanes 1–3 show column MALDI-TOF mass spectrometry. Six tropomodulin peptides fractions on the leading edge, peak, and trail of the minifilament peak, were detected, along with 11 actin peptides, confirming that the respectively. pre, sample before column loading; Tm, tropomyosin. particles contained tropomodulin. The heights of the most prominent tropomodulin peaks (corresponding to residues 315– 325 and 170–189) were similar to tropomodulin peak heights for ticles contained actin, tropomyosin, TnC, TnI, and TnT–gelsolin a control sample containing actin and tropomodulin in a 14:1 in a 7:1:1:1:1 ratio (Materials and Methods). Thus, on average, the ratio (peak heights measured relative to actin peptide peaks). actin, tropomyosin, and troponin composition of the particles However, the observed peak ratios were variable, and therefore resembled the composition of the repeating structural unit of the the amount of tropomodulin per particle could not be measured thin filament. easily. The molecular weight of tropomodulin is nearly identical to Mini-Thin Filaments Activate the MgATPase Activity of Myosin. Con- ventional thin filaments activate the myosin S1 MgATPase rate, and Ca2ϩ regulates this activation. Similarly, minifilaments activated myosin S1 ATPase, and Ca2ϩ controlled this activation (Fig. 5, filled symbols and solid line). Composite data from three preparations showed overall dependence on Ca2ϩ in which the ATPase rate was almost 3-fold higher in the presence of calcium than in its absence: 0.049 Ϯ 0.006 vs. 0.127 Ϯ 0.004 sϪ1 (ATP split per S1͞s). Long filaments, therefore, are not necessary for Ca2ϩ-sensitive control. Fig. 5 also shows that Ca2ϩ sensitivity (i.e., apparent Ca2ϩ affinity) was similar for thin-filament particles (filled circles, Kapp ϭ 1.34 Ϯ 0.14 ϫ 106 MϪ1) and for conventional thin filaments 5 Ϫ1 (open circles, Kapp ϭ 7.4 Ϯ 0.5 ϫ 10 M ). To facilitate long filament vs. minifilament Ca2ϩ-sensitivity comparison, ATPase activation data for conventional filaments was normalized and then rescaled for Fig. 5 to match the maximum and minimum rates for minifilaments. When not so scaled, conventional thin filaments revealed slightly lower rates in the absence of Ca2ϩ Ϯ Ϫ1 Fig. 4. Mini-thin-filament particle length distribution. After size-exclusion (0.03 0.01 s ) and a 2-fold higher maximal rate in the 2ϩ Ϯ Ϫ1 chromatography, filaments were negatively stained and examined by elec- presence of saturating Ca (0.28 0.09 s ). These differences tron microscopy. Particle lengths are shown as binned into 2-nm groups and may reflect the presence of a small fraction of particles either BIOPHYSICS expressed as percent total. The major peak corresponds to a Gaussian distri- shorter or longer than 40 nm (see Fig. 4). Tropomyosin would not bution of 45.7 Ϯ 8.8 nm (SD). be expected to bind properly to the very short particles, and

Gong et al. PNAS ͉ January 18, 2005 ͉ vol. 102 ͉ no. 3 ͉ 659 Downloaded by guest on October 1, 2021 similarly, those with lengths between 40 and 80 nm might bind only one tropomyosin, which in both cases could lead to actins that were unregulated by troponin–tropomyosin. Surprisingly, minifilament ATPase activation by Ca2ϩ was cooperative, as evidenced by a Hill coefficient (nH)of2.4Ϯ 0.5 (Fig. 5, solid line), where nH Ͼ 1 suggests cooperative site–site interactions. Long filaments exhibit very similar cooperativity 2ϩ (and Ca Kapp), as shown in Fig. 5 and as reported (4, 10, 15, 27). Myosin is known to increase TnC Ca2ϩ affinity, and myosin can bind cooperatively to the seven actins contacted by a single troponin–tropomyosin complex (3, 28–31). These properties may have influenced the Fig. 5 data. Could they explain the cooperativity? To evaluate this possible explanation, one can consider a minifilament model in which the above-mentioned properties are postulated in the extreme: (i) myosin binding is maximally cooperative for each actin strand (i.e., either seven myosins are bound or none); (ii) bound myosin increases the Ca2ϩ affinity of that strand’s TnC by 10-fold; (iii) the ATPase rate is proportional to myosin binding and also requires Ca2ϩ Fig. 6. Ca2ϩ binding to the regulatory sites of thin filaments and to regula- ϩ binding to TnC site II; and (iv) the two actin strands do not affect tory sites of thin-filament particles. Ca2 binding was monitored by titration each other. Simulations of this steady-state model (not shown) of thin filaments (open circles) or minifilament particles (filled circles), in each show that the magnitude and Ca2ϩ sensitivity of the ATPase rate case fluorescently labeled on cardiac TnC at Cys-84. Solid lines are best-fit 2ϩ curves and indicate a qualitatively different transition shape for the long thin depend on myosin concentration, and Ca and myosin affect filaments. Unlike the findings for thin-filament particles, the effect of Ca2ϩ on the binding of each other. Nevertheless, the simulations confirm long thin filaments was cooperative, producing a steeper transition. Steady- 2ϩ a noncooperative nH ϭ 1 for ATPase rate vs. Ca concentra- state fluorescence results from three experiments are superimposed. Intensi- tion, which is what cooperativity theory (33) would predict, ties were normalized to the fractional change produced by saturating Ca2ϩ. because there is only one Ca2ϩ-binding site in this illustrative The maximal fluorescence increase for short thin filaments was two-thirds of system. Therefore, returning to the present data, cooperative the maximal increase for conventional thin filaments (i.e., a 24% increase ϩ ϩ myosin binding to the multiple actins within each strand cannot rather than 36% after Ca2 addition). Ca2 binding to the particles was slightly ϭ Ϯ ϫ 6 Ϫ1 Ϯ ϫ 6 Ϫ1 be excluded but nevertheless is disfavored as an explanation for stronger (KCa2ϩ 4.5 0.2 10 M for the particles vs. 2.78 0.05 10 M for thin filaments). nH Ͼ 1 in Fig. 5. An alternative interpretation is that the troponins on opposite strands of the particles interact in some manner. This possibility is discussed further below. troponin–troponin interactions along the longitudinal filament axis affect Ca2ϩ binding in the absence of myosin. Second, 2؉ 2ϩ Ca Binding to Thin Filaments and to Mini-Filaments. Ca binds minifilaments activated the myosin S1 MgATPase rate in a cooperatively to the regulatory sites (TnC site II) of isolated Ca2ϩ-regulated manner, indicating that short-length particles cardiac thin filaments (27), as monitored by the fluorescence are sufficient for Ca2ϩ-mediated control of myosin. Finally, intensity of TnC labeled with IAANS. However, two Cys resi- ATPase regulation by Ca2ϩ was cooperative, which suggests the dues are labeled with this approach (34), introducing ambiguity possibility that troponin–troponin interactions also may occur in interpretation, and results are condition-dependent (27). A across the thin filament (i.e., between its two strands) when potential improvement is to use single-Cys TnC, specifically myosin is present. labeled on Cys-84 (17, 34, 35). Single-Cys TnC has been used to ϩ Although attempts to understand the cooperative regulation monitor Ca2 binding to isolated TnC and to TnC incorporated of muscle contraction lack consensus, they have a common into muscle fibers but has not been used previously to study thin filaments in the absence of myosin. We prepared both conven- theme emphasizing the extended linear organization of the thin tional and mini-thin filaments containing Cys-84–IAANS TnC. filament. Both structural and biochemical data imply that there Ca2ϩ cooperatively increased the fluorescence intensity of are no less than three states of the thin filament (e.g., see refs. Cys-84 IAANS-labeled control thin filaments (Fig. 6, open 30, 36, and 37). In some models (38, 39), the key regulatory phenomenon is a propagated change in the conformation of symbols), with nH ϭ 1.77 Ϯ 0.05. Because there is only one regulatory site on each cardiac TnC, this cooperativity implies many adjacent actins. Functionally important changes in actin that troponins interact with each other in the absence of myosin. may occur, but the regulatory mechanism in our view instead An identical Ca2ϩ titration was performed by using IAANS- should emphasize structural and kinetic evidence that strong labeled thin-filament particles (Fig. 6, filled symbols). In this myosin–actin attachment requires tropomyosin to shift position on actin (30, 37, 40). Therefore, we concur with others who case, the fluorescence transition was noncooperative (nH ϭ 0.999 Ϯ 0.005), which suggests that Ca2ϩ-binding cooperativity consider myosin-binding cooperativity to involve the tendency of in conventional thin filaments reflects interactions along the the tropomyosin strand to shift position not at a single actin or long filament axis, absent in the particles. even at seven actins but rather coordinately on actins within several adjacent regulatory units (31, 37, 41, 42). Such shifts are Discussion consistent with three-state models, because Ca2ϩ and myosin The design and isolation of mini-thin filaments allowed us to have different effects on the position of tropomyosin on actin. distinguish aspects of cooperative thin-filament activation that Transitions among thin-filament states tend to be cooperative. ϩ are inherent in a single regulatory unit (i.e., a double actin helix Results shown in Fig. 6 strongly suggest that the effect of Ca2 with seven actins and a troponin–tropomyosin complex in each on the thin filament involves cooperative effects between tro- strand) from those that depend on end-to-end contacts between ponins located successively along the long axis of the actin multiple contiguous units. There are three principal findings. filament. If, instead, cooperative Ca2ϩ binding to conventional First, in the absence of myosin, Ca2ϩ bound cooperatively to the thin filaments were caused by interactions between troponins regulatory sites of long thin filaments but noncooperatively to opposite each other on the two actin strands, then similar those of mini-thin filaments, which suggests that (indirect) cooperative Ca2ϩ binding to the mini-thin filaments would be

660 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0407225102 Gong et al. Downloaded by guest on October 1, 2021 expected, contrary to observation. Therefore, we conclude that and͞or ATPase results. Ca2ϩ binding by the gelsolin fragments regulatory sites on troponins adjacent to each other along the might have altered troponin-mediated effects of Ca2ϩ or even long axis of (conventional) thin filaments tend to bind Ca2ϩ mediated strand–strand communication at the particle ends. We cooperatively, despite their Ϸ40-nm separation. From the ob- consider such phenomena unlikely to have had major effects. 2ϩ 2ϩ served nH, the cooperative free energy affecting Ca binding Conventional and mini-thin filaments exhibited similar Ca 2 can be estimated as ⌬Gc ϭϪRT ln (nH ) ϭ 0.7 kcal͞mol (1 cal ϭ affinity (Figs. 4 and 5), fluorescence change, and ATPase 4.18 J) (27). regulation. The similar Ca2ϩ affinities suggest that the gelsolin Notably, the cooperative activation of the myosin S1- and troponin Ca2ϩ sites do not interact strongly, and the data ϩ minifilament ATPase rate by Ca2 (Fig. 5) contrasts with the generally suggest that the particles comprise a valid experimental ϩ noncooperative Ca2 binding observed in the absence of myosin model for studying the properties of the thin filament. Also, (Fig. 6). Low ATPase rates and limited material thus far have cooperativity typically reflects quaternary structure shifts that precluded testing whether myosin binding itself is cooperative in involve specific protein–protein contacts. Therefore, we view Ϸ the presence of ATP. Nevertheless, nH 2 suggests that myosin end effects or other spurious features of the particles as unlikely S1 ATPase activity requires that site II of both TnCs on ϩ (albeit plausible) explanations of the ATPase cooperativity. minifilaments bind Ca2 and that the sites strongly affect each Finally, there are two interrelated rationales for isolating other. Previously, we suggested that myosin’s Ϸ10,000-fold thin-filament particles: to investigate function as in the present strengthening of tropomyosin–actin binding reflects an effect of report and to facilitate determination of structure. Current myosin on the actin inner domain, the actin site at which structural models of the thin filament (and of bare F-actin) are tropomyosin binds to myosin-decorated actin. The current re- informative, but these models are not at atomic resolution and sults can be explained if the effects of myosin on actin also alter are notably poor in information about troponin. Also, the the interface between monomers on the two strands of the structure of gelsolin bound to F-actin is less well elucidated than filament. If so, then myosin binding to actin on one strand would is the structure of gelsolin bound to G-actin. Single-particle influence the troponin–tropomyosin on the opposite strand by analysis of electron microscope images of the minifilaments and means of linked actin–actin and actin–tropomyosin effects. potentially x-ray crystallography of minifilaments offer solutions However, additional evidence will be needed to determine to unraveling the structural organization of actin and actin- whether myosin binding to one actin strand affects the actin binding proteins. monomers, tropomyosin, and͞or troponin on the opposite actin strand. If strand–strand interactions are as important as sug- We thank Jianqiang Shao and Carol Butters for technical assistance and gested by the current data, then additional supporting results Drs. D. Kwiatokowski, V. Fowler, and M. Regnier for gifts of plasmids. should emerge. This work was supported by National Institutes of Health Grants It is important to consider whether experimental capping of HL-38834 (to L.S.T.), HL-36153 (to W.L.), and AR-34711 (to R.C.) and the minifilament ends spuriously affected the fluorescence Shared Instrumentation Grant RR08426 (to R.C.).

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