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 tropo- myosin 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.