Tomoki Aihara

The nature of the globular- to fibrous-actin transition.

Actin plays crucial parts in cell motility through a dynamic process driven by polymerization and depolymerization, that is, the globular (G) to fibrous (F) actin transition. Although our knowledge about the actin-based cellular functions and the molecules that regulate the G- to F-actin transition is growing, the structural aspects of the transition remain enigmatic. We created a model of F-actin using X-ray fibre diffraction intensities obtained from well oriented sols of rabbit skeletal muscle F-actin to 3.3 Å in the radial direction and 5.6 Å along the equator. Here we show that the G- to F-actin conformational transition is a simple relative rotation of the two major domains by about 20 degrees. As a result of the domain rotation, the actin molecule in the filament is flat. The flat form is essential for the formation of stable, helical F-actin. Our F-actin structure model provides the basis for understanding actin polymerization as well as its molecular interactions with actin-binding proteins.

Switch Action of Troponin on Muscle Thin Filament as Revealed by Spin Labeling and Pulsed EPR

We have used pulsed electron-electron double resonance (PELDOR) spectroscopy to measure the distance between spin labels at Cys133 of the regulatory region of TnI (TnI133) and a native or genetically substituted cysteine of TnC (TnC44, TnC61, or TnC98). In the +Ca2+ state, the TnC44-TnI133-T distance was 42 Å, with a narrow distribution (half-width of 9 Å), suggesting that the regulatory region binds the N-lobe of TnC. Distances for TnC61-TnI133 and TnC98-TnI133 were also determined to be 38 Å (width of 12 Å) and 22 Å (width of 3.4 Å), respectively. These values were all consistent with recently published crystal structure (Vinogradova, M. V., Stone, D. B., Malanina, G. G., Karatzaferi, C., Cooke, R., Mendelson, R. A., and Fletterick, R. J. (2005) Proc. Natl Acad. Sci. U.S.A. 102, 5038–5043). Similar distances were obtained with the same spin pairs on a reconstituted thin filament in the +Ca2+ state. In the −Ca2+ state, the distances displayed broad distributions, suggesting that the regulatory region of TnI was physically released from the N-lobe of TnC and consequently fluctuated over a variety of distances on a large scale (20–80 Å). The interspin distance appeared longer on the filament than on troponin alone, consistent with the ability of the region to bind actin. These results support a concept that the regulatory region of TnI, as a molecular switch, binds to the exposed hydrophobic patch of TnC and traps the inhibitory region of TnI away from actin in Ca2+ activation of muscle.

Calcium-Dependent Interaction Sites of Tropomyosin on Reconstituted Muscle Thin Filaments with Bound Myosin Heads as Studied by Site-Directed Spin-Labeling

To identify the interaction sites of Tm, we measured the rotational motion of a spin-label covalently bound to the side chain of a cysteine that was genetically incorporated into rabbit skeletal muscle tropomyosin (Tm) at positions 13, 36, 146, 160, 174, 190, 209, 230, 271, or 279. Most of the Tm residues were immobilized on actin filaments with myosin-S1 bound to them. The residues in the mid-portion of Tm, namely, 146, 174, 190, 209, and 230, were mobilized when the troponin (Tn) complex bound to the actin-Tm-S1 filaments. The addition of Ca(2+) ions partially reversed the Tn-induced mobilization. In contrast, residues at the joint region of Tm, 13, 36, 271, and 279 were unchanged or oppositely changed. All of these changes were detected using a maleimide spin label and less obviously using a methanesulfonate label. These results indicated that Tm was fixed on thin filaments with myosin bound to them, although a small change in the flexibility of the side chains of Tm residues, presumably interfaced with Tn, actin and myosin, was induced by the binding of Tn and Ca(2+). These findings suggest that even in the myosin-bound (open) state, Ca(2+) may regulate actomyosin contractile properties via Tm.

Interaction sites of tropomyosin in muscle thin filament as identified by site-directed spin-labeling.

To identify interaction sites we measured the rotational motion of a spin label covalently bound to the side chain of a cysteine genetically incorporated into rabbit skeletal muscle tropomyosin (Tm) at positions 13, 36, 146, 160, 174, 190, 209, 230, 271, and 279. Upon the addition of F-actin, the mobility of all the spin labels, especially at position 13, 271, or 279, of Tm was inhibited significantly. Slow spin-label motion at the C-terminus (at the 230th and 271st residues) was observed upon addition of troponin. The binding of myosin-head S1 fragments without troponin immobilized Tm residues at 146, 160, 190, 209, 230, 271, and 279, suggesting that these residues are involved in a direct interaction between Tm and actin in its open state. As immobilization occurred at substoichiometric amounts of S1 binding to actin (a 1:7 molar ratio), the structural changes induced by S1 binding to one actin subunit must have propagated and influenced interaction sites over seven actin subunits.

Orientation and Motion of Myosin Light Chain and Troponin in Reconstituted Muscle Fibers as Detected by ESR with a New Bifunctional Spin Label

Using electron spin resonance, we have studied dynamic structures of myosin neck domain and troponin C by site-directed spin labeling. We observed two broad but distinct orientations of a spin label attached specifically to a single cysteine (cys156) on the regulatoy light chain (RLC) of myosin in relaxed skeletal muscle fibers. The two probe orientations, separated by a 25 degrees axial rotation, did not change upon muscle activation, but orientational distributions became narrower substantially, indicating that a fraction of myosin heads undergoes a disorder-to-order transition of the myosin light chain domain upon force generation and muscle contraction. These results provide insight into the mechanism how myosin heads move their domains to translocate an actin filament. Site-directed spin-labeling was achieved by cysteine residues of human cardiac troponin C (TnC). Spin dipole-dipole interaction showed that free TnC undergoes a global structural change (extended-to-compact) by Ca2+ or Mg2+. The spectra from the spin labels at N-terminal half domain were broad and almost identical in parallel and perpendicular orientations of fiber, suggesting that the N-terminal of TnC molecule is flexible or disoriented with respect to the filament axis. We also succeeded, for the first time, in fixing the newly-synthesized bifunctional spin label rigidly on TnC molecule in solution (either in +/- Ca2+), giving a promise that we can determine the precise coordinate of the spin principal axis on protein surface.

Cooperative and non-cooperative conformational changes of F-actin induced by cofilin

Cofilin is an actin-binding protein that promotes F-actin depolymerization. It is well-known that cofilin-coated F-actin is more twisted than naked F-actin, and that the protomer is more tilted. However, the means by which the local changes induced by the binding of individual cofilin proteins proceed to the global conformational changes of the whole F-actin molecule remain unknown. Here we investigated the cofilin-induced changes in several parts of F-actin, through site-directed spin-label electron paramagnetic resonance spectroscopy analyses of recombinant actins containing single reactive cysteines. We found that the global, cooperative conformational changes induced by cofilin-binding, which were detected by the spin-label attached to the Cys374 residue, occurred without the detachment of the D-loop in subdomain 2 from the neighboring protomer. The two processes of local and global changes do not necessarily proceed in sequence.

Role of the Actin Ala-108–Pro-112 Loop in Actin Polymerization and ATPase Activities

Background: Upon polymerization, the actin molecule becomes flattened, from the twisted form to the untwisted form. Results: The A108G substitution in the loop crucial for the flattening slowed down polymerization, and the P109A substitution accelerated polymerization. Conclusion: Actin polymerization involves at least two processes, the formation of the collision complex and its conformational change. Significance: This knowledge is important for understanding the polymerization mechanism. Actin plays fundamental roles in a variety of cell functions in eukaryotic cells. The polymerization-depolymerization cycle, between monomeric G-actin and fibrous F-actin, drives essential cell processes. Recently, we proposed the atomic model for the F-actin structure and found that actin was in the twisted form in the monomer and in the untwisted form in the filament. To understand how the polymerization process is regulated (Caspar, D. L. (1991) Curr. Biol. 1, 30–32), we need to know further details about the transition from the twisted to the untwisted form. For this purpose, we focused our attention on the Ala-108–Pro-112 loop, which must play crucial roles in the transition, and analyzed the consequences of the amino acid replacements on the polymerization process. As compared with the wild type, the polymerization of P109A was accelerated in both the nucleation and the elongation steps, and this was attributed to an increase in the frequency factor of the Arrhenius equation. The multiple conformations allowed by the substitution presumably resulted in the effective formation of the collision complex, thus accelerating polymerization. On the other hand, the A108G mutation reduced the rates of both nucleation and elongation due to an increase in the activation energy. In the cases of polymerization acceleration and deceleration, each functional aberration is attributed to a distinct elementary process. The rigidity of the loop, which mediates neither too strong nor too weak interactions between subdomains 1 and 3, might play crucial roles in actin polymerization.

Erratum: The nature of the globular- to fibrous-actin transition

This corrects the article DOI: 10.1038/nature07685

Calcium structural transition of troponin in the complexes, on the thin filament, and in muscle fibres, as studied by site-directed spin-labelling EPR.

We have measured the intersite distance, side-chain mobility and orientation of specific site(s) of troponin (Tn) complex on the thin filaments or in muscle fibres as well as in solution by means of site-directed spin labeling electron paramagnetic resonance (SDSL-EPR). We have examined the Ca(2+)-induced movement of the B and C helices relative to the D helix in a human cardiac (hc)TnC monomer state and hcTnC-hcTnI binary complex. An interspin distance between G42C (B helix) and C84 (D helix) was 18.4 angstroms in the absence of Ca2+. The distance between Q58C (C helix) and C84 (D helix) was 18.3 angstroms. Distance changes were observed by the addition of Ca2+ and by the formation of a complex with TnI. Both Ca2+ and TnI are essential for the full opening -3 angstroms of the N-domain in cardiac TnC. We have determined the in situ distances between C35 and C84 by measuring pulsed electron-electron double resonance (PELDOR) spectroscopy. The distances were 26.0 and 27.2 A in the monomer state and in reconstituted fibres, respectively. The addition of Ca2+ decreased the distance to 23.2 angstroms in fibres but only slightly in the monomer state, indicating that Ca2+ binding to the N-lobe of hcTnC induced a larger structural change in muscle fibres than in the monomer state. We also succeeded in synthesizing a new bifunctional spin labels that is firmly fixed on a central E-helix (94C-101C) of skeletal(sk)TnC to examine its orientation in reconstituted muscle fibres. EPR spectrum showed that this helix is disordered with respect to the filament axis. We have studied the calcium structural transition in skTnI and tropomyosin on the filament by SDSL-EPR. The spin label at a TnI switch segment (C133) showed three motional states depending on Ca2+ and actin. The data suggested that the TnI switch segment binds to TnC N-lobe in +Ca2+ state, and that in -Ca2+ state it is free in TnC-I-T complex alone while fixed to actin in the reconstituted thin filaments. In contrast, the side chain spin labels along the entire tropomyosin molecule showed no Ca(2+)-induced mobility changes.