Takeyuki Wakabayashi

Structural basis for tropomyosin overlap in thin (actin) filaments and the generation of a molecular swivel by troponin-T

Head-to-tail polymerization of tropomyosin is crucial for its actin binding, function in actin filament assembly, and the regulation of actin-myosin contraction. Here, we describe the 2.1 Å resolution structure of crystals containing overlapping tropomyosin N and C termini (TM-N and TM-C) and the 2.9 Å resolution structure of crystals containing TM-N and TM-C together with a fragment of troponin-T (TnT). At each junction, the N-terminal helices of TM-N were splayed, with only one of them packing against TM-C. In the C-terminal region of TM-C, a crucial water in the coiled-coil core broke the local 2-fold symmetry and helps generate a kink on one helix. In the presence of a TnT fragment, the asymmetry in TM-C facilitates formation of a 4-helix bundle containing two TM-C chains and one chain each of TM-N and TnT. Mutating the residues that generate the asymmetry in TM-C caused a marked decrease in the affinity of troponin for actin-tropomyosin filaments. The highly conserved region of TnT, in which most cardiomyopathy mutations reside, is crucial for interacting with tropomyosin. The structure of the ternary complex also explains why the skeletal- and cardiac-muscle specific C-terminal region is required to bind TnT and why tropomyosin homodimers bind only a single TnT. On actin filaments, the head-to-tail junction can function as a molecular swivel to accommodate irregularities in the coiled-coil path between successive tropomyosins enabling each to interact equivalently with the actin helix.

Electron microscopic evidence for the myosin head lever arm mechanism in hydrated myosin filaments using the gas environmental chamber

Muscle contraction results from an attachment-detachment cycle between the myosin heads extending from myosin filaments and the sites on actin filaments. The myosin head first attaches to actin together with the products of ATP hydrolysis, performs a power stroke associated with release of hydrolysis products, and detaches from actin upon binding with new ATP. The detached myosin head then hydrolyses ATP, and performs a recovery stroke to restore its initial position. The strokes have been suggested to result from rotation of the lever arm domain around the converter domain, while the catalytic domain remains rigid. To ascertain the validity of the lever arm hypothesis in muscle, we recorded ATP-induced movement at different regions within individual myosin heads in hydrated myosin filaments, using the gas environmental chamber attached to the electron microscope. The myosin head were position-marked with gold particles using three different site-directed antibodies. The amplitude of ATP-induced movement at the actin binding site in the catalytic domain was similar to that at the boundary between the catalytic and converter domains, but was definitely larger than that at the regulatory light chain in the lever arm domain. These results are consistent with the myosin head lever arm mechanism in muscle contraction if some assumptions are made.

Structural Basis for Calcium-Regulated Relaxation of Striated Muscles at Interaction Sites of Troponin with Actin and Tropomyosin

In summary, we have shown that the TnI-TnC-TnT2 ternary complex (-52 kDa) has a mobile actin-binding domain (-6.1 kDa) that tumbles independently of the core domain. By docking the mobile domain and the core domain into the cryo-EM map obtained for thin filaments at low Ca2+, a model for actin-troponin interaction has been obtained. This model shows the atomic details of interactions of actin with the mobile domain and suggests the mechanism by which troponin generates a shift in the azimuthal position of tropomyosin in response to changes in Ca2+ levels. In this model the mobile domain of troponin interacts with three actins and one troponin interacts with four actin molecules. The relationship between myosin and the mobile domain suggests that the latter may work as a fail-safe latch to secure a relaxed state. The model also provides insights into many mutations associated with human cardiomyopathy and has implications for the function of other actin-binding proteins. Coordinates of the mobile domain have been deposited in the Protein Data Bank under accession codes 1VDI (low Ca2+) and 1VDJ (high Ca2+). Chemical shifts of the mobile domain have been deposited in the BMRB under accession ID 18140.

Evaluation of a 2k CCD camera with an epitaxially grown CsI scintillator for recording energy-filtered electron cryo-micrographs

Zero-loss imaging of frozen-hydrated specimens requires a detector with high sensitivity, a low noise level and high spatial resolution, because more electrons are scattered inelastically than elastically by cryo-specimens and the number of electrons detected is approximately 1/4 of incident electrons after energy filtering. Cameras using charge-coupled devices (CCDs) are good candidates due to their high sensitivity. They have been used mainly to record electron diffraction patterns for electron crystallography due to their limited spatial resolution but recently used for acquiring direct images due to their convenience. The spatial resolution has been limited by the characteristics of a phosphor that is necessary to convert high-energy electrons to photons and the coupling. We adopted a CsI scintillator with good modulation transfer function (MTF), which was epitaxially grown from each of optical fibres. The stripes of carbon graphite with 3.4 A spacing and 1.4 A stripes of gold thin crystals could be recorded with a magnification of 240,000x and 560,000x at 200 kV, respectively. A computed Fourier transform of an image of a frozen-hydrated crystal of catalase containing about 1000 units showed diffraction spots at spatial frequencies of 1/9.6 A(-1) up to 1/8 A(-1) without background subtraction, when the image was recorded at 140,000x. These results show that the resolution of the developed camera was good enough to record images. Our used test method for MTF determination may be useful for others.

Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate.

Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60Å(2) to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (Vmax) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller Kapp) than that of the wild-type actin, with the Vmax being almost unchanged. The Kapp and Vmax of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of Kapp was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA) of the replaced amino acid molecule. Because 1/Kapp reflects the affinity of F-actin for the myosin-ADP-phosphate intermediate (M.ADP.Pi) through the weak binding, these data suggest that the bulkiness or the aromatic nature of the tyrosin-143 is important for the initial binding of the M.ADP.Pi intermediate with F-actin but not for later processes such as the phosphate release.

Use of Cosine Tapered Window to Improve Dynamic Range of OCT without Loss of Resolution

We propose another window function used in production process of A-line by Fourier transform in optical frequency domain imaging. It is shown that cosine tapered window has an advantage over Gaussian window employed conventionally.