Shiho Minakata

Two Distinct Mechanisms for Actin Capping Protein Regulation—Steric and Allosteric Inhibition

A crystallographic study reveals the structural basis for regulation by two different inhibitors of the actin capping protein, a critical factor controlling actin-driven cell motility.

Crystal structures of tropomyosin : Flexible coiled-coil

Tropomyosin (Tm) is a 400 angstroms long coiled coil protein, and with troponin it regulates contraction in skeletal and cardiac muscles in a [Ca2+]-dependent manner. Tm consists of multiple domains with diverse stabilities in the coiled coil form, thus providing Tm with dynamic flexibility. This flexibility must play important roles in the actin binding and the cooperative transition between the calcium regulated states of the entire muscle thin filament. In order to understand the flexibility of Tm in its entirety, the atomic coordinates of Tm are needed. Here we report the two crystal structures of Tm segments. One is rabbit skeletal muscle alpha-Tm encompassing residues 176-284 with an N-terminal extension of 25 residues from the leucine zipper sequence of GCN4, which includes the region that interacts with the troponin core domain. The other is alpha-Tm encompassing residues 176-273 with N- and C-terminal extensions of the leucine zipper sequences. These two crystal structures imply that this molecule is a flexible coiled coil. First, Tms are not homogeneous and smooth coiled coils, but instead they undulate, with highly fluctuating local parameters specifying the coiled coil. Independent fluctuating showed by two crystal structures is important. Second, in the first crystal, the coiled coil is bent by 9 degrees in the region centered about Y214-E218-Y221, where the inter-helical distance has its maximum. On the other hand, no bend is observed at the same region in the second crystal even if its inter-helical distance has also its maximum. E218, an unusual negatively charged residue at the a position in the heptad repeat, seems to play the key role in destabilizing the coiled coil with alanine destabilizing clusters.

Two-crystal structures of tropomyosin C-terminal fragment 176-273: exposure of the hydrophobic core to the solvent destabilizes the tropomyosin molecule.

Tropomyosin (Tm) is a two-stranded alpha-helical coiled-coil protein, and when associated with troponin, it is responsible for the actin filament-based regulation of muscle contraction in vertebrate skeletal and cardiac muscles. It is widely believed that Tm adopts a flexible rod-like structure in which the flexibility must play a crucial role in its functions. To obtain more information about the flexibility of Tm, we solved and compared two crystal structures of the identical C-terminal segments, spanning approximately 40% of the entire length. We also compared these structures with our previously reported crystal structure of an almost identical Tm segment in a distinct crystal form. The parameters specifying the local coiled-coil geometry, such as the separation between two helices and the local helical pitch, undulate along the length of Tm in the same way as among the three crystal structures, indicating that these parameters are defined by the amino acid sequence. In the region of increased separation, around Glu-218 and Gln-263, the hydrophobic core is disrupted by three holes. Moreover, for the first time to our knowledge, for Tm, water molecules have been identified in these holes. In some structures, the B-factors are higher around the holes than in the rest of the molecule. The Tm coiled-coil must be destabilized and therefore may be flexible, not only in the alanine clusters but also in the regions of the broken core. A closer look at the local staggering between the two chains and the local bending revealed that the strain accumulates at the alanine cluster and may be relaxed in the broken core region. Moreover, the strain is distributed over a long range, even when a deformation like bending may occur at a limited number of spots. Thus, Tm should not be regarded as a train of short rigid rods connected by flexible linkers, but rather as a seamless rubber rod patched with relatively more flexible regions.

Actin capping protein and its inhibitor CARMIL: how intrinsically disordered regions function.

The actin capping protein (CP) tightly binds to the barbed end of actin filaments to block further elongation. The β-tentacle in CP is an important region that ensures stable interaction with actin filaments. CARMIL inhibits the interaction of CP with actin filaments via the C-terminal portion containing the CP-binding motif, located in an intrinsically disordered region. We have proposed an allosteric inhibition model in which CARMIL suppresses CP by the population shift mechanism. Here, we solved a crystal structure of CP in complex with a CARMIL-derived peptide, CA32. The new structure clearly represents the α-helical form of the β-tentacle that was invisible in other CP/CARMIL peptide complex structures. In addition, we exhaustively performed a normal mode analysis with the elastic network model on all available crystal structures of the CP/CARMIL peptide complexes, including the new structure. We concluded that the CP-binding motif is necessary and sufficient for altering the fluctuation of CP, which is essential for attenuating the barbed-end-capping activity along the population shift mechanism. The roles and functions of the β-tentacle and the CP-binding motif are discussed in terms of their intrinsically disordered nature.

Structural Alterations of Thin Actin Filaments in Muscle Contraction by Synchrotron X-ray Fiber Diffraction

Strong evidence has been accumulated that the conformational changes of the thin actin filaments are occurring and playing an important role in the entire process of muscle contraction. The conformational changes and the mechanical properties of the thin actin filaments we have found by X-ray fiber diffraction on skeletal muscle contraction are explored. Recent studies on the conformational changes of regulatory proteins bound to actin filaments upon activation and in the force generation process are also described. Finally, the roles of structural alterations and dynamics of the actin filaments are discussed in conjunction with the regulation mechanism and the force generation mechanism.

Simultaneous Imaging of Cryo-Bright Field, Dark Field STEM and SEM Using Unroofed Living Cells with Special Reference to Membrane Cytoskeletons.

Cryo-electron microscopy has been used exclusively so far for single particle analysis or crystallography of purified proteins. Recently, technical improvements on rapid freezing and cryo-microtome permitted structural analysis of organelle or macromolecules in native state using whole cells (1,2) and vitreous cryo-sections (3). However, cryo-sections don’t scatter electrons so much and therefore show quite low contrast in addition to weakness against electron irradiation. They complicate a searching and a focusing of samples. In order to overcome these difficulties, we are improving unroofing techniques for preparation of cryo-electron microscopy of cells together with development of SEM based cryo-STEM. We succeeded simultaneous imaging of cryo-dark field and bright field STEM and secondary electron with extremely high contrast and accuracy for untreated cells.

The reconstitution of the membrane cytoskeleton using a lipid layer

Actin filaments play central roles in shape determination, cytokinesis, and cell motility. Recently, we found a new type of actin filament that is seemed to contact tightly membrane surface and be buried into membrane under electron microscope. However, the function of this actin filament is not obvious. Then, we have reproduced of such actin filaments using a lipid layer instead of the cell membrane cytoskeleton. We confirmed that the several actin filaments extended on the lipid layer while contacting firmly. The appearance of actin filaments on the lipid layer is quite similar to the new type of actin filaments buried in the cell membrane.