Kayo Maeda

Structure of the core domain of human cardiac troponin in the Ca2+-saturated form

Troponin is essential in Ca2+ regulation of skeletal and cardiac muscle contraction. It consists of three subunits (TnT, TnC and TnI) and, together with tropomyosin, is located on the actin filament. Here we present crystal structures of the core domains (relative molecular mass of 46,000 and 52,000) of human cardiac troponin in the Ca2+-saturated form. Analysis of the four-molecule structures reveals that the core domain is further divided into structurally distinct subdomains that are connected by flexible linkers, making the entire molecule highly flexible. The α-helical coiled-coil formed between TnT and TnI is integrated in a rigid and asymmetric structure (about 80 Å long), the IT arm, which bridges putative tropomyosin-anchoring regions. The structures of the troponin ternary complex imply that Ca2+ binding to the regulatory site of TnC removes the carboxy-terminal portion of TnI from actin, thereby altering the mobility and/or flexibility of troponin and tropomyosin on the actin filament.

Crystal structure of CapZ: Structural basis for actin filament barbed end capping

Capping protein, a heterodimeric protein composed of α and β subunits, is a key cellular component regulating actin filament assembly and organization. It binds to the barbed ends of the filaments and works as a ‘cap’ by preventing the addition and loss of actin monomers at the end. Here we describe the crystal structure of the chicken sarcomeric capping protein CapZ at 2.1 Å resolution. The structure shows a striking resemblance between the α and β subunits, so that the entire molecule has a pseudo 2‐fold rotational symmetry. CapZ has a pair of mobile extensions for actin binding, one of which also provides concomitant binding to another protein for the actin filament targeting. The mobile extensions probably form flexible links to the end of the actin filament with a pseudo 21 helical symmetry, enabling the docking of the two in a symmetry mismatch.

Molecular structure of the ParM polymer and the mechanism leading to its nucleotide-driven dynamic instability.

ParM is a prokaryotic actin homologue, which ensures even plasmid segregation before bacterial cell division. In vivo, ParM forms a labile filament bundle that is reminiscent of the more complex spindle formed by microtubules partitioning chromosomes in eukaryotic cells. However, little is known about the underlying structural mechanism of DNA segregation by ParM filaments and the accompanying dynamic instability. Our biochemical, TIRF microscopy and high‐pressure SAX observations indicate that polymerization and disintegration of ParM filaments is driven by GTP rather than ATP and that ParM acts as a GTP‐driven molecular switch similar to a G protein. Image analysis of electron micrographs reveals that the ParM filament is a left‐handed helix, opposed to the right‐handed actin polymer. Nevertheless, the intersubunit contacts are similar to those of actin. Our atomic model of the ParM‐GMPPNP filament, which also fits well to X‐ray fibre diffraction patterns from oriented gels, can explain why after nucleotide release, large conformational changes of the protomer lead to a breakage of intra‐ and interstrand interactions, and thus to the observed disintegration of the ParM filament after DNA segregation.

Enhancement of protein expression in insect cells by a lobster tropomyosin cDNA leader sequence

We describe a cis element that dramatically increases the expression levels of exogenous genes in baculovirus‐infected insect cells. This 21 bp sequence element is derived from a 5′ untranslated leader sequence of a lobster tropomyosin cDNA (L21). By using a transfer vector carrying L21, the expression levels of tropomyosin and luciferase were 20‐ and seven‐fold higher with L21 than without L21, respectively. L21 has both the Kozak sequence and the A‐rich sequence found in the polyhedrin leader sequence. We assume that both sequence elements are essential for the enhancement of protein expression in the baculovirus‐based expression system.

Spinach chloroplast thioredoxins in evolutionary drift

The amino acid sequences surrounding the active sites of spinach chloroplast thioredoxins m and f have been determined. Both types of thioredoxins share common ancestor genes with the E. coli one, demonstrated by invariant active site sequences. The m-type thioredoxins have closer homology with the E. coli one in the sequence analyzed as well as in enzymatic specificity, whereas the f-type is less homologous both in sequence and specificity. It suggests that the m-type gene represents a prototype conserved throughout evolutionary processes whereas the f-type has undergone mutations resulting in a modified specificity.

Filament structure, organization, and dynamics in MreB sheets.

In vivo fluorescence microscopy studies of bacterial cells have shown that the bacterial shape-determining protein and actin homolog, MreB, forms cable-like structures that spiral around the periphery of the cell. The molecular structure of these cables has yet to be established. Here we show by electron microscopy that Thermatoga maritime MreB forms complex, several μm long multilayered sheets consisting of diagonally interwoven filaments in the presence of either ATP or GTP. This architecture, in agreement with recent rheological measurements on MreB cables, may have superior mechanical properties and could be an important feature for maintaining bacterial cell shape. MreB polymers within the sheets appear to be single-stranded helical filaments rather than the linear protofilaments found in the MreB crystal structure. Sheet assembly occurs over a wide range of pH, ionic strength, and temperature. Polymerization kinetics are consistent with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation. Steady-state TIRF microscopy studies of MreB suggest filament treadmilling while high pressure small angle x-ray scattering measurements indicate that the stability of MreB polymers is similar to that of F-actin filaments. In the presence of ADP or GDP, long, thin cables formed in which MreB was arranged in parallel as linear protofilaments. This suggests that the bacterial cell may exploit various nucleotides to generate different filament structures within cables for specific MreB-based functions.

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.

Dual Roles of Gln137 of Actin Revealed by Recombinant Human Cardiac Muscle α-Actin Mutants

The actin filament is quite dynamic in the cell. To determine the relationship between the structure and the dynamic properties of the actin filament, experiments using actin mutants are indispensable. We focused on Gln137 to understand the relationships between two activities: the conformational changes relevant to the G- to F-actin transition and the activation of actin ATPase upon actin polymerization. To elucidate the function of Gln137 in these activities, we characterized Gln137 mutants of human cardiac muscle α-actin. Although all of the single mutants, Q137E, Q137K, Q137P, and Q137A, as well as the wild type were expressed by a baculovirus-based system, only Q137A and the wild type were purified to high homogeneity. The CD spectrum of Q137A was similar to that of the wild type, and Q137A showed the typical morphology of negatively stained Q137A F-actin images. However, Q137A had an extremely low critical concentration for polymerization. Furthermore, we found that Q137A polymerized 4-fold faster, cleaved the γ-phosphate group of bound ATP 4-fold slower, and depolymerized 5-fold slower, as compared with the wild-type rates. These results suggest that Gln137 plays dual roles in actin polymerization, in both the conformational transition of the actin molecule and the mechanism of ATP hydrolysis.

Rabbit skeletal muscle ??-tropomyosin expressed in baculovirus-infected insect cells possesses the authentic N-terminus structure and functions

SummaryWhen expressed in E. coli, skeletal muscle α-tropomyosin has an unacetylated N-terminus. Unacetylated α-tropomyosin lacks important functions; this is non-polymerizable and has a low affinity to actin. In the present work, in order to obtain fully functional recombinant α-tropomyosin, rabbit skeletal muscle α-tropomyosin (α-tropomyosinBV) has been expressed in baculovirus-infected insect cells. α-TropomyosinBV was not distinguishable from the authentic tropomyosin, not only in functional properties but also in blocked N-terminus. To know the N-terminus structure of α-tropomyosinBV, the N-terminal segment six amino acids long, MDAIKK, has been specifically and efficiently removed from α-tropomyosinBV by use of an immobilized proteolytic enzyme system based on E. coli cell bodies which carry the ompT gene product, a proteolytic enzyme localized on the outer cell wall of E. coli. The structure of recombinant α-tropomyosinBV was shown to be identical to the authentic protein by electrospray mass spectrometry and protein sequencing analysis. Additionally, electrospray mass spectometry indicated a single phosphorylation not only in α-but also β-tropomyosin chains in the rabbit skeletal muscle. The differentiated susceptibilities of potential ompT cleavage sites are indicative of a non-coiled-coil conformation of the N-terminus of α-tropomyosin.