Noriyuki Igarashi

The 2.0 A crystal structure of catalase-peroxidase from Haloarcula marismortui.

Catalase-peroxidase is a member of the class I peroxidase superfamily. The enzyme exhibits both catalase and peroxidase activities to remove the harmful peroxide molecule from the living cell. The 2.0 Å crystal structure of the catalase-peroxidase from Haloarcula marismortui (HmCP) reveals that the enzyme is a dimer of two identical subunits. Each subunit is composed of two structurally homologous domains with a topology similar to that of class I peroxidase. The active site of HmCP is in the N-terminal domain. Although the arrangement of the catalytic residues and the cofactor heme b in the active site is virtually identical to that of class I peroxidases, the heme moiety is buried inside the domain, similar to that in a typical catalase. In the vicinity of the active site, novel covalent bonds are formed among the side chains of three residues, including that of a tryptophan on the distal side of the heme. Together with the C-terminal domain, these covalent bonds fix two long loops on the surface of the enzyme that cover the substrate access channel to the active site. These features provide an explanation for the dual activities of this enzyme.

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.

Enhancement in the perfection of orthorhombic lysozyme crystals grown in a high magnetic field (10 T)

Orthorhombic crystals of hen egg-white (HEW) lysozyme were grown in a homogeneous and static magnetic field of 10 T. All crystals grown at 10 T were oriented such that their crystallographic c axes were parallel to the magnetic field direction and showed a narrower average full-width at half-maximum (FWHM) of the rocking curve than those grown at 0 T. Rocking-width measurements were made at the BL-10A station at the Photon Factory, Tsukuba, Japan, using a high-resolution vertical-type four-circle diffractometer. Crystal perfection was evaluated using the FWHM of the rocking curve; the effects of the magnetic field on the quality of the crystals were examined by comparison of the FWHM of seven crystals grown at 10 and 0 T. The FWHMs of the reflections along the a, b and c axes decreased by 23.5, 35.3 and 27.8%, respectively, and those of other general reflections decreased by 17.4-42.2% in the crystals grown at high magnetic field. These results clearly showed that a magnetic field of 10 T improved the crystal perfection of the orthorhombic lysozyme crystals. As a result, the maximum resolution of X-ray diffraction increased from 1.3 A at 0 T to 1.13 A at 10 T. The magnetic field also affected the dimensions of the unit cell, increments being 0.2% for the a and c axes and 0.1% for the b axis, respectively. These facts suggest that the application of a high magnetic field during crystallization might result in remarkable enhancements in the diffraction power of protein crystals having magnetic anisotropy.

High-throughput operation of sample-exchange robots with double tongs at the Photon Factory beamlines

Sample-exchange robots with a double-tongs system that could exchange samples within 10u2005s have been developed.

Structures of the carbohydrate recognition domain of Ca2+-independent cargo receptors Emp46p and Emp47p.

Emp46p and Emp47p are type I membrane proteins, which cycle between the endoplasmic reticulum (ER) and the Golgi apparatus by vesicles coated with coat protein complexes I and II (COPI and COPII). They are considered to function as cargo receptors for exporting N-linked glycoproteins from the ER. We have determined crystal structures of the carbohydrate recognition domains (CRDs) of Emp46p and Emp47p of Saccharomyces cerevisiae, in the absence and presence of metal ions. Both proteins fold as a β-sandwich, and resemble that of the mammalian ortholog, p58/ERGIC-53. However, the nature of metal binding is distinct from that of Ca2+-dependent p58/ERGIC-53. Interestingly, the CRD of Emp46p does not bind Ca2+ ion but instead binds K+ ion at the edge of a concave β-sheet whose position is distinct from the corresponding site of the Ca2+ ion in p58/ERGIC-53. Binding of K+ ion to Emp46p appears essential for transport of a subset of glycoproteins because the Y131F mutant of Emp46p, which cannot bind K+ ion fails to rescue the transport in disruptants of EMP46 and EMP47 genes. In contrast the CRD of Emp47p binds no metal ions at all. Furthermore, the CRD of Emp46p binds to glycoproteins carrying high mannosetype glycans and the is promoted by binding not the addition of Ca2+ or K+ ion in These results suggest that Emp46p can be regarded as a Ca2+-independent intracellular lectin at the ER exit sites.

Impurity effects on lysozyme crystal growth.

Atomic force microscopy (AFM) and X-ray diffraction experiments have been combined to study the correlation between impurity incorporation, crystal surface morphology and crystal quality. Hen egg-white lysozyme has been used as a model protein, and covalently bound lysozyme dimer as a model impurity. AFM observation of the [101] crystal face revealed that the crystal surface clearly became rough when 5% impurity was added, and the steps disappeared as the impurity concentration increased to 10%. The crystal quality was evaluated by four factors: maximum resolution limit, /, Rmerge, and overall B factor. In every index, the crystal quality tended to degrade as the impurity concentration increased. The B-factor dropped significantly at 5% impurity; at the same time the step roughening was observed. This strongly suggested that the impurity incorporation affected the step growth mechanism and degraded the crystal quality.

Systematic analysis of supersaturation and lysozyme crystal quality

A systematic study of the correlation between supersaturation and protein crystal quality was carried out employing atomic force microscopy (AFM) and X-ray crystallography with synchrotron radiation (SR). The surface morphology and growth rates of hen egg-white (HEW) lysozyme crystals soaked in various supersaturated solutions were first investigated by AFM. The results showed that the formation of two-dimensional islands increased as a function of supersaturation. The growth rate (molecule intake speed) also increased as a function of supersaturation. In order to examine the correlation between the surface morphology, growth rate and the crystal quality, X-ray diffraction experiments were performed. It was confirmed that crystals grown at lower supersaturations diffracted better with higher signal-to-noise ratios, including better agreement between symmetry-related reflections. The results strongly suggested that the molecular misorientation at high supersaturation affected the crystal quality.

Improvement in diffraction maxima in orthorhombic HEWL crystal grown under high magnetic field

Orthorhombic crystals of hen egg-white lysozyme (HEWL) were grown under a homogeneous magnetic field of 10 T (B = 10 T) In the magnetic field, crystals were oriented such that their crystallographic c-axes were parallel to the magnetic field, and gave narrower average rocking widths than those grown under 0 T. Crystal quality was evaluated using the full-width at half-maximum (FWHM) of the rocking curve, and the effects of a magnetic field were examined by comparing FWHM of seven crystals grown at 10 T with an equal number grown at OT. FWHM values in (1000), (080), and (006) reflections decreased by 25%, 38%, and 32%, respectively, and those of general reflections decreased by 7-45% in crystals grown under high magnetic field. These results show that a magnetic field of 10 T improved the crystal perfection of the orthorhombic lysozyme crystals. At the same time, the maximum resolution limit of X-ray diffraction increased from 1.33 A for 0 T to 1.13 A for 10 T. These facts suggest that the application of magnetic field for crystallization might generally have a striking effect in enhancing the diffraction power of protein crystals.

Crystallization and preliminary X-ray analysis of catalase–peroxidase from the halophilic archaeon Haloarcula marismortui

Catalase-peroxidases are bifunctional enzymes found in many microorganisms. Crystals of catalase-peroxidase from the halophilic archaeon Haloarcula marismortui were obtained using the hanging-drop vapour-diffusion method. The rhombic plate-shaped crystals were grown from purified protein solution using (NH(4))(2)SO(4) as precipitant at 293 K. The crystal belongs to the monoclinic system, space group C2, and diffracted beyond 2.0 A resolution.

Systematic analysis of the effect of supersaturation on protein crystal quality

We proved the correlation between supersaturation and the quality of Hen Egg White (HEW) lysozyme crystal by X-ray crystallography and confirmed that crystals grown in lower supersaturated solution diffracted better with higher signal-to-noise ratios. The surface morphologies of lysozyme crystals grown in various supersaturated solutions were investigated by AFM. The observations revealed that no major crystal defects existed in tetragonal lysozyme crystals. We also investigated the step growth rates in various supersaturated conditions. The step kinetic constant β tended to increase as the supersaturation increased. The results strongly suggested that the molecular misorientation brought about at high supersaturation affected the crystal quality.