Masayoshi Nakasako

Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 A resolution

Calcium ATPase is a member of the P-type ATPases that transport ions across the membrane against a concentration gradient. Here we have solved the crystal structure of the calcium ATPase of skeletal muscle sarcoplasmic reticulum (SERCA1a) at 2.6 Å resolution with two calcium ions bound in the transmembrane domain, which comprises ten α-helices. The two calcium ions are located side by side and are surrounded by four transmembrane helices, two of which are unwound for efficient coordination geometry. The cytoplasmic region consists of three well separated domains, with the phosphorylation site in the central catalytic domain and the adenosine-binding site on another domain. The phosphorylation domain has the same fold as haloacid dehalogenase. Comparison with a low-resolution electron density map of the enzyme in the absence of calcium and with biochemical data suggests that large domain movements take place during active transport.

Fe-type nitrile hydratase

The characteristic features of Fe-type nitrile hydratase (NHase) from Rhodococcus sp. N-771 are described. Through the biochemical analyses, we have found that nitric oxide (NO) regulates the photoreactivity of this enzyme by association with the non-heme iron center and photoinduced dissociation from it. The regulation is realized by a unique structure of the catalytic non-heme iron center composed of post-translationally modified cysteine-sulfinic (Cys-SO2H) and -sulfenic acids (Cys-SOH). To understand the biogenic mechanism and the functional role of these modifications, we constructed an over-expression system of whole NHase and individual subunits in Escherichia coli. The results of the studies on several recombinant NHases have shown that the Cys-SO2H oxidation of alphaC112 is indispensable for the catalytic activity of Fe-type NHase.

Structural basis of the LOV1 dimerization of Arabidopsis phototropins 1 and 2

Phototropin (phot) is a blue-light receptor protein that triggers phototropic responses, chloroplast relocation, and stomata opening to maximize the efficiency of photosynthesis in higher plants. Phot is composed of three functional domains. The N-terminal half folds into two light-oxygen-voltage-sensing domains called LOV1 and LOV2, each binding a flavin mononucleotide to absorb blue light. The C-terminal half is a serine/threonine kinase domain that causes light-dependent autophosphorylation leading to cellular signaling cascades. LOV2 domain is primarily responsible for activation of the kinase, and LOV1 domain is thought to act as a dimerization site and to regulate sensitivity to activation by blue light. Here we show the crystal structures of LOV1 domains of Arabidopsis phot1 and phot2 in the dark at resolutions of 2.1 A and 2.0 A, respectively. Either LOV1 domain forms a dimer through face-to-face association of beta-scaffolds in the crystallographic asymmetric unit. Three types of interactions stabilizing the dimer structures found are as follows: contacts of side chains in their beta-scaffolds, hydrophobic interactions of a short helix found in the N-terminus of a subunit with the beta-scaffolds of both subunits, and hydrogen bonds mediated by hydration water molecules filling the dimer interface. The critical residues for dimerization are Cys261, forming a disulfide bridge between subunits in phot1-LOV1 domain, and Thr217 and Met232 in phot2-LOV1. The topology in homodimeric associations of the LOV1 domains is discussed when referring to those of homodimers or heterodimers of light-oxygen-voltage-sensing or Per-ARNT-Sim domains. The present results also provide clues to understanding structural basis in dimeric interactions of Per-ARNT-Sim protein modules in cellular signaling.

Specific lipid–protein interactions in a novel honeycomb lattice structure of bacteriorhodopsin

In the purple membrane of Halobacterium salinarium, bacteriorhodopsin trimers are arranged in a hexagonal lattice. When purple membrane sheets are incubated at high temperature with neutral detergent, membrane vesicularization takes place, yielding inside-out vesicles with a diameter of 50 nm. The vesicular structure becomes unstable at low temperature, where successive fusion of the vesicles yields a crystal which is composed of stacked planar membranes. X-ray crystallographic analysis reveals that the bacteriorhodopsin trimers are arranged in a honeycomb lattice in each membrane layer and that neighbouring membranes orient in opposite directions. The native structure of the trimeric unit is preserved in the honeycomb lattice, irrespective of alterations in the in-plane orientation of the trimer. One phospholipid tightly bound to a crevice between monomers in the trimeric unit is suggested to act as a glue in the formation of the trimer.

Hydration structure of human lysozyme investigated by molecular dynamics simulation and cryogenic X-ray crystal structure analyses: On the correlation between crystal water sites, solvent density, and solvent dipole

The hydration structure of human lysozyme was studied with cryogenic X‐ray diffraction experiment and molecular dynamics simulations. The crystal structure analysis at a resolution of 1.4 Å provided 405 crystal water molecules around the enzyme. In the simulations at 300 K, the crystal structure was immersed in explicit water molecules. We examined correlations between crystal water sites and two physical quantities calculated from the 1‐ns simulation trajectories: the solvent density reflecting the time‐averaged distribution of water molecules, and the solvent dipole measuring the orientational ordering of water molecules around the enzyme. The local high solvent density sites were consistent with the crystal water sites, and better correlation was observed around surface residues with smaller conformational fluctuations during the simulations. Solvent dipoles around those sites exhibited coherent and persistent ordering, indicating that the hydration water molecules at the crystal water sites were highly oriented through the interactions with hydrophilic residues. Those water molecules restrained the orientational motions of adjoining water molecules and induced a solvent dipole field, which was persistent during the simulations around the enzyme. The coherent ordering was particularly prominent in and around the active site cleft of the enzyme. Because the ordering was significant up to the third to fourth solvent layer region from the enzyme surface, the coherently ordered solvent dipoles likely contributed to the molecular recognition of the enzyme in a long‐distance range. The present work may provide a new approach combining computational and the experimental studies to understand protein hydration.

Single-shot three-dimensional structure determination of nanocrystals with femtosecond X-ray free-electron laser pulses

Conventional three-dimensional (3D) structure determination methods require either multiple measurements at different sample orientations or a collection of serial sections through a sample. Here we report the experimental demonstration of single-shot 3D structure determination of an object; in this case, individual gold nanocrystals at ~5.5 nm resolution using ~10 fs X-ray free-electron laser pulses. Coherent diffraction patterns are collected from high-index-faceted nanocrystals, each struck by an X-ray free-electron laser pulse. Taking advantage of the symmetry of the nanocrystal and the curvature of the Ewald sphere, we reconstruct the 3D structure of each nanocrystal from a single-shot diffraction pattern. By averaging a sufficient number of identical nanocrystals, this method may be used to determine the 3D structure of nanocrystals at atomic resolution. As symmetry exists in many virus particles, this method may also be applied to 3D structure studies of such particles at nanometer resolution on femtosecond time scales.

Structure of a thermophilic F1-ATPase inhibited by an ε-subunit: deeper insight into the ε-inhibition mechanism

F1‐ATPase (F1) is the catalytic sector in FoF1‐ATP synthase that is responsible for ATP production in living cells. In catalysis, its three catalytic β‐subunits undergo nucleotide occupancy‐dependent and concerted open–close conformational changes that are accompanied by rotation of the γ‐subunit. Bacterial and chloroplast F1 are inhibited by their own ε‐subunit. In the ε‐inhibited Escherichia coli F1 structure, the ε‐subunit stabilizes the overall conformation (half‐closed, closed, open) of the β‐subunits by inserting its C‐terminal helix into the α3β3 cavity. The structure of ε‐inhibited thermophilic F1 is similar to that of E. coli F1, showing a similar conformation of the ε‐subunit, but the thermophilic ε‐subunit stabilizes another unique overall conformation (open, closed, open) of the β‐subunits. The ε‐C‐terminal helix 2 and hook are conserved between the two structures in interactions with target residues and in their positions. Rest of the ε‐C‐terminal domains are in quite different conformations and positions, and have different modes of interaction with targets. This region is thought to serve ε‐inhibition differently. For inhibition, the ε‐subunit contacts the second catches of some of the β‐ and α‐subunits, the N‐ and C‐terminal helices, and some of the Rossmann fold segments. Those contacts, as a whole, lead to positioning of those β‐ and α‐ second catches in ε‐inhibition‐specific positions, and prevent rotation of the γ‐subunit. Some of the structural features are observed even in IF1 inhibition in mitochondrial F1.

Coherent diffraction imaging analysis of shape-controlled nanoparticles with focused hard X-ray free-electron laser pulses

We report the first demonstration of the coherent diffraction imaging analysis of nanoparticles using focused hard X-ray free-electron laser pulses, allowing us to analyze the size distribution of particles as well as the electron density projection of individual particles. We measured 1000 single-shot coherent X-ray diffraction patterns of shape-controlled Ag nanocubes and Au/Ag nanoboxes and estimated the edge length from the speckle size of the coherent diffraction patterns. We then reconstructed the two-dimensional electron density projection with sub-10 nm resolution from selected coherent diffraction patterns. This method enables the simultaneous analysis of the size distribution of synthesized nanoparticles and the structures of particles at nanoscale resolution to address correlations between individual structures of components and the statistical properties in heterogeneous systems such as nanoparticles and cells.

KOTOBUKI-1 apparatus for cryogenic coherent X-ray diffraction imaging

We have developed an experimental apparatus named KOTOBUKI-1 for use in coherent X-ray diffraction imaging experiments of frozen-hydrated non-crystalline particles at cryogenic temperature. For cryogenic specimen stage with small positional fluctuation for a long exposure time of more than several minutes, we here use a cryogenic pot cooled by the evaporation cooling effect for liquid nitrogen. In addition, a loading device is developed to bring specimens stored in liquid nitrogen to the specimen stage in vacuum. The apparatus allows diffraction data collection for frozen-hydrated specimens at 66 K with a positional fluctuation of less than 0.4 μm and provides an experimental environment to easily exchange specimens from liquid nitrogen storage to the specimen stage. The apparatus was developed and utilized in diffraction data collection of non-crystalline particles with dimensions of μm from material and biological sciences, such as metal colloid particles and chloroplast, at BL29XU of SPring-8. Recently, it has been applied for single-shot diffraction data collection of non-crystalline particles with dimensions of sub-μm using X-ray free electron laser at BL3 of SACLA.

Quaternary structure of LOV‐domain containing polypeptide of Arabidopsis FKF1 protein

Flavin‐binding, Kelch repeat, F‐box (FKF1) protein is a photoreceptor to regulate flowering of Arabidopsis. The protein has a light, oxygen and voltage (LOV)‐sensing domain binding a flavin mononucleotide. The photo‐activation of the domain is an indispensable step to initiate the cellular signaling for flowering. In the present study, a LOV‐containing polypeptide of FKF1 was prepared by an overexpression system, and the quaternary structure of it was studied by size exclusion chromatography and small‐angle X‐ray scattering. The apparent molecular weight from chromatography suggested a globular trimeric or an anisotropic‐shaped dimeric association of the polypeptide in solution. The scattering experiment demonstrated a dimeric association of the polypeptides with an elongated molecular shape displaying the radius of gyration of 27 Å and the maximum dimension of 94 Å. The molecular shape simulated from scattering profiles suggests an antiparallel association of the LOV domains in the dimer. Though the absorption spectrum of blue‐light irradiated polypeptide was stable in the photoactivated state for a long period, the scattering profiles showed very small changes between the dark and light conditions. Based on the homologies in the amino‐acid sequences and the scattering profiles, these results are discussed in connection with the structures and function of LOV domains of phototropin.