Toshihiko Akiba

Crystal Structure of the Parasporin-2 Bacillus thuringiensis Toxin That Recognizes Cancer Cells

Parasporin-2 is a protein toxin that is isolated from parasporal inclusions of the Gram-positive bacterium Bacillus thuringiensis. Although B. thuringiensis is generally known as a valuable source of insecticidal toxins, parasporin-2 is not insecticidal, but has a strong cytocidal activity in liver and colon cancer cells. The 37-kDa inactive nascent protein is proteolytically cleaved to the 30-kDa active form that loses both the N-terminal and the C-terminal segments. Accumulated cytological and biochemical observations on parasporin-2 imply that the protein is a pore-forming toxin. To confirm the hypothesis, we have determined the crystal structure of its active form at a resolution of 2.38 A. The protein is unusually elongated and mainly comprises long beta-strands aligned with its long axis. It is similar to aerolysin-type beta-pore-forming toxins, which strongly reinforce the pore-forming hypothesis. The molecule can be divided into three domains. Domain 1, comprising a small beta-sheet sandwiched by short alpha-helices, is probably the target-binding module. Two other domains are both beta-sandwiches and thought to be involved in oligomerization and pore formation. Domain 2 has a putative channel-forming beta-hairpin characteristic of aerolysin-type toxins. The surface of the protein has an extensive track of exposed side chains of serine and threonine residues. The track might orient the molecule on the cell membrane when domain 1 binds to the target until oligomerization and pore formation are initiated. The beta-hairpin has such a tight structure that it seems unlikely to reform as postulated in a recent model of pore formation developed for aerolysin-type toxins. A safety lock model is proposed as an inactivation mechanism by the N-terminal inhibitory segment.

The dynamin A ring complex: molecular organization and nucleotide-dependent conformational changes

Here we show that Dictyostelium discoideum dynamin A is a fast GTPase, binds to negatively charged lipids, and self‐assembles into rings and helices in a nucleotide‐dependent manner, similar to human dynamin‐1. Chemical modification of two cysteine residues, positioned in the middle domain and GTPase effector domain (GED), leads to altered assembly properties and the stabilization of a highly regular ring complex. Single particle analysis of this dynamin A* ring complex led to a three‐dimensional map, which shows that the nucleotide‐free complex consists of two layers with 11‐fold symmetry. Our results reveal the molecular organization of the complex and indicate the importance of the middle domain and GED for the assembly of dynamin family proteins. Nucleotide‐dependent changes observed with the unmodified and modified protein support a mechanochemical action of dynamin, in which tightening and stretching of a helix contribute to membrane fission.

Nontoxic crystal protein from Bacillus thuringiensis demonstrates a remarkable structural similarity to β-pore-forming toxins

Toshihiko Akiba, Kazuhiko Higuchi, Eiichi Mizuki, Keisuke Ekino, Takashi Shin, Michio Ohba, Ryuta Kanai, and Kazuaki Harata* Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, Kurume, Fukuoka, Japan Department of Applied Microbial Technology, Sojo University, Kumamoto, Japan Graduate School of Agriculture, Kyushu University, Fukuoka, Japan

X‐ray structure of a membrane‐bound β‐glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii

The β‐glycosidase of the hyperthermophilic Archaeon Pyrococcus horikoshii is a membrane‐bound enzyme with the preferred substrate of alkyl‐β‐glycosides. In this study, the unusual structural features that confer the extreme thermostability and substrate preferences of this enzyme were investigated by X‐ray crystallography and docking simulation. The enzyme was crystallized in the presence of a neutral surfactant, and the crystal structure was solved by the molecular replacement method and refined at 2.5 Å. The main‐chain fold of the enzyme belongs to the (βα)8 barrel structure common to the Family 1 glycosyl hydrolases. The active site is located at the center of the C‐termini of the barrel β‐strands. The deep pocket of the active site accepts one sugar unit, and a hydrophobic channel extending radially from there binds the nonsugar moiety of the substrate. The docking simulation for oligosaccharides and alkylglucosides indicated that alkylglucosides with a long aliphatic chain are easily accommodated in the hydrophobic channel. This sparingly soluble enzyme has a cluster of hydrophobic residues on its surface, situated at the distal end of the active site channel and surrounded by a large patch of positively charged residues. We propose that this hydrophobic region can be inserted into the membrane while the surrounding positively charged residues make favorable contacts with phosphate groups on the inner surface of the membrane. The enzyme could thus adhere to the membrane in the proximity of its glycolipid substrate. Proteins 2004.

Structure of an orthorhombic form of xylanase II from Trichoderma reesei and analysis of thermal displacement.

An orthorhombic crystal of xylanase II from Trichoderma reesei was grown in the presence of sodium iodide. Crystal structures at atomic resolution were determined at 100 and 293 K. Protein molecules were aligned along a crystallographic twofold screw axis, forming a helically extended polymer-like chain mediated by an iodide ion. The iodide ion connected main-chain peptide groups between two adjacent molecules by an N-H...I-...H-N hydrogen-bond bridge, thus contributing to regulation of the molecular arrangement and suppression of the rigid-body motion in the crystal with high diffraction quality. The structure at 293 K showed considerable thermal motion in the loop regions connecting the beta-strands that form the active-site cleft. TLS model analysis of the thermal motion and a comparison between this structure and that at 100 K suggest that the fluctuation of these loop regions is attributable to the hinge-like movement of the beta-strands.

Structure of RadB recombinase from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1: an implication for the formation of a near-7-fold helical assembly

The X-ray crystal structure of RadB from Thermococcus kodakaraensis KOD1, an archaeal homologue of the RecA/Rad51 family proteins, have been determined in two crystal forms. The structure represents the core ATPase domain of the RecA/Rad51 proteins. Two independent molecules in the type 1 crystal were roughly related by 7-fold screw symmetry whereas non-crystallographic 2-fold symmetry was observed in the type 2 crystal. The dimer structure in the type 1 crystal is extended to construct a helical assembly, which resembles the filamentous structures reported for other RecA/Rad51 proteins. The molecular interface in the type 1 dimer is formed by facing a basic surface patch of one monomer to an acidic one of the other. The empty ATP binding pocket is located at the interface and barely concealed from the outside similarly to that in the active form of the RecA filament. The model assembly has a positively charged belt on one surface bordering the helical groove suitable for facile binding of DNA. Electron microscopy has revealed that, in the absence of ATP and DNA, RadB forms a filament with a similar diameter to that of the hypothetical assembly, although its helical properties were not confirmed.

Structural phase transition of monoclinic crystals of hen egg-white lysozyme

Two monoclinic crystals (space group P2(1)) of hen egg-white lysozyme, a type I crystal grown at room temperature in a D2O solution with pD 4.5 containing 2%(w/v) sodium nitrate and a type II crystal grown at 313 K in a 10%(w/v) sodium chloride solution with pH 7.6, were each transformed into another monoclinic crystal with the same space group by dehydration-induced phase transition. Changes in X-ray diffraction were recorded to monitor the progress of the crystal transformation, which started with the appearance of diffuse streaks. In both crystals, the intensity of h + l odd reflections gradually weakened and finally disappeared on completion of the transformation. X-ray diffraction in the intermediate state indicated the presence of lattices of both the native and transformed crystals. In the native type I crystal, two alternate conformations were observed in the main chain of the region Gly71-Asn74. One conformer bound a sodium ion which was replaced with a water molecule in the other conformer. In the transformed crystal, the sodium ion was removed and the main-chain conformation of this region was converted to that of the water-bound form. The transformed crystal diffracted to a higher resolution than the native crystal, while the peak width of the diffraction spots increased. Analysis of the thermal motion of protein molecules using the TLS model has shown that the enhancement of the diffraction power in the transformed crystal is mainly ascribable to the suppression of rigid-body motion owing to an increase in intermolecular contacts as a result of the loss of bulk solvent.

Phase transition of triclinic hen egg-white lysozyme crystal associated with sodium binding.

A triclinic crystal of hen egg-white lysozyme obtained from a D2O solution at 313 K was transformed into a new triclinic crystal by slow release of solvent under a temperature-regulated nitrogen-gas stream. The progress of the transition was monitored by X-ray diffraction. The transition started with the appearance of strong diffuse streaks. The diffraction spots gradually fused and faded with the emergence of diffraction from the new lattice; the scattering power of the crystal fell to a resolution of 1.5 A from the initial 0.9 A resolution. At the end of the transition, the diffuse streaks disappeared and the scattering power recovered to 1.1 A resolution. The transformed crystal contained two independent molecules and the solvent content had decreased to 18% from the 32% solvent content of the native crystal. The structure was determined at 1.1 A resolution and compared with the native structure refined at the same resolution. The backbone structures of the two molecules in the transformed crystal were superimposed on the native structure with root-mean-square deviations of 0.71 and 0.96 A. A prominent structural difference was observed in the loop region of residues Ser60-Leu75. In the native crystal, a water molecule located at the centre of this helical loop forms hydrogen bonds to main-chain peptide groups. In the transformed crystal, this water molecule is replaced by a sodium ion with octahedral coordination that involves water molecules and a nitrate ion. The peptide group connecting Arg73 and Asn74 is rotated by 180 degrees so that the CO group of Arg73 can coordinate to the sodium ion. The change in the X-ray diffraction pattern during the phase transition suggests that the transition proceeds at the microcrystal level. A mechanism is proposed for the crystal transformation.

Role of Trp140 at subsite -6 on the maltohexaose production of maltohexaose-producing amylase from alkalophilic Bacillus sp.707

Maltohexaose‐producing amylase (G6‐amylase) from alkalophilic Bacillus sp.707 predominantly produces maltohexaose (G6) in the yield of >30% of the total products from short‐chain amylose (DP = 17). Our previous crystallographic study showed that G6‐amylase has nine subsites, from −6 to +3, and pointed out the importance of the indole moiety of Trp140 in G6 production. G6‐amylase has very low levels of hydrolytic activities for oligosaccharides shorter than maltoheptaose. To elucidate the mechanism underlying G6 production, we determined the crystal structures of the G6‐amylase complexes with G6 and maltopentaose (G5). In the active site of the G6‐amylase/G5 complex, G5 is bound to subsites −6 to −2, while G1 and G6 are found at subsites + 2 and −7 to −2, respectively, in the G6‐amylase/G6 complex. In both structures, the glucosyl residue located at subsite −6 is stacked to the indole moiety of Trp140 within a distance of 4Å. The measurement of the activities of the mutant enzymes when Trp140 was replaced by leucine (W140L) or by tyrosine (W140Y) showed that the G6 production from short‐chain amylose by W140L is lower than that by W140Y or wild‐type enzyme. The face‐to‐face short contact between Trp140 and substrate sugars is suggested to regulate the disposition of the glucosyl residue at subsite −6 and to govern product specificity for G6 production.

Role of Phe283 in enzymatic reaction of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp.1011: Substrate binding and arrangement of the catalytic site.

Cyclodextrin glycosyltransferase (CGTase) belonging to the α‐amylase family mainly catalyzes transglycosylation and produces cyclodextrins from starch and related α‐1,4‐glucans. The catalytic site of CGTase specifically conserves four aromatic residues, Phe183, Tyr195, Phe259, and Phe283, which are not found in α‐amylase. To elucidate the structural role of Phe283, we determined the crystal structures of native and acarbose‐complexed mutant CGTases in which Phe283 was replaced with leucine (F283L) or tyrosine (F283Y). The temperature factors of the region 259–269 in native F283L increased >10 Å2 compared with the wild type. The complex formation with acarbose not only increased the temperature factors (>10 Å2) but also changed the structure of the region 257–267. This region is stabilized by interactions of Phe283 with Phe259 and Leu260 and plays an important role in the cyclodextrin binding. The conformation of the side‐chains of Glu257, Phe259, His327, and Asp328 in the catalytic site was altered by the mutation of Phe283 with leucine, and this indicates that Phe283 partly arranges the structure of the catalytic site through contacts with Glu257 and Phe259. The replacement of Phe283 with tyrosine decreased the enzymatic activity in the basic pH range. The hydroxyl group of Tyr283 forms hydrogen bonds with the carboxyl group of Glu257, and the pKa of Glu257 in F283Y may be lower than that in the wild type.