Kazuaki Harata

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.

Conformation of permethylated cyclodextrins and the host-guest geometry of their inclusion complexes

Macrocylic conformation of permethylated cyclodextrins and the geometry of their inclusion complexes were examined on the basis of the X-ray data of three permethylated α-cyclodextrin complexes and two permethylated β-cyclodextrin complexes. The host macrocyclic ring is remarkably distorted owing to steric hindrance involving the methyl groups and the inability to form intramolecular hydrogen bonds. The guest molecules are included within the host cavity, but their position and orientation are quite different from those found in the corresponding cyclodextrin complexes.

X-ray structure of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011. Comparison of two independent molecules at 1.8 A resolution.

Cyclodextrin glucanotransferase (CGTase) is an enzyme which produces cyclodextrins by the degradation of starch. The enzyme from alkalophilic Bacillus sp. 1011, consisting of 686 amino acid residues, was crystallized from the solution containing 20% PEG 3000 and 20% 2-propanol at pH 5.6 adjusted with citrate buffer. The space group was P1 and the unit cell contained two molecules (V(m) = 2.41 A(3) Da(-1)). The structure was solved by the molecular replacement method and refined to a conventional R value of 0.161 (R(free) = 0.211) for the reflections in the resolution range 1.8-10 A by energy minimization combined with simulated annealing. The molecule consists of five domains, designated A-E, and its backbone structure is similar to the structure of other bacterial CGTases. The molecule has two calcium binding sites where calcium ions are coordinated by seven ligands, forming a distorted pentagonal bipyramid. The two independent molecules are related by a pseudotwofold symmetry and are superimposed with an r.m.s. deviation value of 0.32 A for equivalent C(alpha) atoms. Comparison of these molecules indicated the relatively large mobility of domains C and E with respect to domain A. The active site is filled with water molecules forming a hydrogen-bond network with polar side-chain groups. Two water molecules commonly found in the active center of both molecules link to several catalytically important residues by hydrogen bonds and participate in maintaining a similar orientation of side chains in the two independent molecules.

Interactions of wheat-germ agglutinin with GlcNAcβ1,6Gal sequence

The interactions of wheat-germ agglutinin (WGA) with the GlcNAc beta 1,6Gal sequence, a characteristic component of branched poly-N-acetyllactosaminoglycans, were investigated using isothermal titration calorimetry and X-ray crystallography. GlcNAc beta 1,6Gal exhibited an affinity greater than GlcNAc beta 1,4GlcNAc to all WGA isolectins, whereas Gal beta 1,6GlcNAc showed much less affinity than GlcNAc beta 1,4GlcNAc. X-ray structural analyses of the glutaraldehyde-crosslinked WGA isolectin 3 crystals in complex with GlcNAc beta 1,6Gal, GlcNAc beta 1,4GlcNAc and GlcNAc beta 1,6Gal beta 1,4Glc were performed at 2.4, 2.2 and 2.2 A resolution, respectively. In spite of different glycosidic linkages, GlcNAc beta 1,6Gal and GlcNAc beta 1,4GlcNAc exhibited basically similar binding modes to each other, in contact with side chains of two aromatic residues, Tyr64 and His66. However, the conformations of the ligands in the two primary binding sites were not always identical. GlcNAc beta 1,6Gal showed more extensive variation in the parameters defining the glycosidic linkage structure compared to GlcNAc beta 1,4GlcNAc, demonstrating large conformational flexibility of the former ligand in the interaction with WGA. The difference in the ligand binding conformation was accompanied by alterations of the side chain conformation of the amino acid residues involved in the interactions. The hydrogen bond between Ser62 and the non-reducing end GlcNAc was always observed regardless of the ligand type, indicating the key role of this interaction. In addition to the hydrogen bonding and van der Waals interactions, CH--pi interactions involving Tyr64, His66 and Tyr73 are suggested to play an essential role in determining the ligand binding conformation in all complexes. One of the GlcNAc beta 1,6Gal ligands had no crystal packing contact with another WGA molecule, therefore the conformation might be more relevant to the interaction mode in solution.

The Structure of the Cyclodextrin Complex. XIX. Crystal Structures of Hexakis(2,3,6-tri-O-methyl)-α-cyclodextrin Complexes with (S)- and (R)-Mandelic Acid. Chiral Recognition through the Induced-Fit Conformational Change of the Macrocyclic Ring

Hexakis(2,3,6-tri-O-methyl)-α-cyclodextrin (methyl-α-CDx) forms crystalline inclusion complexes with (S)-mandelic acid (S-MA) and (R)-mandelic acid (R-MA). Both crystals are monoclinic, and the space group is P21 with cell dimensions: a=13.123(2), b=23.187(4), c=13.113(2) A, β=107.19(1)° for the S-MA complex, a=11.624(2), b=23.739(4), c=13.786(2) A, β=106.56(1)° for the R-MA complex. The structures were solved by inspection of a Patterson map and the R-map method, and refined by the block-diagonal least-squares method to the R-values of 0.087 for the S-MA complex and 0.055 for the R-MA complex. In the S-MA complex, the methyl-α-CDx molecule, which has a pseudo two-fold symmetry, loosely includes the phenyl group of S-MA. The hydroxyl and carboxyl groups of S-MA protrude from the O(2), O(3) side of the methyl-α-CDx cavity and form hydrogen bonds with water molecules located outside the host cavity. The methyl-α-CDx molecule in the R-MA complex tightly binds the R-MA molecule which is deeply inserted into t...

Implication for buried polar contacts and ion pairs in hyperthermostable enzymes

Understanding the structural basis of thermostability and thermoactivity, and their interdependence, is central to the successful future exploitation of extremophilic enzymes in biotechnology. However, the structural basis of thermostability is still not fully characterized. Ionizable residues play essential roles in proteins, modulating protein stability, folding and function. The dominant roles of the buried polar contacts and ion pairs have been reviewed by distinguishing between the inside polar contacts and the total intramolecular polar contacts, and by evaluating their contribution as molecular determinants for protein stability using various protein structures from hyperthermophiles, thermophiles and mesophilic organisms. The analysis revealed that the remarkably increased number of internal polar contacts in a monomeric structure probably play a central role in enhancing the melting temperature value up to 120 °C for hyperthermophilic enzymes from the genus Pyrococcus. These results provide a promising contribution for improving the thermostability of enzymes by modulating buried polar contacts and ion pairs.

Molecular Structure and Novel DNA Binding Sites Located in Loops of Flap Endonuclease-1 from Pyrococcus horikoshii

The crystal structure of flap endonuclease-1 fromPyrococcus horikoshii (phFEN-1) was determined to a resolution of 3.1 Å. The active cleft of the phFEN-1 molecule is formed with one large loop and four small loops. We examined the function of the conserved residues and positively charged clusters on these loops by kinetic analysis with 45 different mutants. Arg40 and Arg42 on small loop 1, a cluster Lys193–Lys195 on small loop 2, and two sites, Arg94 and Arg118-Lys119, on the large loop were identified as binding sites. Lys87 on the large loop may play significant roles in catalytic reaction. Furthermore, we successfully elucidated the function of the four DNA binding sites that form productive ES complexes specific for each endo- or exo-type hydrolysis, probably by bending the substrates. For the endo-activity, Arg94 and Lys193–Lys195 located at the top and bottom of the molecule were key determinants. For the exo-activity, all four sites were needed, but Arg118-Lys119 was dominant. The major binding sites for both the nick substrate and double-stranded DNA might be the same.

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

Full-matrix least-squares refinement of lysozymes and analysis of anisotropic thermal motion.

Crystal structures of turkey egg lysozyme (TEL) and human lysozyme (HL) were refined by full‐matrix least‐squares method using anisotropic temperature factors. The refinement converged at the conventional R‐values of 0.104 (TEL) and 0.115 (HL) for reflections with Fo > 0 to the resolution of 1.12 Å and 1.15 Å, respectively. The estimated r.m.s. coordinate errors for protein atoms were 0.031 Å (TEL) and 0.034 Å (HL). The introduction of anisotropic temperature factors markedly reduced the R‐value but did not significantly affect the main chain coordinates. The degree of anisotropy of atomic thermal motion has strong positive correlation with the square of distance from the molecular centroid. The ratio of the radial component of thermal ellipsoid to the r.m.s. magnitude of three principal components has negative correlation with the distance from the molecular centroid, suggesting the domination of libration rather than breathing motion. The TLS model was applied to elucidate the characteristics of the rigid‐body motion. The TLS tensors were determined by the least‐squares fit to observed temperature factors. The profile of the magnitude of reproduced temperature factors by the TLS method well fitted to that of observed Beqv. However, considerable disagreement was observed in the shape and orientation of thermal ellipsoid for atoms with large temperature factors, indicating the large contribution of local motion. The upper estimate of the external motion, 67% (TEL) and 61% (HL) of Beqv, was deduced from the plot of the magnitude of TLS tensors determined for main chain atoms which were grouped into shells according to the distance from the center of libration. In the external motion, the translational portion is predominant and the contribution of libration and screw motion is relatively small. The internal motion, estimated by subtracting the upper estimate of the external motion from the observed temperature factor, is very similar between TEL and HL in spite of the difference in 54 of 130 amino acid residues and in crystal packing, being suggested to reflect the intrinsic internal motion of chicken‐type lysozymes. Proteins 30:232–243, 1998.

Crystal structure of 6-O-[(R)-2-hydroxypropyl]cyclomaltoheptaose and 6-O-[(S)-2-hydroxypropyl]cyclomaltoheptaose

Crystal structures of 6-O-[(R)-2-hydroxypropyl]- and 6-O-[(S)-2-hydroxypropyl]-cyclomaltoheptaose were determined by X-ray analysis. In both structures, the 2-hydroxypropyl group is inserted into the macrocyclic cavity of the next molecule related by the two-fold screw axis, and a helically extended polymeric structure is formed by repetition of the intermolecular inclusion. The hydroxyl group of the substituent group penetrates through the macrocyclic ring from the secondary hydroxyl side and is linked to an HO-6 group by a hydrogen bond. Comparison of intermolecular contacts of the substituent group indicates that the (S)-2-hydroxypropyl group is better fitted to the cavity than the (R)-2-hydroxy-propyl group.