Ryuta Kanai

Crystal structure of west nile virus envelope glycoprotein reveals viral surface epitopes.

ABSTRACT West Nile virus, a member of the Flavivirus genus, causes fever that can progress to life-threatening encephalitis. The major envelope glycoprotein, E, of these viruses mediates viral attachment and entry by membrane fusion. We have determined the crystal structure of a soluble fragment of West Nile virus E. The structure adopts the same overall fold as that of the E proteins from dengue and tick-borne encephalitis viruses. The conformation of domain II is different from that in other prefusion E structures, however, and resembles the conformation of domain II in postfusion E structures. The epitopes of neutralizing West Nile virus-specific antibodies map to a region of domain III that is exposed on the viral surface and has been implicated in receptor binding. In contrast, we show that certain recombinant therapeutic antibodies, which cross-neutralize West Nile and dengue viruses, bind a peptide from domain I that is exposed only during the membrane fusion transition. By revealing the details of the molecular landscape of the West Nile virus surface, our structure will assist the design of antiviral vaccines and therapeutics.

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.

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

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.

Crystallographic dissection of the thermal motion of protein‐sugar complex

The crystal structure of turkey egg lysozyme (TEL) complexed with di‐N‐acetylchitobiose (NAG2) was refined at 1.19 Å resolution by the full‐matrix least‐squares method with anisotropic temperature factors, and its thermal motion was evaluated by the TLS method. The average ESDs of atomic parameters of nonhydrogen atoms were 0.030 Å for coordinates and 0.025 Å2 for anisotropic temperature factors. The active site cleft of TEL binds the α‐anomer of NAG2 in a nonproductive binding mode with its pyranose rings parallel to a β‐sheet. The TEL structure was compared with the re‐refined 1.12 Å structure of native TEL. The RMS difference for equivalent Cα atoms was 0.103 Å and a relatively large difference was observed in the region of residues 104–125 rather than in the β‐sheet region where NAG2 was bound. In contrast, the temperature factor of the β‐sheet region was significantly decreased by the NAG2 binding. The TLS model that describes the rigid body motion in translation, libration, and screw motion was adopted for the evaluation of the molecular motion of TEL and NAG2, and the TLS parameters were determined by the least‐squares fit to Uij. The contribution of the external motion of TEL was estimated to be 55.8% of the observed temperature factor for the native structure and 45.9% for the NAG2 complex. The internal motion of TEL represented with atomic thermal ellipsoids was very similar between the native and complex structures except the NAG2 binding region. In the structure of NAG2, the rigid body motion dominates the thermal motion. The center of rotation of NAG2, 4.45Å far from the center of gravity, is on the nitrogen atom of the acetylamino group that is hydrogen bonded to the main‐chain peptide groups of Asn49 and Ala107. The rigid body motion of NAG2 indicates that the acetylamino group is most strongly bound to the active site, and the recognition of this group is a crucial step of the substrate binding. Proteins 2002;48:53–62.

Crystal Structure of Cyclodextrin Glucanotransferase from Alkalophilic Bacillus sp. 1011 Complexed with 1-Deoxynojirimycin at 2.0 Å Resolution

1-Deoxynojirimycin, a pseudo-monosaccharide, is a strong inhibitor of glucoamylase but a relatively weak inhibitor of cyclodextrin glucanotransferase (CGTase). To elucidate this difference, the crystal structure of the CGTase from alkalophilic Bacillus sp. 1011 complexed with 1-deoxynojirimycin was determined at 2.0 A resolution with the crystallographic R value of 0.154 (R(free) = 0.214). The asymmetric unit of the crystal contains two CGTase molecules and each molecule binds two 1-deoxynojirimycins. One 1-deoxynojirimycin molecule is bound to the active center by hydrogen bonds with catalytic residues and water molecules, but its binding mode differs from that expected in the substrate binding. Another 1-deoxynojirimycin found at the maltose-binding site 1 is bound to Asn-667 with a hydrogen bond and by stacking interaction with the indole moiety of Trp-662 of molecule 1 or Trp-616 of molecule 2. Comparison of this structure with that of the acarbose-CGTase complex suggested that the lack of stacking interaction with the aromatic side chain of Tyr-100 is responsible for the weak inhibition by 1-deoxynojirimycin of the enzymatic action of CGTase.

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.

Crystallization of parasporin-2, a Bacillus thuringiensis crystal protein with selective cytocidal activity against human cells.

Bacillus thuringiensis is a valuable source of protein toxins that are specifically effective against certain insects and worms but harmless to mammals. In contrast, a protein toxin obtained from B. thuringiensis strain A1547, designated parasporin-2, is not insecticidal but has a strong cytocidal activity against human cells with markedly divergent target specificity. The 37 kDa inactive protein is proteolytically activated to a 30 kDa active form. The active form of the recombinant protein toxin was crystallized in the presence of ethylene glycol and polyethylene glycol 8000 at neutral pH. The crystals belong to the hexagonal space group P6(1) or P6(5), with unit-cell parameters a = b = 134.37, c = 121.24 A. Diffraction data from a native crystal were collected to 2.75 A resolution using a synchrotron-radiation source.