JAXA protein crystallization in space: ongoing improvements for growing high-quality crystals

Takahashi, Sachiko; Ohta, Kazunori; Furubayashi, Naoki; Yan, Bangbo; Koga, Misako; Wada, Yoshio; Yamada, Michihiro; Inaka, Koji; Tanaka, Hiroaki; Miyoshi, Hiroshi; Kobayashi, Tomoyuki; Kamigaichi, Shigeki

doi:10.1107/s0909049513021596

Cited by 26 publications

(22 citation statements)

References 20 publications

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“…The protein solutions were microseeded with crushed and diluted crystals of the corresponding PPL3 isoform in the precipitant solution. The capillaries were packed in the JAXA Crystallization Box (JCB‐SLG) . The crystallization condition was fixed to start after the samples had been placed in the microgravity environment (about 10 days after sample loading) by adjusting the precipitant concentration and the gel tube length.…”

Section: Methodsmentioning

confidence: 99%

Structures of jacalin‐related lectin PPL3 regulating pearl shell biomineralization

et al. 2018

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The nacreous layer of pearl oysters is one of the major biominerals of commercial and industrial interest. Jacalin-related lectins, including PPL3 isoforms, are known to regulate biomineralization of the Pteria penguin pearl shell, although the molecular mechanisms are largely unknown. The PPL3 crystal structures were determined partly by utilizing microgravity environments for 3 isoforms, namely, PPL3A, PPL3B, and PPL3C. The structures revealed a tail-to-tail dimer structure established by forming a unique inter-subunit disulfide bond at C-termini. The N-terminal residues were found in pyroglutamate form, and this was partly explained by the post-translational modification of PPL3 isoforms implied from the discrepancy between amino acid and gene sequences. The complex structures with trehalose and isomaltose indicated that the novel specificity originated from the unique α-helix of PPL3 isoforms. Docking simulations of PPL3B to various calcite crystal faces suggested the edge of a β-sheet and the carbohydrate-binding site rich in charged residues were the interface to the biomineral, and implied that the isoforms differed in calcite interactions.

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Section: Methodsmentioning

confidence: 99%

Structures of jacalin‐related lectin PPL3 regulating pearl shell biomineralization

et al. 2018

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“…The sitting drops were prepared by mixing 3 μl of the enzyme solution with an equal volume of reservoir solution containing 0.8–1.3 M (NH 4 ) 2 HPO 4 and 0.1 M sodium citrate buffer, pH 4.5–5.5, and equilibrated at 20°C with 0.5 ml of the reservoir solution. The counter-diffusion crystallization method was carried out under a microgravity environment in the Japanese Experiment Module “Kibo” at the International Space Station (ISS) [21] with the same crystallization solutions (launch date of ISS: September 26, 2014, return date to Earth: November 10, 2014). The rod-shaped crystals grew to a maximum of 0.1 × 0.1 × 1.0 mm.…”

Section: Methodsmentioning

confidence: 99%

Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery

et al. 2016

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The Gram-positive bacterium Paenibacillus sp. str. FPU-7 effectively hydrolyzes chitin by using a number of chitinases. A unique chitinase with two catalytic domains, ChiW, is expressed on the cell surface of this bacterium and has high activity towards various chitins, even crystalline chitin. Here, the crystal structure of ChiW at 2.1 Å resolution is presented and describes how the enzyme degrades chitin on the bacterial cell surface. The crystal structure revealed a unique multi-modular architecture composed of six domains to function efficiently on the cell surface: a right-handed β-helix domain (carbohydrate-binding module family 54, CBM-54), a Gly-Ser-rich loop, 1st immunoglobulin-like (Ig-like) fold domain, 1st β/α-barrel catalytic domain (glycoside hydrolase family 18, GH-18), 2nd Ig-like fold domain and 2nd β/α-barrel catalytic domain (GH-18). The structure of the CBM-54, flexibly linked to the catalytic region of ChiW, is described here for the first time. It is similar to those of carbohydrate lyases but displayed no detectable carbohydrate degradation activities. The CBM-54 of ChiW bound to cell wall polysaccharides, such as chin, chitosan, β-1,3-glucan, xylan and cellulose. The structural and biochemical data obtained here also indicated that the enzyme has deep and short active site clefts with endo-acting character. The affinity of CBM-54 towards cell wall polysaccharides and the degradation pattern of the catalytic domains may help to efficiently decompose the cell wall chitin through the contact surface. Furthermore, we clarify that other Gram-positive bacteria possess similar cell-surface-expressed multi-modular enzymes for cell wall polysaccharide degradation.

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“…Peptide-free crystals were also obtained using a counter-diffusion crystallisation method40 under a microgravity environment in the Japanese Experimental Module “Kibo” at the International Space Station (ISS)41. Although autolysis was not observed for DAP BII, a mutant (S657A, the catalytic serine was mutated to alanine) was used just to be safe for the prolonged (4 to 6 months) crystallisation experiment at the ISS.…”

Section: Methodsmentioning

confidence: 99%

S46 Peptidases are the First Exopeptidases to be Members of Clan PA

Sakamoto

Suzuki

Iizuka

et al. 2014

Sci Rep

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The dipeptidyl aminopeptidase BII (DAP BII) belongs to a serine peptidase family, S46. The amino acid sequence of the catalytic unit of DAP BII exhibits significant similarity to those of clan PA endopeptidases, such as chymotrypsin. However, the molecular mechanism of the exopeptidase activity of family S46 peptidase is unknown. Here, we report crystal structures of DAP BII. DAP BII contains a peptidase domain including a typical double β-barrel fold and previously unreported α-helical domain. The structures of peptide complexes revealed that the α-helical domain covers the active-site cleft and the side chain of Asn330 in the domain forms hydrogen bonds with the N-terminus of the bound peptide. These observations indicate that the α-helical domain regulates the exopeptidase activity of DAP BII. Because S46 peptidases are not found in mammals, we expect that our study will be useful for the design of specific inhibitors of S46 peptidases from pathogens.

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JAXA protein crystallization in space: ongoing improvements for growing high-quality crystals

Cited by 26 publications

References 20 publications

Structures of jacalin‐related lectin PPL3 regulating pearl shell biomineralization

Structures of jacalin‐related lectin PPL3 regulating pearl shell biomineralization

Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery

S46 Peptidases are the First Exopeptidases to be Members of Clan PA

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