Crystal structure analysis, overexpression and refolding behaviour of a DING protein with single mutation.

After crystallization of a certain protein-RNA complex, well diffracting crystals were obtained. However, the asymmetric unit of the crystal was too small to locate any components. Mass spectrometry and X-ray crystal structure analysis showed that it was a member of the DING protein family (HPBP). Surprisingly, the structure of HPBP reported previously was also determined accidentally as a contaminant, suggesting that HPBP has a strong tendency to crystallize. Furthermore, DING proteins were reported to relate in disease. These observations suggest that DING has potential for application in a wide range of research fields. To enable further analyses, a system for preparation of HPBP was constructed. As HPBP was expressed in insoluble form in Escherichia coli, it was unfolded chemically and refolded. Finally, a very high yield preparation method was constructed, in which 43 mg of HPBP was obtained from 1 L of culture. Furthermore, to evaluate the validity of refolding, its crystal structure was determined at 1.03 Å resolution. The determined structure was identical to the native structure, in which two disulfide bonds were recovered correctly and a phosphate ion was captured. Based on these results, it was concluded that the refolded HPBP recovers its structure correctly.

Crystal structure of a putative methyltransferase SAV1081 from Staphylococcus aureus.

Cluster of Orthologous Groups (COG) 1092 contains two distinct types of methylation enzyme from Escherichia coli, YccW and YcbY. YccW is a 5-methylcytosine methyltransferase (m5C MTase) responsible for m5C 1962 in 23S rRNA, whereas YcbY is a dimethyltransferase, of which N- and C-terminal domains are responsible for N2- methylguanosine (m2G) 2445 and 7-methylguanosine (m7G) 2069 in 23S rRNA, respectively. However, proteins in COG1092 other than YccW and YcbY remain functionally unidentified. SAV1081 from Staphylococcus aureus is one of the functionally unassigned proteins of COG1092. Although SAV1081 has an identical domain organization to YccW with 26% sequence identity, it lacks the catalytic cysteine residue essential for m5C formation activity. In the present study, we determined the crystal structure of SAV1081 and compared it with those of other COG1092 proteins. Based on the structure characteristics, such as the presence or absence of the catalytic cysteine residue, ß-hairpin structure, and oligomeric state, as well as domain organization, we propose a functional classification of COG1092 proteins.

Molecular basis of dihydrouridine formation on tRNA.

Dihydrouridine (D) is a highly conserved modified base found in tRNAs from all domains of life. Dihydrouridine synthase (Dus) catalyzes the D formation of tRNA through reduction of uracil base with flavin mononucleotide (FMN) as a cofactor. Here, we report the crystal structures of Thermus thermophilus Dus (TthDus), which is responsible for D formation at positions 20 and 20a, in complex with tRNA and with a short fragment of tRNA (D-loop). Dus interacts extensively with the D-arm and recognizes the elbow region composed of the kissing loop interaction between T- and D-loops in tRNA, pulling U20 into the catalytic center for reduction. Although distortion of the D-loop structure was observed upon binding of Dus to tRNA, the canonical D-loop/T-loop interaction was maintained. These results were consistent with the observation that Dus preferentially recognizes modified rather than unmodified tRNAs, indicating that Dus introduces D20 by monitoring the complete L-shaped structure of tRNAs. In the active site, U20 is stacked on the isoalloxazine ring of FMN, and C5 of the U20 uracil ring is covalently cross linked to the thiol group of Cys93, implying a catalytic mechanism of D20 formation. In addition, the involvement of a cofactor molecule in uracil ring recognition was proposed. Based on a series of mutation analyses, we propose a molecular basis of tRNA recognition and D formation catalyzed by Dus.

Crystal structure of the octameric pore of staphylococcal γ-hemolysin reveals the ß-barrel pore formation mechanism by two components.

Staphylococcal γ-hemolysin is a bicomponent pore-forming toxin composed of LukF and Hlg2. These proteins are expressed as water-soluble monomers and then assemble into the oligomeric pore form on the target cell. Here, we report the crystal structure of the octameric pore form of γ-hemolysin at 2.5 Å resolution, which is the first high-resolution structure of a ß-barrel transmembrane protein composed of two proteins reported to date. The octameric assembly consists of four molecules of LukF and Hlg2 located alternately in a circular pattern, which explains the biochemical data accumulated over the past two decades. The structure, in combination with the monomeric forms, demonstrates the elaborate molecular machinery involved in pore formation by two different molecules, in which interprotomer electrostatic interactions using loops connecting ß2 and ß3 (loop A: Asp43-Lys48 of LukF and Lys37-Lys43 of Hlg2) play pivotal roles as the structural determinants for assembly through unwinding of the N-terminal ß-strands (amino-latch) of the adjacent protomer, releasing the transmembrane stem domain folded into a ß-sheet in the monomer (prestem), and interaction with the adjacent protomer.

Crystallization and preliminary X-ray crystallographic analysis of dihydrouridine synthase from Thermus thermophilus and its complex with tRNA.

Dihydrouridine synthase (Dus) is responsible for catalyzing dihydrouridine formation in RNA by the reduction of uridine. To elucidate its RNA-recognition mechanism, Dus from Thermus thermophilus (TthDus) and its complex with tRNA were crystallized. Diffraction data sets were collected from crystals of native and selenomethionine-substituted TthDus to resolutions of 1.70 and 2.30 Å, respectively. These crystals belonged to space group P1. Preliminary X-ray crystallographic analysis showed that two molecules of TthDus were contained in an asymmetric unit. In addition, diffraction data were collected to 3.51 Šresolution from a crystal of selenomethionine-substituted TthDus in complex with tRNA, which belonged to space group P4(1)2(1)2. Preliminary structural analysis showed that the asymmetric unit contained two TthDus-tRNA complexes.

2-Methyl-2,4-pentanediol induces spontaneous assembly of staphylococcal α-hemolysin into heptameric pore structure.

Staphylococcal α-hemolysin is expressed as a water-soluble monomeric protein and assembles on membranes to form a heptameric pore structure. The heptameric pore structure of α-hemolysin can be prepared from monomer in vitro only in the presence of deoxycholate detergent micelles, artificially constructed phospholipid bilayers, or erythrocytes. Here, we succeeded in preparing crystals of the heptameric form of α-hemolysin without any detergent but with 2-methyl-2,4-pentanediol (MPD), and determined its structure. The structure of the heptameric pore was similar to that reported previously. In the structure, two molecules of MPD were bound around Trp179, around which phospholipid head groups were bound in the heptameric pore structure reported previously. Size exclusion chromatography showed that α-hemolysin did not assemble spontaneously even when stored for 1 year. SDS-PAGE analysis revealed that, among the compounds in the crystallizing buffer, MPD could induce heptamer formation. The concentration of MPD that most efficiently induced oligomerization was between 10 and 30%. Based on these observations, we propose MPD as a reagent that can facilitate heptameric pore formation of α-hemolysin without membrane binding.