title: Crystallographic study of the 2-thioribothymidine-synthetic complex TtuA-TtuB from Thermus thermophilus.
abstract: The ubiquitin-like protein TtuB is a sulfur carrier for the biosynthesis of 2-thioribothymidine (s2T) at position 54 in some thermophilic bacterial tRNAs. TtuB captures a S atom at its C-terminus as a thiocarboxylate and transfers it to tRNA by the transferase activity of TtuA. TtuB also functions to suppress s2T formation by forming a covalent bond with TtuA. To explore how TtuB interacts with TtuA and switches between these two different functions, high-resolution structure analysis of the TtuA-TtuB complex is required. In this study, the TtuA-TtuB complex from Thermus thermophilus was expressed, purified and crystallized. To mimic the thiocarboxylated TtuB, the C-terminal Gly residue was replaced with Cys (G65C) to obtain crystals of the TtuA-TtuB complex. A Zn-MAD data set was collected to a resolution of 2.5 Å. MAD analysis successfully determined eight Zn sites, and a partial structure model composed of four TtuA-TtuB complexes in the asymmetric unit was constructed.
authors.affiliation: Graduate School of Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan.
authors.forename: Minghao
authors.initials: M
authors.lastname: Chen
authors.affiliation: Graduate School of Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan.
authors.forename: Shun
authors.initials: S
authors.lastname: Narai
authors.affiliation: Graduate School of Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Omura
authors.affiliation: Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Shigi
authors.affiliation: Faculty of Advanced Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan.
authors.forename: Sarin
authors.initials: S
authors.lastname: Chimnaronk
authors.affiliation: Graduate School of Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Tanaka
authors.affiliation: Graduate School of Life Science, Hokkaido University, Kita 10, Nishi 8, Kita-ku, Sapporo, Hokkaido 060-0810, Japan.
authors.forename: Min
authors.initials: M
authors.lastname: Yao
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002413:Cations, Divalent; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D005998:Glycine / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D012343:RNA, Transfer / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016963:Thermus thermophilus / Q000737:chemistry* / Q000201:enzymology; D013891:Thiouridine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D014961:X-Ray Diffraction; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D011994:Recombinant Proteins; D013891:Thiouridine; C022856:2-thioribothymidine; D012343:RNA, Transfer; D015032:Zinc; D003545:Cysteine; D005998:Glycine
keywords: TtuA; TtuB; Zn-MAD; post-transcriptional modification; sulfur transfer; ubiquitin-like protein