1XRK

Crystal structure of a mutant bleomycin binding protein from Streptoalloteichus hindustanus displaying increased thermostability

1XRK の概要

• エントリーDOI: 10.2210/pdb1xrk/pdb
• 関連するPDBエントリー: 1BYL
• 分子名称: Bleomycin resistance protein, SULFATE ION, BLEOMYCIN A2, ... (4 entities in total)
• 機能のキーワード: arm exchange, ligand binding protein, thermostable mutant, antibiotic inhibitor
• 由来する生物種: Streptoalloteichus hindustanus
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 30960.05

構造登録者

Brouns, S.J.J.,Wu, H.,Akerboom, J.,Turnbull, A.P.,de Vos, W.M.,Van der Oost, J. (登録日: 2004-10-15, 公開日: 2005-01-11, 最終更新日: 2023-10-25)

主引用文献

Brouns, S.J.J.,Wu, H.,Akerboom, J.,Turnbull, A.P.,de Vos, W.M.,van der Oost, J.
Engineering a selectable marker for hyperthermophiles
J.Biol.Chem., 280:11422-11431, 2005

PubMed Abstract: Limited thermostability of antibiotic resistance markers has restricted genetic research in the field of extremely thermophilic Archaea and bacteria. In this study, we used directed evolution and selection in the thermophilic bacterium Thermus thermophilus HB27 to find thermostable variants of a bleomycin-binding protein from the mesophilic bacterium Streptoalloteichus hindustanus. In a single selection round, we identified eight clones bearing five types of double mutated genes that provided T. thermophilus transformants with bleomycin resistance at 77 degrees C, while the wild-type gene could only do so up to 65 degrees C. Only six different amino acid positions were altered, three of which were glycine residues. All variant proteins were produced in Escherichia coli and analyzed biochemically for thermal stability and functionality at high temperature. A synthetic mutant resistance gene with low GC content was designed that combined four substitutions. The encoded protein showed up to 17 degrees C increased thermostability and unfolded at 85 degrees C in the absence of bleomycin, whereas in its presence the protein unfolded at 100 degrees C. Despite these highly thermophilic properties, this mutant was still able to function normally at mesophilic temperatures in vivo. The mutant protein was co-crystallized with bleomycin, and the structure of the binary complex was determined to a resolution of 1.5 A. Detailed structural analysis revealed possible molecular mechanisms of thermostabilization and enhanced antibiotic binding, which included the introduction of an intersubunit hydrogen bond network, improved hydrophobic packing of surface indentations, reduction of loop flexibility, and alpha-helix stabilization. The potential applicability of the thermostable selection marker is discussed.

PubMed: 15640151
DOI: 10.1074/jbc.M413623200

実験手法

X-RAY DIFFRACTION (1.5 Å)