1DFA

CRYSTAL STRUCTURE OF PI-SCEI IN C2 SPACE GROUP

1DFA の概要

• エントリーDOI: 10.2210/pdb1dfa/pdb
• 関連するPDBエントリー: 1VDE
• 分子名称: PI-SCEI ENDONUCLEASE (2 entities in total)
• 機能のキーワード: intein, homing endonuclease, hydrolase
• 由来する生物種: Saccharomyces cerevisiae (baker's yeast)
• 細胞内の位置: Endomembrane system: P17255
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 51003.89

構造登録者

Hu, D.,Crist, M.,Duan, X.,Quiocho, F.A.,Gimble, F.S. (登録日: 1999-11-18, 公開日: 1999-12-08, 最終更新日: 2024-02-07)

主引用文献

Hu, D.,Crist, M.,Duan, X.,Quiocho, F.A.,Gimble, F.S.
Probing the structure of the PI-SceI-DNA complex by affinity cleavage and affinity photocross-linking.
J.Biol.Chem., 275:2705-2712, 2000

PubMed Abstract: The PI-SceI protein is an intein-encoded homing endonuclease that initiates the mobility of its gene by making a double strand break at a single site in the yeast genome. The PI-SceI protein splicing and endonucleolytic active sites are separately located in each of two domains in the PI-SceI structure. To determine the spatial relationship between bases in the PI-SceI recognition sequence and selected PI-SceI amino acids, the PI-SceI-DNA complex was probed by photocross-linking and affinity cleavage methods. Unique solvent-accessible cysteine residues were introduced into the two PI-SceI domains at positions 91, 97, 170, 230, 376, and 378, and the mutant proteins were modified with either 4-azidophenacyl bromide or iron (S)-1-(p-bromoacetamidobenzyl)-ethylenediaminetetraacetate (FeBABE). The phenyl azide-coupled proteins cross-linked to the PI-SceI target sequence, and the FeBABE-modified proteins cleaved the DNA proximal to the derivatized amino acid. The results suggest that an extended beta-hairpin loop in the endonuclease domain that contains residues 376 and 378 contacts the major groove near the PI-SceI cleavage site. Conversely, residues 91, 97, and 170 in the protein splicing domain are in close proximity to a distant region of the substrate. To interpret our results, we used a new PI-SceI structure that is ordered in regions of the protein that bind DNA. The data strongly support a model of the PI-SceI-DNA complex derived from this structure.

PubMed: 10644733
DOI: 10.1074/jbc.275.4.2705

実験手法

X-RAY DIFFRACTION (2 Å)