ジャーナル: Sci Rep / 年: 2016 タイトル: Structural insights into the Escherichia coli lysine decarboxylases and molecular determinants of interaction with the AAA+ ATPase RavA. 著者: Eaazhisai Kandiah / Diego Carriel / Julien Perard / Hélène Malet / Maria Bacia / Kaiyin Liu / Sze W S Chan / Walid A Houry / Sandrine Ollagnier de Choudens / Sylvie Elsen / Irina Gutsche / 要旨: The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. A unique ...The inducible lysine decarboxylase LdcI is an important enterobacterial acid stress response enzyme whereas LdcC is its close paralogue thought to play mainly a metabolic role. A unique macromolecular cage formed by two decamers of the Escherichia coli LdcI and five hexamers of the AAA+ ATPase RavA was shown to counteract acid stress under starvation. Previously, we proposed a pseudoatomic model of the LdcI-RavA cage based on its cryo-electron microscopy map and crystal structures of an inactive LdcI decamer and a RavA monomer. We now present cryo-electron microscopy 3D reconstructions of the E. coli LdcI and LdcC, and an improved map of the LdcI bound to the LARA domain of RavA, at pH optimal for their enzymatic activity. Comparison with each other and with available structures uncovers differences between LdcI and LdcC explaining why only the acid stress response enzyme is capable of binding RavA. We identify interdomain movements associated with the pH-dependent enzyme activation and with the RavA binding. Multiple sequence alignment coupled to a phylogenetic analysis reveals that certain enterobacteria exert evolutionary pressure on the lysine decarboxylase towards the cage-like assembly with RavA, implying that this complex may have an important function under particular stress conditions.
名称: 25 MM HEPES, 100 MM NACL, 0.2 MM PLP, 1 MM DTT, PH 7.2 pH: 7.2
試料
濃度: 2 mg/ml / 包埋: NO / シャドウイング: NO / 染色: NO / 凍結: YES
試料支持
詳細: HOLEY CARBON
急速凍結
詳細: VITRIFICATION 1 -- CRYOGEN- ETHANE, HUMIDITY- 100, TEMPERATURE- 91, INSTRUMENT- FEI VITROBOT MARK III, METHOD- BLOT FOR 2.5 SECONDS BEFORE PLUNGING,
モード: BRIGHT FIELD / 倍率(公称値): 59000 X / 倍率(補正後): 59000 X / 最大 デフォーカス(公称値): 4290 nm / 最小 デフォーカス(公称値): 540 nm / Cs: 2 mm
試料ホルダ
温度: 91 K
撮影
電子線照射量: 25 e/Å2 / フィルム・検出器のモデル: KODAK SO-163 FILM
画像スキャン
デジタル画像の数: 206
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解析
CTF補正
詳細: INDIVIDUAL PARTICLE WAS APPLIED FULL CTF CORRECTION AFTER FIRST PEAK
対称性
点対称性: D5 (2回x5回 2面回転対称)
3次元再構成
手法: PROJECTION MATCHING / 解像度: 5.5 Å / ピクセルサイズ(公称値): 1.186 Å / ピクセルサイズ(実測値): 1.186 Å 詳細: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-3205. (DEPOSITION ID: 13909).
原子モデル構築
手法: CROSS-CORRELATION, ENERGY / 空間: REAL / Target criteria: Cross-correlation coefficient / 詳細: X-ray