3IQY

Active site mutants of B. subtilis SecA

3IQY の概要

• エントリーDOI: 10.2210/pdb3iqy/pdb
• 関連するPDBエントリー: 3IQM
• 分子名称: Protein translocase subunit secA, SULFATE ION (2 entities in total)
• 機能のキーワード: alpha beta, atp-binding, cell membrane, membrane, metal-binding, nucleotide-binding, protein transport, translocation, transport
• 由来する生物種: Bacillus subtilis
• 細胞内の位置: Cell membrane; Peripheral membrane protein; Cytoplasmic side (By similarity): P28366
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 96019.03

構造登録者

Kim, D.,Hunt, J.F. (登録日: 2009-08-21, 公開日: 2010-08-11, 最終更新日: 2023-09-06)

主引用文献

Kim, D.M.,Zheng, H.,Huang, Y.J.,Montelione, G.T.,Hunt, J.F.
ATPase Active-Site Electrostatic Interactions Control the Global Conformation of the 100 kDa SecA Translocase.
J.Am.Chem.Soc., 135:2999-3010, 2013

PubMed Abstract: SecA is an intensively studied mechanoenzyme that uses ATP hydrolysis to drive processive extrusion of secreted proteins through a protein-conducting channel in the cytoplasmic membrane of eubacteria. The ATPase motor of SecA is strongly homologous to that in DEAD-box RNA helicases. It remains unclear how local chemical events in its ATPase active site control the overall conformation of an ~100 kDa multidomain enzyme and drive protein transport. In this paper, we use biophysical methods to establish that a single electrostatic charge in the ATPase active site controls the global conformation of SecA. The enzyme undergoes an ATP-modulated endothermic conformational transition (ECT) believed to involve similar structural mechanics to the protein transport reaction. We have characterized the effects of an isosteric glutamate-to-glutamine mutation in the catalytic base, a mutation which mimics the immediate electrostatic consequences of ATP hydrolysis in the active site. Calorimetric studies demonstrate that this mutation facilitates the ECT in Escherichia coli SecA and triggers it completely in Bacillus subtilis SecA. Consistent with the substantial increase in entropy observed in the course of the ECT, hydrogen-deuterium exchange mass spectrometry demonstrates that it increases protein backbone dynamics in domain-domain interfaces at remote locations from the ATPase active site. The catalytic glutamate is one of ~250 charged amino acids in SecA, and yet neutralization of its side chain charge is sufficient to trigger a global order-disorder transition in this 100 kDa enzyme. The intricate network of structural interactions mediating this effect couples local electrostatic changes during ATP hydrolysis to global conformational and dynamic changes in SecA. This network forms the foundation of the allosteric mechanochemistry that efficiently harnesses the chemical energy stored in ATP to drive complex mechanical processes.

PubMed: 23167435
DOI: 10.1021/ja306361q

実験手法

X-RAY DIFFRACTION (3.3 Å)