3iqm | pdb_00003iqm
Active site mutants of B. subtilis SecA
Structural highlights
Function[SECA_BACSU] Part of the Sec protein translocase complex. Interacts with the SecYEG preprotein conducting channel. Has a central role in coupling the hydrolysis of ATP to the transfer of proteins into and across the cell membrane, serving as an ATP-driven molecular motor driving the stepwise translocation of polypeptide chains across the membrane (By similarity).[HAMAP-Rule:MF_01382] Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMedSecA 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 approximately 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 approximately 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. ATPase Active-Site Electrostatic Interactions Control the Global Conformation of the 100 kDa SecA Translocase.,Kim DM, Zheng H, Huang YJ, Montelione GT, Hunt JF J Am Chem Soc. 2013 Feb 27;135(8):2999-3010. doi: 10.1021/ja306361q. Epub 2013, Feb 14. PMID:23167435[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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