Abstract:
:The SecA molecular nanomachine in bacteria uses energy from ATP hydrolysis to drive post-translational secretion of preproteins through the SecYEG translocon. Cytosolic SecA exists in a dimeric, "closed" state with relatively low ATPase activity. After binding to the translocon, SecA undergoes major conformational rearrangement, leading to a state that is structurally more "open", has elevated ATPase activity, and is active in translocation. The structural details underlying this conformational change in SecA remain incompletely defined. Most SecA crystal structures report on the cytosolic form; only one structure sheds light on a form of SecA that has engaged the translocon. We have used mild destabilization of SecA to trigger conformational changes that mimic those in translocation-active SecA and thus study its structural changes in a simplified, soluble system. Results from circular dichroism, tryptophan fluorescence, and limited proteolysis demonstrate that the SecA conformational reorganization involves disruption of several domain-domain interfaces, partial unfolding of the second nucleotide binding fold (NBF) II, partial dissociation of the helical scaffold domain (HSD) from NBF I and II, and restructuring of the 30 kDa C-terminal region. These changes account for the observed high translocation SecA ATPase activity because they lead to the release of an inhibitory C-terminal segment (called intramolecular regulator of ATPase 1, or IRA1) and of constraints on NBF II (or IRA2) that allow it to stimulate ATPase activity. The observed conformational changes thus position SecA for productive interaction with the SecYEG translocon and for transfer of segments of its passenger protein across the translocon.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Maki JL,Krishnan B,Gierasch LMdoi
10.1021/bi201793esubject
Has Abstractpub_date
2012-02-21 00:00:00pages
1369-79issue
7eissn
0006-2960issn
1520-4995journal_volume
51pub_type
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