Using a Low Denaturant
Model To Explore the Conformational Features of Translocation-Active
SecA
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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