ATPase Active-Site Electrostatic Interactions Control the Global Conformation of the 100 kDa SecA Translocase

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.