Abstract
In bacteria, the SecA protein associates with a ubiquitous protein channel SecYEG where it drives the post-translational secretion of pre-proteins across the plasma membrane. The high-resolution structures of both proteins have been determined in their resting states; however, the mechanism that couples ATP hydrolysis to active transport of substrate proteins through the membrane is not well understood. An analysis of the steady-state ATPase activity of the enzyme reveals that there is an allosteric binding site for magnesium distinct from that associated with hydrolysis of ATP. We have demonstrated that this regulation involves a large conformational change to the SecA dimer, which exerts a strong influence on the turnover and affinity for ATP, as well as the affinity for ADP. The strong inhibitory influence of magnesium on the ATPase activity can be countered by cardiolipin and conditions that promote protein translocation.
Highlights
Chemo-mechanical protein machines have evolved to couple energy derived from chemical reactions to mechanical motion
Several studies reported that a dimer with two active subunits was required for active translocation [17] and in support, a later study demonstrated that covalently cross-linked dimers were able to sustain ATP hydrolysis and protein translocation [18, 19]
An analysis of complexes formed between SecYEG and SecA by analytical ultracentrifugation showed that in the absence of nucleotide one SecA bound to the channel, whereas two bound in the presence of the ATP analogue AMPPNP2 [7]
Summary
SecA associates with the proteinconducting channel, the SecYEG complex, where it harnesses the energy from ATP binding and hydrolysis to actively push it across the membrane (9 –11). Four of the dimers revealed the SecA protomers packed head-to-tail in the crystal lattice, all in different antiparallel conformations [22,23,24, 26], another has a parallel arrangement of the subunits [25] These differences obviously add further confusion to our picture of the nature of the oligomeric state and the conformation of the active form of the protein. The basal ATPase activity of SecA is partially increased by the presence of acidic phospholipids and fully stimulated by addition of vesicles containing SecYEG and precursor protein [28, 29] The mechanism for this regulation is not clear.
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