Protein translocation by the SecA ATPase occurs by a power‐stroke mechanism

Abstract

SecA belongs to the large class of ATPases that use the energy of ATP hydrolysis to perform mechanical work resulting in protein translocation across membranes, protein degradation, and unfolding. SecA translocates polypeptides through the SecY membrane channel during protein secretion in bacteria, but how it achieves directed peptide movement is unclear. Here, we use single‐molecule FRET to derive a model that couples ATP hydrolysis‐dependent conformational changes of SecA with protein translocation. Upon ATP binding, the two‐helix finger of SecA moves toward the SecY channel, pushing a segment of the polypeptide into the channel. The finger retracts during ATP hydrolysis, while the clamp domain of SecA tightens around the polypeptide, preserving progress of translocation. The clamp opens after phosphate release and allows passive sliding of the polypeptide chain through the SecA‐SecY complex until the next ATP binding event. This power‐stroke mechanism may be used by other ATPases that move polypeptides.

The SecA motor generates mechanical force during protein translocation

“…A wealth of genetic, biochemical and structural studies of the Sec translocation machinery has yielded a comprehensive understanding of its constituent parts and their activities. Mechanical aspects have been proposed to be important for Sec translocon function 7,9,14 , but their experimental investigation is challenging. Here, we have mechanically calibrated a widely employed probe for protein transport studies, mDHFR, using optical tweezers (Figures 2 and 3).…”

“…"Power stroke" models posit that a structural element in SecA, termed the two-helix finger, inserts into the SecY channel upon ATP binding, dragging the translocating polypeptide along with it 7,8 . Two other SecA domains, the polypeptide crosslinking domain and the nucleotide binding domain 2, form a clamp that subsequently closes around the substrate, preventing backsliding as the two-helix finger resets after ATP hydrolysis 9 .…”

Interaction of the Motor Protein SecA and the Bacterial Protein Translocation Channel SecYEG in the Absence of ATP

“…The observation is in line with single molecule FRET experiments in which the movement of the two helix finger was interpreted in terms of a power stroke. 43 The model envisions the finger to push the polypeptide into the channel. Its retrograde movement does not lead to backsliding, because the clamp domain of SecA tightens around the polypeptide during ATP hydrolysis.…”

“…The presence of a translocating polypeptide would not preserve the SecYEG-SecA complex, because phosphate release opens SecA's clamp so that the polypeptide chain passively slides through SecYEG and through SecA. 8,43 In the interval between the release of one SecA molecule and the binding of a new SecA molecule, the translocating polypeptide would slide back, thereby significantly reducing the translocation efficiency. In contrast, the now observed K app = 10 nM ensures a SecA residence time that easily bridges the comparatively short time window between ADP release and ATP capture.…”

Energy landscape steering mediates dynamic coupling in ATP-driven protein translocation by the bacterial Sec machinery

“…8 The two-helix finger (2HF) domain of SecA has been proposed to act as a sensor regulating nucleotide exchange, 9,10 or alternatively, to directly push the polypeptide across the membrane. 11,12 The precise nature of the translocation mechanism has divided opinion owing to the inherent complexity of the system. 9,[11][12][13] As for other molecular machines which convert the chemical potential of NTP hydrolysis into directional motion, there are two limiting cases of energy transduction: power stroke and Brownian ratchet.…”