Structure of the substrate-engaged SecA-SecY protein translocation machine

Abstract

The Sec61/SecY channel allows the translocation of many proteins across the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. In bacteria, most secretory proteins are transported post-translationally through the SecY channel by the SecA ATPase. How a polypeptide is moved through the SecA-SecY complex is poorly understood, as structural information is lacking. Here, we report an electron cryo-microscopy (cryo-EM) structure of a translocating SecA-SecY complex in a lipid environment. The translocating polypeptide chain can be traced through both SecA and SecY. In the captured transition state of ATP hydrolysis, SecA’s two-helix finger is close to the polypeptide, while SecA’s clamp interacts with the polypeptide in a sequence-independent manner by inducing a short β-strand. Taking into account previous biochemical and biophysical data, our structure is consistent with a model in which the two-helix finger and clamp cooperate during the ATPase cycle to move a polypeptide through the channel.

Atomic structures of anthrax toxin protective antigen channels bound to partially unfolded lethal and edema factors

“…Recently, structures of the substrate-engaged SecY protein translocon have been determined using X-ray crystallography and cryoEM 34,35 . The SecY system is one of the few other protein translocation systems where structural information is available.…”

“…In addition, a polypeptide clamp has been identified in SecA which would position the translocating polypeptide right above the SecY pore 36 . The recent structure of the clamp bound to the translocating substrate indicates that it engages with the polypeptide in a sequence-independent manner by inducing short β strand conformations in the polypeptide 35 . This action would allow a broad range of polypeptides to be bound and translocated by the SecA.…”

The SecA motor generates mechanical force during protein translocation

“…Structural studies 11,52,53 suggest that tension on the translocating polypeptide arises when the SecA two-helix finger pulls the folded domain against the clamp. The axis of force application depends on how the domain abuts the clamp, which is not known.…”

A snapshot of membrane protein insertion

“…The flexibility of the substrate represents an obstacle in structure determination. This can be resolved by fixing the substrate onto the Sec translocon, e.g., by disulfide bond formation, a strategy that recently allowed the determination of an intermediate step of protein translocation across the membrane by cryo‐EM . Ma et al reported the structure of lipid‐embedded SecYEG in complex with SecA ATPase, and the substrate.…”

“…This can be resolved by fixing the substrate onto the Sec translocon, e.g., by disulfide bond formation, a strategy that recently allowed the determination of an intermediate step of protein translocation across the membrane by cryo‐EM . Ma et al reported the structure of lipid‐embedded SecYEG in complex with SecA ATPase, and the substrate. In this context, the SecYEG complex interacts with the motor protein SecA ATPase, instead of the ribosome, through the same cytoplasmic loops.…”