Crystal structure of a substrate-engaged SecY protein-translocation channel

Abstract

Hydrophobic signal sequences target secretory polypeptides to a protein-conducting channel formed by a heterotrimeric membrane protein complex, the prokaryotic SecY or eukaryotic Sec61 complex. How signal sequences are recognized is poorly understood, particularly because they are diverse in sequence and length. Structures of the inactive channel show that the largest subunit, SecY or Sec61α, consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces lipid1-10. The cytoplasmic funnel is empty, while the extracellular funnel is filled with a plug domain. In bacteria, the SecY channel associates with the translating ribosome in co-translational translocation, and with the SecA ATPase in post-translational translocation 11. How a translocating polypeptide inserts into the channel is uncertain, as cryo-EM structures of the active channel have a relatively low resolution (~10Å) or are of insufficient quality 6-8. Here we report a crystal structure of the active channel, assembled from SecY complex, the SecA ATPase, and a segment of a secretory protein fused into SecA. The translocating protein segment inserts into the channel as a loop, displacing the plug domain. The hydrophobic core of the signal sequence forms a helix that sits in a groove outside the lateral gate, while the following polypeptide segment intercalates into the gate. The C-terminal section of the polypeptide loop is located in the channel, surrounded by residues of the pore ring. Thus, during translocation, the hydrophobic segments of signal sequences, and probably bilayer-spanning domains of nascent membrane proteins, exit the lateral gate and dock at a specific site that faces the lipid phase.

A central cavity within the holo-translocon suggests a mechanism for membrane protein insertion

“…While crystal structures of SecYEG, SecDF and YidC are available814151735, the subunit organization in the HTL complex remained unclear. Here, we combined a range of biophysical data to provide mechanistic and structural insights into the HTL membrane protein complex in an integrative approach.…”

Alignment of the protein substrate hairpin along the SecA two-helix finger primes protein transport in Escherichia coli

“…SecA is shown in light gray, SecYE is in dark gray, and the OmpA peptide substrate inserted at the end of the THF is shown in pink. For clarity, the nanobody crystallized with the complex has been omitted (10). Generation of FRET-mapped regions and their associated colors in the presence of ATP-γS was done as described in Fig.…”

“…These channels display remarkably similar hourglass-shaped structures that are doubly gated: they open vertically to allow protein transport across the membrane or open laterally to allow insertion of integral membrane proteins into the lipid bilayer (2)(3)(4)(5)(6)(7)(8)(9)(10). The SecYEG channel consists of 15 transmembrane helices with a short helical region that blocks the channel, referred to as the plug domain.…”

Driving Forces of Translocation Through Bacterial Translocon SecYEG

“…In contrast to most of the structures with a translocation intermediate such as 3J46 (Park et al 2013), 5GAE (Jomaa et al 2016), and 5EUL (Li et al 2016), the reconstituted SecY-signal peptide, SecY-proOmpA complex, or SecY-ribosome complex appear to be ion permeable at small transmembrane potentials (Knyazev et al 2013; Knyazev et al 2014). The complexes exclude ions when physiological values of membrane potential (Fig.…”