Conformational transition of Sec machinery inferred from bacterial SecYE structures

Abstract

Over 30% of proteins are secreted across or integrated into membranes. Their newly synthesized forms contain either cleavable signal sequences or non-cleavable membrane anchor sequences, which direct them to the evolutionarily conserved Sec translocon (SecYEG in prokaryotes and Sec61, comprising alpha-, gamma- and beta-subunits, in eukaryotes). The translocon then functions as a protein-conducting channel. These processes of protein localization occur either at or after translation. In bacteria, the SecA ATPase drives post-translational translocation. The only high-resolution structure of a translocon available so far is that for SecYEbeta from the archaeon Methanococcus jannaschii, which lacks SecA. Here we present the 3.2-A-resolution crystal structure of the SecYE translocon from a SecA-containing organism, Thermus thermophilus. The structure, solved as a complex with an anti-SecY Fab fragment, revealed a 'pre-open' state of SecYE, in which several transmembrane helices are shifted, as compared to the previous SecYEbeta structure, to create a hydrophobic crack open to the cytoplasm. Fab and SecA bind to a common site at the tip of the cytoplasmic domain of SecY. Molecular dynamics and disulphide mapping analyses suggest that the pre-open state might represent a SecYE conformational transition that is inducible by SecA binding. Moreover, we identified a SecA-SecYE interface that comprises SecA residues originally buried inside the protein, indicating that both the channel and the motor components of the Sec machinery undergo cooperative conformational changes on formation of the functional complex.

Figure 1. Overall structure of T. thermophilus SecYE

a, b, The SecYE complex viewed from the lateral gate side (a) and the cytoplasm (b). The SecY transmembranes are coloured light blue to red from the N to C termini, and SecE is coloured pink. Arg 351 (ref. 17) is coloured red and is shown in stick representation. The residues coloured green in stick representation were mutated to cysteine for intermolecular crosslinking experiments.

Figure 2. Comparison of the T. thermophilus SecYE and M. jannaschii SecYEβ structures

a, b, Molecular surfaces of SecYE (a) and SecYEβ (b), coloured as in Fig. 1a. Transmembrane regions are numbered. c, d, The cytoplasmic regions of TM2, TM8 and TM9 of SecY. Pre-open (c, crystal structure of Fab–SecYE) and closed forms (d, without Fab, molecular dynamics analysis at 72.93 ns) are shown. Numbers show distances between α carbons. e, Intramolecular disulphide bond formation in SecY as assessed by quantitative carboxymethylation. Averages of three analyses are shown with s.d. f, SecA-mediated inhibition of SecY (Thr92Cys–Val329Cys) intramolecular disulphide bond formation in the presence of AMP-PNP.

Figure 3. Contacting residues between T. thermophilus SecA and SecYE

a, The SecA structure (Protein Data Bank 2IPC) is colour-coded for its domains, except that light pink indicates SecA-specific regions. SecY contact residues identified in this study are space filled in green and purple. b, Close-up view of the α-helix in motif IV; evolutionarily conserved regions are shown in red, and the positions replaced by cysteines are indicated in stick representation. c, Disulphide crosslinking between SecA and SecY. The disulphide complex is indicated by solid circles. d, e, Disulphide and BMOE crosslinking of SecA and SecY.

Figure 4. Multiple modes of SecA–SecY interactions

According to the dimer model, one copy of SecY serves as a SecA-docking site, and the other functions as a translocation pore. The SecA–SecY interaction observed here should represent the one between the non-translocating copy of SecY and SecA, and is crucial for the SecA ATPase activation. Both the SecA and SecY components undergo conformational changes on their interaction, as shown by bidirectional arrows. The orientation of SecY protomers in the dimeric assembly is shown arbitrarily.