Immobilization of the plug domain inside the SecY channel allows unrestricted protein translocation.
The SecYEG complex forms a protein-conducting channel in the inner membrane of Escherichia coli to support the translocation of secretory proteins in their unfolded state. The SecY channel is closed at the periplasmic face of the membrane by a small re-entrance loop that connects transmembrane segment 1 with 2b. This helical domain 2a is termed the plug domain. By the introduction of pairs of cysteines and crosslinkers, the plug domain was immobilized inside the channel and connected to transmembrane segment 10. Translocation was inhibited to various degrees depending on the position and crosslinker spacer length. With one of the crosslinked mutants translocation occurred unrestricted. Biochemical characterization of this mutant as well as molecular dynamics simulations suggest that only a limited movement of the plug domain suffices for translocation.
Lycklama A Nijeholt
Jelger A
JA
Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands.
Bulacu
Monica
M
Marrink
Siewert Jan
SJ
Driessen
Arnold J M
AJ
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003198:Computer Simulation; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D000069816:SEC Translocation Channels
D001425:Bacterial Outer Membrane Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D000069816:SEC Translocation Channels; C057841:SecY protein, E coli; C499588:ompT protein, E coli; D010447:Peptide Hydrolases; D003545:Cysteine