Students: Wouter Spoor, Martijn van Rosmalen, Jim EijsermansSupervisor: Prof. M. Egmond
Global overview protein channel SecA (motor) and SecYE (channel)
Hydrophobic crack Two-helix finger Clamp Plug
SecA Motor protein
T. maritima B. subtilis (PDB) M. jannaschii
ATP binding domain (NBD1 & 2) Polypeptide crosslinking domain (PPXD)
SecA - Topview (cytoplasmic) Fig A. X-ray of SecA Fig B. X-ray of SecA with ADP
SecA - Clamp Hydrophobic residues show where
polypeptide can bind
Orange: conserved hydrophobic residues
Blue shows high conserved regions,red the lowest
SecY Red: SecA/Fab binding domain Blue: used for intramolecular crosslinking Green: used for intermolecular crosslinking
SecYE – Hydrophobic crack Binding to SecYE results in opening
SecYE – Hydrophobic crack due to conformational change V329 and T92 cannot form disulfide bonds
after conformational change: Fig F. Addition of SecA results in no disulfide bond
between V329C and T92C
SecYE – MD analysis MD analysis show closing of hydrophobic crack
SecYE – Comparison of M. jannaschii and T. maritima Pore ring of SecYE opens by
conformational change of TM8 & 9 Plug opens also by this conformational
change
SecYE – Hydrophobic residues in Pore ring (cytoplasmic-/topview)
Remember SecYE and SecA How do SecYE and SecA fit together?
SecYE (sideview) SecA (topview)
Alignment of the clamp of SecA and the lateral gate of SecYRed line: signal
polypeptide
Yellow/blue: SecA
Red/green/gray: SecYEG
Conclusion ATP necessary for closing of clamp
This to hold polypeptide in place
Clamp SecA positioned above lateral gate SecYE Channel which goes through SecA and SecYE
Hydrophobic crack occurs in SecYE where the two-finger helix is positioned
Two-finger helix “pushes” polypeptide through hydrophobic crack
Model 1: the dimer model Results brought in
consideration: Polypeptide “holded” in clamp
of SecA SecA positioned central above
SecYE No evidence for dimer
formation of SecYE
Suggestion for different model
Model 2: based on results
Discussion Fab antibody
How much comparable with SecA
Some other crystals as proof X-ray structure is not representative for
dynamics
Visualize interactions between SecA and SecYE
Stability of the SecA-SecY complex
Grey: ADP Blue: ADP-BeFx
Fab and SecA bind at the same residues