1. Students:Wouter Spoor, Martijn van Rosmalen, Jim Eijsermans Supervisor:Prof. M. Egmond

2. Global overview protein channel  SecA (motor) and SecYE (channel) Hydrophobic crack Two-helix finger Clamp Plug

3. SecA  Motor protein T. maritima B. subtilis (PDB) M. jannaschii  ATP binding domain (NBD1 & 2)  Polypeptide crosslinking domain (PPXD)

4. SecA - Topview (cytoplasmic)  Fig A. X-ray of SecA  Fig B. X-ray of SecA with ADP

5. SecA - Clamp  Hydrophobic residues show where polypeptide can bind Orange: conserved hydrophobic residues Blue shows high conserved regions, red the lowest

6. SecY  Red: SecA/Fab binding domain  Blue: used for intramolecular crosslinking  Green: used for intermolecular crosslinking

7. SecYE – Hydrophobic crack  Binding to SecYE results in opening

8. 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

9. SecYE – MD analysis  MD analysis show closing of hydrophobic crack

10. 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

11. SecYE – Hydrophobic residues in Pore ring (cytoplasmic-/topview)

12. Remember SecYE and SecA  How do SecYE and SecA fit together? SecYE (sideview)SecA (topview)

13. Alignment of the clamp of SecA and the lateral gate of SecY Red line: signal polypeptide Yellow/blue: SecA Red/green/gray: SecYEG

14. 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

15. 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

16. Model 2: based on results

17. 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

19. Stability of the SecA-SecY complex

20.  Grey: ADP  Blue: ADP-BeFx

21. Fab and SecA bind at the same residues