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