Albert BondtTessa Sinnige

Laurens Vehmeijer

Introduction Experiments

◦ Structural studies◦ Functional studies

Conclusion Discussion

Membrane proteins: mostly α-helices

Outer membranes proteins Gram(-) bacteria, mitochondria and chloroplasts: mostly β-barrels◦ OMPs: Outer Membrane Proteins

Synthesized in cytoplasm

Transported to periplasm by SecYEG

Transported to β-barrel assembly sites on OM ◦ OMP structure probably

recognized by assembly complex

Folded and inserted by conserved process involving a multiprotein machine◦ Four lipoproteins: YfgL, YfiO, NlpB and SmpA◦ Conserved β –barrel: YaeT in E.coli, Sam50 in

mitochondria, Toc75 in chloroplasts

YaeT◦ Essential for viability ◦ Reported to bind C-terminal peptides of OMPs ◦ Large region in the intermembrane space contains POTRA

domains.

POlypeptide TRansport-Associated (POTRA) domains◦ Implicated role assembling other beta-barrel proteins in

mitochondria◦ Implicated role as docking sites for proteins to be transported

over membrane in chloroplasts

What is the structure of periplasmic part of YaeT?

Which POTRA domains are essential?

How do they bind different peptide sequences?

Complete periplasmic fragment: YaeT21-420

◦ All five POTRA domains◦ Crystallization unsuccessful

Partial periplasmic fragment: YaeT21-351

◦ Only first four POTRA domains◦ Crystallization successful

Fishhook-like shape

Successive POTRA domains rotated inright-handed fashion

Similar secondary structures despite low sequence similarity◦ Order: β1-1-2-β2-β3

Three β-strands β-sheet◦ β1 and β2: edges◦ β3: center

Two antiparallel -helices

Only hydrophobic core and loop regions conserved between POTRA domains◦ Implicates importance for structure

YaeT21-351: dimer in crystal◦ Intertwined monomers◦ Solvent-accessible part is buried

H-bonds at edge of P3 and first residues of P5 “stump” ◦ Only major contact area monomers◦ Formation β-strand parallel to β2 of P3 causes

dimerization

Formation β-stranded interface may be needed for successful crystallization

Dimer not physiologically relevant◦ YaeT21-351 elutes as a monomer from SEC◦ N-terminus P5 needed for

β-interface in YaeT21-351 not available in wt-protein

Dimerization shows possible interaction of other proteins with POTRA domains◦ β-augmentation: addition of β-strands to β-sheet

through H-bonds

Similar highly ordered contacts at interfaces all POTRA domains fishhook confirmation in monomer

P5 crucial for interactions with lipoproteins

OMP assembly complex functions as monomer◦ Blue-Native PAGE◦ Ni2+-affinity chromatography

All POTRA domains required for proper function

β-bulge P3 involved in interaction with YfgL◦ Evidence for β-augmentation

P3 loop might interact with Imp

POTRA domains have fold Domains form a “fishhook” arrangement POTRA domains can interact by

augmentation P3 and P5 crucial for interactions

Fishhook conformation native?◦ Extensive hydrophobic and polar inter-domain

contacts

Fishhook conformation native?◦ Probably not!◦ More extended conformation shown by NMR,

SAXS and X-ray

Mechanism of YaeT?◦ Monomer or oligomer◦ Interactions with lipoproteins◦ Recognition of substrate