1. Albert Bondt Tessa Sinnige Laurens Vehmeijer

2.  Introduction  Experiments ◦ Structural studies ◦ Functional studies  Conclusion  Discussion

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

4.  Synthesized in cytoplasm  Transported to periplasm by SecYEG  Transported to β-barrel assembly sites on OM ◦ OMP structure probably recognized by assembly complex

5.  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

6.  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

7.  What is the structure of periplasmic part of YaeT?  Which POTRA domains are essential?  How do they bind different peptide sequences?

8.  Complete periplasmic fragment: YaeT 21-420 ◦ All five POTRA domains ◦ Crystallization unsuccessful  Partial periplasmic fragment: YaeT 21-351 ◦ Only first four POTRA domains ◦ Crystallization successful

9.  Fishhook-like shape  Successive POTRA domains rotated in right-handed fashion

10.  Similar secondary structures despite low sequence similarity ◦ Order: β 1 -  1 -  2 -β 2 -β 3  Three β-strands  β-sheet ◦ β 1 and β 2 : edges ◦ β 3 : center  Two antiparallel  -helices

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

12.  YaeT 21-351 : dimer in crystal ◦ Intertwined monomers ◦ Solvent-accessible part is buried

13.  H-bonds at edge of P3 and first residues of P5 “stump” ◦ Only major contact area monomers ◦ Formation β-strand parallel to β 2 of P3 causes dimerization

14.  Formation β-stranded interface may be needed for successful crystallization  Dimer not physiologically relevant ◦ YaeT 21-351 elutes as a monomer from SEC ◦ N-terminus P5 needed for β-interface in YaeT 21-351 not available in wt-protein

15.  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

16.  P5 crucial for interactions with lipoproteins

17.  OMP assembly complex functions as monomer ◦ Blue-Native PAGE ◦ Ni 2+ -affinity chromatography

18.  All POTRA domains required for proper function

19.  β-bulge P3 involved in interaction with YfgL ◦ Evidence for β-augmentation  P3 loop might interact with Imp

20.  POTRA domains have  fold  Domains form a “fishhook” arrangement  POTRA domains can interact by  augmentation  P3 and P5 crucial for interactions

21.  Fishhook conformation native? ◦ Extensive hydrophobic and polar inter-domain contacts

22.  Fishhook conformation native? ◦ Probably not! ◦ More extended conformation shown by NMR, SAXS and X-ray

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