Albert Bondt Tessa 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: YaeT ◦ All five POTRA domains ◦ Crystallization unsuccessful  Partial periplasmic fragment: YaeT ◦ Only first four POTRA domains ◦ Crystallization successful

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

 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

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

 YaeT : 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 β 2 of P3 causes dimerization

 Formation β-stranded interface may be needed for successful crystallization  Dimer not physiologically relevant ◦ YaeT elutes as a monomer from SEC ◦ N-terminus P5 needed for β-interface in YaeT 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 ◦ Ni 2+ -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