Albert Bondt
Tessa Sinnige
Laurens Vehmeijer
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Introduction
Experiments
◦ Structural studies
◦ Functional studies
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Conclusion
Discussion
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Membrane proteins: mostly α-helices
Outer membranes proteins Gram(-) bacteria,
mitochondria and chloroplasts: mostly βbarrels
◦ OMPs: Outer Membrane Proteins
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Synthesized in cytoplasm
Transported to periplasm
by SecYEG
Transported to β-barrel
assembly sites on OM
◦ OMP structure probably
recognized by assembly
complex
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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
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YaeT
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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
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What is the structure of periplasmic part
of YaeT?
Which POTRA domains are essential?
How do they bind different peptide
sequences?
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Complete periplasmic fragment: YaeT21-420
◦ All five POTRA domains
◦ Crystallization unsuccessful
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Partial periplasmic fragment: YaeT21-351
◦ Only first four POTRA domains
◦ Crystallization successful
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Fishhook-like shape
Successive POTRA
domains rotated in
right-handed fashion
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Similar secondary structures despite low
sequence similarity
◦ Order: β1-1-2-β2-β3
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Three β-strands  β-sheet
◦ β1 and β2: edges
◦ β3: center
Two antiparallel
-helices
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Only hydrophobic core and loop regions
conserved between POTRA domains
◦ Implicates importance for structure
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YaeT21-351: dimer in crystal
◦ Intertwined monomers
◦ Solvent-accessible part is buried
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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
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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
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Dimerization shows possible interaction of
other proteins with POTRA domains
◦ β-augmentation: addition of β-strands to β-sheet
through H-bonds
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Similar highly ordered contacts at interfaces
all POTRA domains  fishhook confirmation
in monomer
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P5 crucial for
interactions with
lipoproteins
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OMP assembly complex functions as monomer
◦ Blue-Native PAGE
◦ Ni2+-affinity chromatography
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All POTRA domains required for proper function
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β-bulge P3 involved in interaction with YfgL
◦ Evidence for β-augmentation
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P3 loop might interact with Imp
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POTRA domains have b---b-b fold
Domains form a “fishhook” arrangement
POTRA domains can interact by b
augmentation
P3 and P5 crucial for interactions
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Fishhook conformation native?
◦ Extensive hydrophobic and polar inter-domain
contacts
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Fishhook conformation native?
◦ Probably not!
◦ More extended conformation shown by NMR,
SAXS and X-ray
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Mechanism of YaeT?
◦ Monomer or oligomer
◦ Interactions with lipoproteins
◦ Recognition of substrate