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Search for a suitable cargo prototype

Search for a suitable cargo prototype. Sterol carrier protein-2 preSCP2 (15.4 kDa), mSCP2 (13.4 kDa), SCP-X (bifunctional) Structures of SCP2 have been determined (NMR & X-ray) [Choinowski et al ., 2000; Garcia et al ., 2000]

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Search for a suitable cargo prototype

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  1. Search for a suitable cargo prototype Sterol carrier protein-2 • preSCP2 (15.4 kDa), mSCP2 (13.4 kDa), SCP-X (bifunctional) • Structures of SCP2 have been determined (NMR & X-ray) [Choinowski et al., 2000; Garcia et al., 2000] • Some evidence that SCP2 function may be affected by Pex5p binding [Lensink et al., 2002] SCP2 Pre sequence AKL-COOH

  2. In vitro binding Pex5p - cargo (SCP2) by ITC • SCP2 recognizes the Pex5p receptor, irrespective of its functional state. • Binding of complete cargo (SCP2) > Binding of PTS1 motif. • Structure-based mutations in the receptor that affect binding

  3. NMR shift analysis of SCP2 +/- Pex5p receptor • SCP2 remains functional and folded when bound to the Pex5p receptor. • Evidence by NMR (shown), ITC (shown), ESI-MS (not shown). • Data support translocation of functional PTS1 cargos through peroxisome membrane.

  4. Crystallisation of Pex5p +/- SCP2 cargo

  5. Residue mobilities in the Pex5p +/- SCP2 structures Pex5p (apo): grey, black Pex5p-SCP2: red 1 2 3 4 5 6 7 C

  6. Protein translocation into peroxisomes EMBL-Grenoble Nov 07, 2006 Matthias Wilmanns, EMBL-Hamburg

  7. Consecutive TPR elements build up a superhelical coil structure, with 7 TPRs / turn TPR motif architecture D’Andrea & Regan (2003) TIBS 28, 665-662

  8. Structure of the Pex5 receptor - SCP2 cargo complex Pex5p • 7 TPR domains (blue, cyan) • Distorted TPR4 domain (green) • C-terminal 3-helical bundle (violet) • Flexible ‘7C loop’ (red) SCP2 • Extended C-terminal PTS1 motif Pex5p/SCP2 binding • 1. PTS1 (SCP2) - central hole (Pex5p) • 2. Core domain (SCP2) -C-terminal bundle (Pex5p)

  9. Structure of the Pex5 receptor - SCP2 cargo complex

  10. SCP2 Linker PTS1-COOH Structure of the cargo PTS1 motif, bound to Pex5p

  11. 7C loop PTS-1 Ser600 2nd site

  12. Structural changes in the Pex5p receptor +/- SCP2 apo bound to SCP2

  13. Structural changes in the Pex5p receptor +/- SCP2 apo (snail) bound to SCP2 (ring)

  14. Structural changes in the Pex5p receptor +/- SCP2 Structure superposition based on N-terminal TPR triplet: • Pex5p (apo): coloured • Pex5p (cargo): grey Comparison • Hinge region: TPR-5/6 (orange) • Largest changes at 7C loop (red)

  15. Residue mobilities in the Pex5p +/- SCP2 structures 7C Loop 1 2 3 4 5 6 7 C

  16. In vitro binding Pex5p - cargo (SCP2) by ITC • SCP2 recognizes the Pex5p receptor, irrespective of its functional state. • Binding of complete cargo (SCP2) > Binding of PTS1 motif. • Structure-based mutations in the receptor that affect binding.

  17. In vivo translocation of PTS1 targets • Mutations in the 7C loop lead to severe PTS-1 import defects (SCP2, catalase): S600W, Q586R > S589Y, N382A. • PTS-2 import (CAcT, control) is not affected. 7C loop PTS1 (control)

  18. Structure of the SCP2 cargo +/- Pex5p receptor PTS-1(C) PTS-1(C) N N apo bound to Pex5p

  19. More conformational changes ? Cargo bound T = 25 Å Apo T = 35 Å Extended ? T = 55 Å Distorted TPR-4 domain

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