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Search for a suitable cargo prototype SCP2 AKL-COOHPre sequence Sterol carrier protein-2  preSCP2 (15.4 kDa), mSCP2 (13.4 kDa), SCP-X (bifunctional) 

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Presentation on theme: "Search for a suitable cargo prototype SCP2 AKL-COOHPre sequence Sterol carrier protein-2  preSCP2 (15.4 kDa), mSCP2 (13.4 kDa), SCP-X (bifunctional) "— Presentation transcript:

1 Search for a suitable cargo prototype SCP2 AKL-COOHPre sequence 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]

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 C Pex5p (apo): grey, black Pex5p-SCP2: red

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

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

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 SCP2LinkerPTS1-COOH Structure of the cargo PTS1 motif, bound to Pex5p

11 PTS-1 2 nd site 7C loop Ser600

12 Structural changes in the Pex5p receptor +/- SCP2 apobound to SCP2

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

14 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) Structural changes in the Pex5p receptor +/- SCP2

15 Residue mobilities in the Pex5p +/- SCP2 structures C 7C Loop

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 apobound to Pex5p PTS-1(C) N N

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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