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]

In vitro binding Pex5p - cargo (SCP2) by ITC

Pex5p receptor CargoΔH

(kJ /mo )l

TΔS

(kJ /mo )l

ΔG

(kJ /mo )l

Kd

(nM)

wild-type mSCP2 -42.4 -1.2 -41.2 109 ± 34

wild-type mSCP2(SC )oA -31.8 8.9 -40.8 124 ± 17

wild-type preSCP2 -35.9 6.2 -42.1 74 ± 9

wild-type PGNAKL -45.1 -8.7 -36.4 664 ± 37

382N A mSCP2 -27.3 10.8 -38.1 348 ± 54

586Q R mSCP2 -17.4 17.3 -34.5 1343 ± 321

S589Y mSCP2 -38.7 1.20 -39.9 173 ± 23

S600W mSCP2 no binding

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

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.

Crystallisation of Pex5p +/- SCP2 cargo

Pex5p(C)-SCP2 Pex5p(C) apoX-ray data collectionSpace group P212121 P1

Unit cell dimensions [Å] 40.5, 68.6, 137.453.5, 85.6, 88.9,

71.2°, 90.0°, 73.4°Resolution range [Å] 25.0-2.3 (2.4-2.3) 20.0-2.5 (2.6-2.5)Rsym [%] 9.4 (49.8) 13.7 (53.8)I/σ(I) 14.1 (3.8) 6.3 (1.7)Completeness [%] 99.8 (100.0) 95.9 (85.9)Data redundancy 6.0 (6.1) 2.2 (2.1)Unique reflections 17,692 47,257Refinement statisticsResolution range [ ]Å 20.0-2.3 20.0-2.5R-fac /torR-free [%] 20.2/25.6 26.3/30.9Protein atoms 3209 9483Solvent atoms 99 147Rmsd bond distance [s Å] 0.006 0.013Rmsd bondangles [°] 1.0 1.4

Residue mobilities in the Pex5p +/- SCP2 structures

0

20

40

60

80

100

120

1 2 3 4 5 6 7 C

Pex5p (apo): grey, blackPex5p-SCP2: red

Protein translocation into peroxisomes

EMBL-Grenoble

Nov 07, 2006

Matthias Wilmanns, EMBL-Hamburg

TPR motif architecture

D’Andrea & Regan (2003) TIBS 28, 665-662

Consecutive TPR elements

build up a superhelical coil

structure, with 7 TPRs / turn

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)

Structure of the Pex5 receptor - SCP2 cargo complex

SCP2 Linker PTS1-COOH

Structure of the cargo PTS1 motif, bound to Pex5p

PTS-1

2nd site

7C loop

Ser600

Structural changes in the Pex5p receptor +/- SCP2

apo bound to SCP2

apo (snail) bound to SCP2 (ring)

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)

Structural changes in the Pex5p receptor +/- SCP2

Residue mobilities in the Pex5p +/- SCP2 structures

0

20

40

60

80

100

120

1 2 3 4 5 6 7 C

7C

Lo

op

In vitro binding Pex5p - cargo (SCP2) by ITC

Pex5p receptor CargoΔH

(kJ /mo )l

TΔS

(kJ /mo )l

ΔG

(kJ /mo )l

Kd

(nM)

wild-type mSCP2 -42.4 -1.2 -41.2 109 ± 34

wild-type mSCP2(SC )oA -31.8 8.9 -40.8 124 ± 17

wild-type preSCP2 -35.9 6.2 -42.1 74 ± 9

wild-type PGNAKL -45.1 -8.7 -36.4 664 ± 37

382N A mSCP2 -27.3 10.8 -38.1 348 ± 54

586Q R mSCP2 -17.4 17.3 -34.5 1343 ± 321

S589Y mSCP2 -38.7 1.20 -39.9 173 ± 23

S600W mSCP2 no binding

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.

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)

Structure of the SCP2 cargo +/- Pex5p receptor

apo bound to Pex5p

PTS-1(C)

PTS-1(C)

NN

More conformational changes ?

Cargo bound

T = 25 Å

Apo

T = 35 Å

Extended ?

T = 55 Å

Distorted TPR-4 domain