Solution Structure of Human Pex5·Pex14·PTS1 Protein Complexes Obtained by Small Angle X-ray Scattering

Abstract

The Pex5p receptor recognizes newly synthesized peroxisomal matrix proteins which have a C-terminal peroxisomal targeting signal to the peroxisome. After docking to protein complexes on the membrane, these proteins are translocated across the membrane. The docking mechanism remains unclear, as no structural data on the multicomponent docking complex are available. As the interaction of the cargo-loaded Pex5p receptor and the peroxisomal membrane protein Pex14p is the essential primary docking step, we have investigated the solution structure of these complexes by small angle x-ray scattering and static light scattering. Titration studies yielded a 1:6 stoichiometry for the Pex5p⅐Pex14p complex, and low resolution structural models were reconstructed from the x-ray scattering data. The free full-length human Pex5p is monomeric in solution, with an elongated, partially unfolded N-terminal domain. The model of the complex reveals that the N terminus of Pex5p remains extended in the presence of cargo and Pex14p, the latter proteins being significantly intermingled with the Pex5p moiety. These results suggest that the extended structure of Pex5p may play a role in interactions with other substrates such as lipids and membrane proteins during the formation of functional multiprotein complexes.

Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes

“…PEX14 can homo-oligomerize and a stable complex with the stoichiometry of 1:5 for PEX5-PEX14 was isolated from rat liver (Gouveia et al, 2000). Consistent with this, titration studies with purified recombinant proteins gave a stoichiometry of 1:6 for PEX5-PEX14 (Shiozawa et al, 2009). …”

“…Controversy exists as to whether PEX5 binds cargo as a monomer (Costa-Rodrigues et al, 2005), dimer or tetramer (Madrid et al, 2004;Moscicka et al, 2007). However, more recent studies are consistent with a monomeric solution structure (Shiozawa et al, 2009).…”

A Novel Pex14 Protein-interacting Site of Human Pex5 Is Critical for Matrix Protein Import into Peroxisomes

“…It was shown that each of the seven WXXX(F/Y) motifs of the human PTS1 receptor can interact with the highly conserved N-terminal domain (NTD) of Pex14 with equilibrium dissociation constants in the nanomolar range (12,13). A recent biophysical study showed that the presence of multiple WXXX(F/Y) motifs in the N terminus of Pex5 allows the formation of higher order complexes with the Pex14-NTD in vitro, although it is not clear whether this also reflects the situation in peroxisomes in vivo (14,15). Interestingly, human Pex19, which is supposed to act as an import receptor/chaperone for peroxisomal membrane proteins, binds competitively to the same surface in Pex14-NTD via an amphipathic helix-forming pentapeptide sequence (16).…”

“…All spectra were processed with NMRPipe (27) and analyzed in Sparky (33). NMR spectra for assignment and structural determination of the Pex14-(16 -80)⅐Pex5-(57-71) complex were acquired on a sample containing 13 C, 15 N-labeled Pex14-(16 -80) bound to unlabeled Pex5-(57-71). Protein backbone and side chain assignments were obtained using standard tripleresonance experiments (28).…”

Identification of Ubiquitin-specific Protease 9X (USP9X) as a Deubiquitinase Acting on Ubiquitin-Peroxin 5 (PEX5) Thioester Conjugate

“…Structurally, it can be divided into two domains: a C-terminal half containing seven tetratricopeptide repeats organized into a ring-like structure (13,14) and a natively unfolded N-terminal half (11,15). Although it has been shown that the C-terminal half of PEX5 has a crucial role in the interaction with the PTS1 (13,14), it is becoming increasingly apparent that the N-terminal half of PEX5 also contributes to the interaction with cargo proteins (16 -20).…”