Insights into Peroxisome Function from the Structure of PEX3 in Complex with a Soluble Fragment of PEX19

Friederike Schmidt,Nora Treiber,Georg Zocher,Sasa Bjelic,Michel O Steinmetz,Hubert Kalbacher,Thilo Stehle,Gabriele Dodt

doi:10.1074/jbc.m110.138503

Abstract

The human peroxins PEX3 and PEX19 play a central role in peroxisomal membrane biogenesis. The membrane-anchored PEX3 serves as the receptor for cytosolic PEX19, which in turn recognizes newly synthesized peroxisomal membrane proteins. After delivering these proteins to the peroxisomal membrane, PEX19 is recycled to the cytosol. The molecular mechanisms underlying these processes are not well understood. Here, we report the crystal structure of the cytosolic domain of PEX3 in complex with a PEX19-derived peptide. PEX3 adopts a novel fold that is best described as a large helical bundle. A hydrophobic groove at the membrane-distal end of PEX3 engages the PEX19 peptide with nanomolar affinity. Mutagenesis experiments identify phenylalanine 29 in PEX19 as critical for this interaction. Because key PEX3 residues involved in complex formation are highly conserved across species, the observed binding mechanism is of general biological relevance.

Highlights

Peroxisomes are single membrane-bound organelles that carry out a variety of metabolic processes
The peroxins PEX3,5 PEX16, and PEX19 are known to be essential for peroxisomal membrane biogenesis as a loss of any of these proteins leads to the complete absence of detectable peroxisomal membrane structures [11]
A PEX19-derived Peptide Binds sPEX3 with High Affinity— The interaction of the two peroxins PEX3 and PEX19 is required for the import of all peroxisomal membrane proteins

Summary

EXPERIMENTAL PROCEDURES

Protein Expression—Two human PEX3 fragments, comprising residues 26 –373 and 41–373, were expressed in Escherichia coli. Site-directed mutagenesis (QuikChange௡, Stratagene) was used to generate Cys-Ser mutations at position 235 in both cases These constructs were transformed into E. coli Rosetta (DE3) cells and grown at 37 °C to an A600 of 0.6 before protein expression was induced with 1 mM isopropyl-␤-thiogalactopyranoside. ITC experiments with PEX19-derived peptides were performed at 25 °C in buffer D (10 mM Na2HPO4, 1.8 mM KH2PO4, 140 mM NaCl, 2.7 mM KCl, 0.5 mM Tris-(2-carboxyethyl)phosphine, pH 7.4). Molecular replacement was carried out with PHASER [38, 39] using a highly twinned structure of PEX326–373(C235S) as the search model [40] This produced a clear solution and unambiguous difference electron density for the bound PEX19 peptide (supplemental Fig. S3) and regions of sPEX3 that had not been included in the search model. Coordinates and structure factors have been deposited with the RCSB Protein Data Bank (accession code 3MK4)

RESULTS

Interfaces between the helices are mostly hydrophobic and

DISCUSSION