Structural basis for competitive interactions of Pex14 with the import receptors Pex5 and Pex19

Abstract

Protein import into peroxisomes depends on a complex and dynamic network of protein-protein interactions. Pex14 is a central component of the peroxisomal import machinery and binds the soluble receptors Pex5 and Pex19, which have important function in the assembly of peroxisome matrix and membrane, respectively. We show that the N-terminal domain of Pex14, Pex14(N), adopts a three-helical fold. Pex5 and Pex19 ligand helices bind competitively to the same surface in Pex14(N) albeit with opposite directionality. The molecular recognition involves conserved aromatic side chains in the Pex5 WxxxF/Y motif and a newly identified F/YFxxxF sequence in Pex19. The Pex14-Pex5 complex structure reveals molecular details for a critical interaction in docking Pex5 to the peroxisomal membrane. We show that mutations of Pex14 residues located in the Pex5/Pex19 binding region disrupt Pex5 and/or Pex19 binding in vitro. The corresponding full-length Pex14 variants are impaired in peroxisomal membrane localisation in vivo, showing that the molecular interactions mediated by the N-terminal domain modulate peroxisomal targeting of Pex14.

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

“…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). In contrast to typical Pex5 WXXX(F/Y) motifs, the identified FFXXXF of Pex19 binds with opposite directionality.…”

“…Pex14-(16 -80) and Pex5-(1-110) for NMR studies were expressed as fusion proteins with a His 6 tag followed by a tobacco etch virus cleavage site as described previously for Pex14-(16 -80) (16). 13 affinity chromatography followed by tobacco etch virus cleavage and removal of the His 6 tag by a second Ni 2ϩ affinity chromatography step.…”

“…The library representing Pex5-(1-343) consisted of 15-mer peptides with a 13-amino acid overlapping region that were immobilized on a cellulose membrane. The library was incubated with purified His 6 -tagged Pex14-(1-78) as described previously (16). Bound Pex14-(1-78) was immunodetected using monoclonal anti-His antibodies (Qiagen).…”

“…The major difference between the novel LVAEF and canonical WXXX(F/Y) motifs concerns the two first residues. Whereas in WXXX(F/Y) motifs only the tryptophan is invariant for interaction with Pex14 (12,16), in the monoaromatic 62 LVAEF 66 binding sequence, two hydrophobic residues Leu-62 and Val-63 are critical for Pex14 binding. However, the leucine residue in the new motif is interchangeable with the aromatic residues tryptophan, phenylalanine, and tyrosine (Fig.…”

“…To elucidate molecular details for the recognition of the novel Pex5 motif by Pex14-NTD, we determined the solution structure of the complex. The structure of the complex was determined based on the previously reported structure of Pex14-NTD (16,17), using a protocol that we have recently established (30), and included 47 intermolecular NOE-derived distance restraints between Pex14-NTD and Pex5-(57-71) ( Table 2). The Pex14-NTD adopts a three-helical bundle in which helices ␣1 and ␣2 constitute the main binding interface.…”