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.

Figure 1

NMR titrations of Pex14(N) with Pex19 and Pex5 peptides. (A) Schematic overview of the domain composition of human Pex5, Pex19 and Pex14. The N-terminal domain of Pex14 is coloured green, the binding motifs are shown in brown (Pex19) and gold (Pex5), respectively. (B) ¹H,¹⁵N correlation spectra of ¹⁵N-labelled recombinant Pex14(N) free (black), and in complex with Pex19 (residues 66–77) (brown; left) and Pex5 (gold; right) (residues 116–124). (C) NMR chemical shift changes (Δδ=(δ_15N²+δ_1H²)^1/2) of Pex14(N) in the presence of saturated concentrations of the Pex5 (gold) and the Pex19 (brown) ligands. Secondary structure elements are indicated underneath. (D) ¹⁵N-labelled recombinant Pex14(N) (black) was titrated with Pex19(66–77) (brown) to the point of saturation and was then cross-titrated with Pex5(116–124) (gold).

Figure 2

Solution structures of the Pex14(N)–Pex5 and Pex14(N)–Pex19 complexes. (A) Stereo view of the backbone atoms of Pex14 (residues 20–76) in complex with Pex5(108–127). An NMR ensemble of the 10 lowest-energy structures (out of 100 calculated) is shown. Secondary structure elements in Pex14 (helices α1, α2, α3 and the helical linker connecting α1 and α2) are coloured in green. The peptide is shown in gold. (B) Ribbon diagram of the lowest-energy structure in (A). (C) Superposition of the backbone atoms of Pex14 (residues 19–76) in complex with Pex19(66–77). The ensemble shows 10 lowest-energy structures (out of 100 calculated). The peptide is shown in brown. (D) Ribbon presentation of the lowest-energy structure in (C).

Figure 3

Electrostatic surface representation of the Pex14–Pex5 and Pex14–Pex19 complexes. The Pex14(N)–Pex5 and Pex14(N)–Pex19 complex structures are shown in the top and bottom rows, respectively. (A) View onto the ligand-binding surface. The Pex5 and Pex19 ligand helices are shown as transparent ribbons. Blue and red colours indicate positive and negative electrostatic surface potential in Pex14. Positively charged residues in Pex14(N) surrounding the binding interface are labelled. (B) Ribbon representation of the Pex14(N)–Pex5 (top) and Pex14(N)–Pex19 complexes. The aromatic residues in the two ligands are shown and labelled. Pex14(N), Pex5 and Pex19 are coloured in green, gold and brown, respectively. (C) Surface representation of the two complexes shown in the same orientation as in (B). Blue and red colours indicate positive and negative electrostatic surface potential, respectively. (D) Surface representation of the Pex5 (top) and Pex19 (bottom) ligands bound to Pex14, as viewed from the Pex14 interaction surface.

Figure 4

Structural details of the Pex14–Pex5 and Pex14–Pex19 interactions. Ribbon (left), surface (middle) and ensemble (right) representations of the molecular interface between Pex14 and Pex5(108–127) (A) and Pex19(66–77) (B). Left: Pex14 is shown in grey, the Pex5 and Pex19 peptides are coloured gold and brown, respectively. Residues showing intermolecular NOEs are shown in stick representation. Pex14 side chains are coloured in green, peptide backbone and side chains are in gold (Pex5) or brown (Pex19). Positively charged Pex14(N) residues are labelled white. An intermolecular salt bridge between Pex14(N) Lys56 and Pex5 Glu121 is indicated by a dashed red line. Labelling of Pex14 residues that were altered for mutational analysis are underlined. (C) Sequences of Pex5 and Pex19 peptide ligands used for binding and structural studies. The peptide fragments used for structure determination are shown with grey background. The Pex5 and Pex19 sequences are aligned based on the structural analysis and are shown with opposite directionality. Smaller Pex5 peptides are indicated by the lines above the sequence (see Supplementary Table 1). Pex5 Glu121, which forms an electrostatic contact with Pex14 Lys56, is highlighted in red. Conserved hydrophobic residues are highlighted in green, the two critical aromatic residues in both peptide motifs are marked with an asterisk.

Figure 5

Mutational analysis of Pex14 and its ligands. N-terminal Pex14 was tested for interactions with variants of Pex5 (A) and Pex19 (B) peptides. Peptides comprising systematic variations of Pex5 (residues 113–127; ALSENWAQEFLAAGD) and Pex19 (residues 66–80; SQEKFFQELFDSELA) ligands were synthesised on cellulose membranes and incubated with purified His₆-Pex14(1–80). Bound Pex14 was visualised immunochemically with monoclonal anti-His₆ antibodies. Spots with reduced intensities represent peptides with reduced binding affinities for Pex14. Amino acids that retain the interaction are shown by their single letter code on the right, where the letter size indicates the relative contribution to the interaction at each sequence position. Green and magenta colours indicate hydrophobic or polar/small residues. Red and blue colours display negatively or positively charged residues, respectively.

Figure 6

In vitro and in vivo effects of single-site mutations within Pex14(N). (A) Left: Pex14 Phe52 and Lys56 are critical for Pex19 binding. His-tagged Pex14(N) variants harbouring the indicated mutations were expressed in E. coli, purified, incubated at two different concentrations (1 μM, upper rows; 45 nM, lower row) with immobilised Pex5(113–127) and Pex19(66–80) peptides and detected by anti-Pex14 antibodies. Right: The location of the residues used for the mutational analysis is schematically indicated on the Pex14(N) structure. (B, C) Full-length Pex14 mutants show various patterns of cellular staining. (B) All single-point mutations lead to partial mitochondrial mislocalisation of full-length Pex14. A Zellweger patient Pex14-deficient fibroblast cell line was transfected with plasmids encoding Pex14 full-length proteins and analysed by immunofluorescence microscopy using antibodies against Pex14 (green, Alexa Fluor 488) and against a mitochondrial marker protein TRAP1 (red, Alexa Fluor 594). Most of the cells expressing Pex14(K56E) and Pex14(F52W), shown as representative examples, display a congruent pattern (yellow colour), showing mitochondrial mislocalisation of Pex14. (C) Pex14 variants can also associate with peroxisomes. Pex14-deficient fibroblasts were co-transfected with plasmids encoding the peroxisomal marker protein GFP-PTS1 and Pex14(K56E) and analysed by fluorescence microscopy (green, EGFP) and immunofluorescence microscopy using antibodies against Pex14 (red, Alexa Fluor 594), respectively. About 10% of the double-transfected cells exhibit a congruent punctuate staining pattern (yellow colour), showing peroxisomal localisation of this Pex14 variant. Bar: 10 μm. (D) Quantitative analysis of the mutational effects on the subcellular localisation of Pex14 has been performed for each mutation. Between 75 and 100 double-transfected cells expressing both GFP-PTS1 and one of the Pex14 mutants were inspected for particulate co-localisation of both proteins. Numbers of cells with a peroxisome localisation of Pex14 are given in percent for each mutant.