Crystal structure of the conserved N-terminal domain of the peroxisomal matrix protein import receptor, Pex14p.
Pex14p is a central component of the peroxisomal protein import machinery, in which the conserved N-terminal domain mediates dynamic interactions with other peroxins including Pex5p, Pex13p, and Pex19p. Here, we report the crystal structure of the conserved N-terminal domain of Pex14p with a three-helix bundle. A hydrophobic surface is composed of the conserved residues, of which two phenylalanine residues (Phe-35 and Phe-52) protrude to the solvent. Consequently, two putative binding pockets suitable for recognizing the helical WXXXF/Y motif of Pex5p are formed on the surface by the two phenylalanine residues accompanying with positively charged residues. The structural feature agrees well with our earlier findings where F35A/L36A and F52A/L53A mutants were impaired in the interactions with other peroxins such as Pex5p and Pex13p. Pex14p variants each with Phe-to-Ala mutation at positions 35, 52, and 35/52, respectively, were defective in restoring the impaired peroxisomal protein import in pex14 Chinese hamster ovary mutant ZP161 cells. Moreover, in GST pull-down assays His(6)-Pex5pL bound only to GST-Pex14p(25-70), not to any of GST-Pex14p(25-70)F35A, GST-Pex14p(25-70)F52A, and GST-Pex14p(25-70)F35A/F52A. Endogenous Pex5p was recruited to FLAG-Pex14p on peroxisomes in vivo but barely to FLAG-Pex14pF35A, FLAG-Pex14pF52A, and FLAG-Pex14pF35A/F52A. Collectively, Phe-35 and Phe-52 are essential for the Pex14p functions, including the interaction between Pex14p and Pex5p.
Su
Jian-Rong
JR
Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Takeda
Kazuki
K
Tamura
Shigehiko
S
Fujiki
Yukio
Y
Miki
Kunio
K
D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry*; D000074435:Peroxisome-Targeting Signal 1 Receptor; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry*; D012997:Solvents
D008024:Ligands; D008565:Membrane Proteins; D000074435:Peroxisome-Targeting Signal 1 Receptor; C499194:Pex14 protein, rat; D018160:Receptors, Cytoplasmic and Nuclear; D012097:Repressor Proteins; D012997:Solvents