title: Characterization of the interaction between recombinant human peroxin Pex3p and Pex19p: identification of TRP-104 IN Pex3p as a critical residue for the interaction.
abstract: Proteins required for peroxisome biogenesis are termed peroxins. The peroxin Pex3p is a peroxisomal membrane protein (PMP), involved in peroxisomal membrane biogenesis. It acts as a docking receptor for another peroxin Pex19p, which is a specific carrier protein for newly synthesized PMPs. Here we have determined the physicochemical properties and binding manners of Pex3p-Pex19p interaction, in terms of the affinity, the stoichiometry, and the binding site in Pex3p. The cytosolic domain of human Pex3p was overproduced, using an Escherichia coli expression system and was highly purified by two chromatography steps. Gel filtration chromatography analyses and intrinsic tryptophan fluorescence titrations revealed that a one-to-one complex is formed between monomeric Pex3p and monomeric Pex19p. The tryptophan fluorescence spectrum of Pex3p showed a large 18-nm blue shift of the maximum emission wavelength by the binding of Pex19p. This result indicates that either one or two tryptophan residues of Pex3p (Trp-104 and Trp-224) are directly involved in binding to Pex19p. We investigated the binding activities of the wild-type and tryptophan mutants of Pex3p by pull-down assays and surface plasmon resonance analyses. As a result, the wild-type and the W104A and W104F mutants showed K(D) values of 3.4 nm, 1080 nm, and 66.2 nm, respectively. The affinity differences with mutation affected their peroxisome restoring activities in pex3 ZPG208 cells. These findings suggest that the indole ring of Trp-104 directly interacts with Pex19p to facilitate the specific peroxisomal translocation of the Pex19p-PMP complexes.
authors.affiliation: Department of Structural Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida Shimoadachi-cho, Sakyo-ku, Kyoto, Japan.
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Sato
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Shibata
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Nakano
authors.forename: Yuji
authors.initials: Y
authors.lastname: Matsuzono
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kashiwayama
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kobayashi
authors.forename: Yukio
authors.initials: Y
authors.lastname: Fujiki
authors.forename: Tsuneo
authors.initials: T
authors.lastname: Imanaka
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Kato
mesh_terms: D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D000074401:Peroxins; D020675:Peroxisomes / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*
chemical_list: D008074:Lipoproteins; D008565:Membrane Proteins; D000074401:Peroxins; C113138:Pex3 protein, human; D011994:Recombinant Proteins; C087343:PEX19 protein, human; D014364:Tryptophan