The peroxisomal membrane protein Pex13p shows a novel mode of SH3 interaction

• Published in: The EMBO journal 2000-12, Vol.19 (23), p.6382-6391
• Main Authors: Barnett, Phil, Bottger, Gina, Klein, André T.J., Tabak, Henk F., Distel, Ben
• Format: Article
• Language: English
• Subjects: Alanine - metabolism; Amino Acid Motifs; Amino Acid Sequence; Binding Sites; Binding, Competitive; Cell Division; Dose-Response Relationship, Drug; Escherichia coli - metabolism; Glutathione Transferase - metabolism; Ligands; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Peroxisome-Targeting Signal 1 Receptor; peroxisomes; Peroxisomes - metabolism; Pex13p; Pex5p; Proline - metabolism; Protein Binding; Protein Structure, Secondary; protein-protein interaction; Receptors, Cytoplasmic and Nuclear - chemistry; Receptors, Cytoplasmic and Nuclear - genetics; Receptors, Cytoplasmic and Nuclear - metabolism; Recombinant Fusion Proteins - metabolism; Sequence Homology, Amino Acid; SH3; src Homology Domains; Suppression, Genetic; Two-Hybrid System Techniques
• ISSN: 0261-4189; 1460-2075; 1460-2075
• DOI: 10.1093/emboj/19.23.6382

Src homology 3 (SH3) domains are small non‐catalytic protein modules capable of mediating protein–protein interactions by binding to proline‐X‐X‐proline (P‐X‐X‐P) motifs. Here we demonstrate that the SH3 domain of the integral peroxisomal membrane protein Pex13p is able to bind two proteins, one of which, Pex5p, represents a novel non‐P‐X‐X‐P ligand. Using alanine scanning, two‐hybrid and in vitro interaction analysis, we show that an α‐helical element in Pex5p is necessary and sufficient for SH3 interaction. Sup pressor analysis using Pex5p mutants located in this α‐helical element allowed the identification of a unique site of interaction for Pex5p on the Pex13p‐SH3 domain that is distinct from the classical P‐X‐X‐P binding pocket. On the basis of a structural model of the Pex13p‐SH3 domain we show that this interaction probably takes place between the RT‐ and distal loops. Thus, the Pex13p‐SH3–Pex5p interaction establishes a novel mode of SH3 interaction.