The peroxisomal membrane protein Pex13p shows a novel mode of SH3 interaction.
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 alpha-helical element in Pex5p is necessary and sufficient for SH3 interaction. Sup pressor analysis using Pex5p mutants located in this alpha-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.
Barnett
P
P
Department of Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
Bottger
G
G
Klein
A T
AT
Tabak
H F
HF
Distel
B
B
D000409:Alanine / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002455:Cell Division; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D011392:Proline / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013489:Suppression, Genetic; D020798:Two-Hybrid System Techniques; D018909:src Homology Domains
D008024:Ligands; D008565:Membrane Proteins; C101884:PEX13 protein, human; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D011392:Proline; D005982:Glutathione Transferase; D000409:Alanine