Interactions between Cdc42 and the scaffold protein Scd2: requirement of SH3 domains for GTPase binding.
The multi-domain protein Scd2 acts as a scaffold upon which the small GTPase Cdc42 (cell division cycle 42), its nucleotide-exchange factor Scd1 and the p21-activated kinase Shk1 assemble to regulate cell polarity and the mating response in fission yeast. In the present study, we show using isothermal titration calorimetry that Scd2 binds two molecules of active GTP-bound Cdc42 simultaneously, but independently of one another. The two binding sites have significantly different affinities, 21 nM and 3 microM, suggesting that they play distinct roles in the Shk1 signalling network. Each of the Cdc42-binding sites includes one of the SH3 (Src homology 3) domains of Scd2. Our data indicate that complex formation does not occur in a conventional manner via the conserved SH3 domain ligand-binding surface. Neither of the isolated SH3 domains is sufficient to interact with the GTPase, and they both require adjacent regions to either stabilize their conformations or contribute to the formation of the Cdc42-binding surface. Furthermore, we show that there is no evidence for an intramolecular PX-SH3 domain interaction, which could interfere with SH3 domain function. This work suggests that SH3 domains might contribute directly to signalling through small GTPases and thereby adds another aspect to the diverse nature of SH3 domains as protein-protein-interaction modules.
Wheatley
Edward
E
Division of Protein Structure, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK.
Rittinger
Katrin
K
D019943:Amino Acid Substitution; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry*; D015398:Signal Transduction; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry*; D018909:src Homology Domains
D018797:Cell Cycle Proteins; D029702:Schizosaccharomyces pombe Proteins; C089770:scd2 protein, S pombe; D020764:cdc42 GTP-Binding Protein