Mapping of synergistic components of weakly interacting protein-protein motifs using arrays of paired peptides.
Protein-protein recognition usually involves multiple interactions among different motifs that are scattered over protein surfaces. To identify such weak interactions, we have developed a novel double peptide synthesis (DS) method. This method allows us to map protein-protein interactions that involve two linear dis- continuous components from a polypeptide by the use of spatially addressable synergistic pairs of synthetic peptides. The DS procedure is based on the "SPOT" membrane-bound peptide synthesis technique, but to synthesize a mixture of two peptides, it uses both Fmoc (N-(9-fluorenyl)methoxycarbonyl))-alanine and Alloc-alanine at the first cycle. This allows their selective deprotection by either piperidine or tributyltin/palladium treatment, respectively. Using SPOT DS, we confirmed as a proof of principle that Elk-1 Ser(383) phosphorylation by ERK-2 kinase is stimulated by the presence of the Elk-1-docking domain. SPOT DS can also be used to dissect protein-protein motifs that define phosphatase substrate affinity. Using this technique, we identified three new regions in the insulin receptor that stimulate the dephosphorylation of the receptor by protein-tyrosine phosphatase (PTP) 1B and presumably increase the selectivity of PTP for this substrate. These data demonstrate that the SPOT DS technique allows the identification of non-linear weakly interacting protein motifs, which are an important determinant of protein kinase and phosphatase substrate specificity and of protein-protein interactions in general.
Espanel
Xavier
X
Serono Pharmaceutical Research Institute, Geneva 1228, Switzerland.
Wälchli
Sébastien
S
Rückle
Thomas
T
Harrenga
Axel
A
Huguenin-Reggiani
Martine
M
Hooft van Huijsduijnen
Rob
R
D020650:Combinatorial Chemistry Techniques; D004268:DNA-Binding Proteins; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D009154:Mutation; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1
D004268:DNA-Binding Proteins; C494112:ELK1 protein, human; D009842:Oligopeptides; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D012694:Serine; D011972:Receptor, Insulin; D019950:Mitogen-Activated Protein Kinase 1; C516607:PTPN1 protein, human; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D017027:Protein Tyrosine Phosphatases