Differentiation of peptide molecular recognition by phospholipase Cγ‐1 Src homology‐2 domain and a mutant Tyr phosphatase PTP1b C215S

• Published in: Protein science 1995-01, Vol.4 (1), p.13-20
• Main Authors: Maclean, Derek, Sefler, Andrea M., Zhu, Guochang, Decker, Stuart J., Saltiel, Alan R., Singh, Juswinder, Mcnamara, Dennis, Dobrusin, Ellen M., Sawyer, Tomi K.
• Format: Article
• Language: English
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1002/pro.5560040103

Activated epidermal growth factor receptor (EGFR) undergoes autophosphorylation on several cytoplasmic tyrosine residues, which may then associate with the src homology‐2 (SH2) domains of effector proteins such as phospholipase Cγ‐1 (PLCγ‐1). Specific phosphotyrosine (pTyr)‐modified EGFR fragment peptides can inhibit this intermolecular binding between activated EGFR and a tandem amino‐ and carboxy‐terminal (N/C) SH2 protein construct derived from PLCγ‐1. In this study, we further explored the molecular recognition of phosphorylated EGFR 988–998 (Asp‐Ala‐Asp‐Glu‐pTyr‐Leu‐Ile‐Pro‐Gln‐Gln‐Gly, I) by PLCγ‐1 N/C SH2 in terms of singular Ala substitutions for amino acid residues N‐ and C‐terminal to the pTyr (P site) of phosphopeptide I. Comparison of the extent to which these phosphopeptides inhibited binding of PLCγ‐1 N/C SH2 to activated EGFR showed the critical importance of amino acid side chains at positions P +2 (Ile 994 ), P +3 (Pro 995 ), and P +4 (Gln 996 ). Relative to phosphopeptide 1, multiple Ala substitution throughout the N‐terminal sequence, N‐terminal truncation, or dephosphorylation of pTyr each resulted in significantly decreased binding to PLCγ‐1 N/C SH2. These structure‐activity results were analyzed by molecular modeling studies of the predicted binding of phosphopeptide 1 to each the N‐ and C‐terminal SH2 domains of PLCγ‐1. In the case of the PLCγ‐1 C‐terminal SH2, the coordinates of a phosphopeptide SH2 domain complex recently determined by NMR studies (Pascal SM, et al., 1994, Cell 77 :461–472) were used directly, whereas the N‐terminal SH2 domain model was developed from homology model building using the NMR coordinates of the PLCγ‐1 C‐terminal SH2 as a template. Relative to PLCγ‐1 N/C SH2 domain molecular recognition, the binding affinities of phosphorylated EGFR 988–998 and its Ala‐scan analog series to a catalytically inert mutant protein tyrosine phosphatase, PTPlb C215S , showed only a slight loss in binding at the P +1 (Leu) site. Therefore, peptide molecular recognition by two functionally distinct signal transduction proteins, PLCγ‐1 and PTP1b, may be differentiated in terms of systematic chemical modifications of EGFR 988 _ 998 and comparative structure‐activity analysis.