Skip to main content
. 2010 Jul 13;9(11):2391–2404. doi: 10.1074/mcp.M110.001586

Fig. 6.

Fig. 6.

Ligands are distinguished by both permissive and non-permissive residues. A, three pYXXPX consensus peptides that were all array-negative for the Crk SH2 were chosen for combinatorial mutagenesis to probe the effects of manipulating permissive and non-permissive residues on binding selectivity. The changes made at each position are summarized to the left of the blots and highlighted as red letters within the sequence to the right. Peptides were synthesized as described and probed with 250 nm GST-SH2 domains of Crk or Brk. In some cases, the equilibrium dissociation constants were calculated from fluorescence polarization saturation binding experiments in solution and are noted to confirm the binding results indicated on the arrays. N.D. indicates not determined. The three parent peptides fail to bind the Crk SH2, but two of the three bind the Brk SH2 with submicromolar affinities. At positions +1, +2, and +4 with respect to Tyr(P), mutations were made based on differences between the OPAL pYXXP blots probed with Crk and Brk SH2 domains (see B). Manipulation of residues that negatively impact Crk SH2 binding results in peptides that are able to interact with both the Crk and Brk SH2 domains. When combined with substitution of +2 Asp for Glu that is non-permissive for Brk binding this switches selectivity to the Crk SH2 and away from the Brk SH2. B, relative differences between the OPAL pYXXP blots probed with Crk and Brk SH2 domains highlight specific residues at each of the +1, +2, and +4 positions that may distinguish binding between these two SH2 domains. C, permissive and non-permissive residue contributions that distinguish binding between Crk and Brk are illustrated for various peptides in A. Black arrows and red lines indicate permissive and non-permissive factors, respectively. The weight of the line indicates the relative intensity of these factors. The text size for each SH2 domain indicates the preference for that particular peptide to a particular SH2 domain (large text, stronger preference; small text, weak preference).