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

Fig. 4.

Fig. 4.

Non-permissive factors modulate binding of Crk SH2 domain. A and B, OPAL blots fixing both the essential Tyr(P) and either Pro or Leu at the +3 position reveal a number of residues that are non-permissive for binding by the Crk SH2. C, a Clustal alignment of various Crk family members across multiple species. Residues within the SH2 domain that make contact with a high affinity ligand (pYAQP) based on Protein Data Bank code 1JU5 are highlighted in colors corresponding to the residues noted in the structure of the SH2 domain of Crk (Protein Data Bank code 1JU5) Highly conserved contacts are indicated (*). (D) modeled bound to a pYAVPR peptide to illustrate the contacts that contribute to certain permissive and non-permissive residues noted in Fig. 6. Tyr-60 of the Crk SH2 may contribute to the observed loss of binding to peptides containing charged residues at the +1 position of the ligand. A basic ridge created by Arg-88 at the end of a hydrophobic channel created by three Ile residues may disfavor Arg or His at +2, whereas Asp-91 may contribute to acidic residues being non-permissive at +4 as well as an observed preference for Arg at +4 among the physiological binding partners. E–G, quadrant plots representing permissive and non-permissive elements for Crk SH2 binding in a binary manner for the 192 physiological peptides on the InsR/IGF-1R/FGFR SPOT arrays. Each peptide is indicated by ♦. Peptides that contain permissive factors such as Pro or Leu at +3 are noted in the top half of the plot. Array-positive peptides reside in the upper left quadrant, whereas those that are apparently blocked from binding by non-permissive (NP) residues are shifted to the upper right quadrant. Any peptide lacking the critical permissive residue of Pro or Leu at +3 was clustered into the lower left quadrant. F and G, data points representing peptides are colored according to their predicted binding score according to Scansite (F) or SMALI (G), revealing that the majority of apparent false positives are attributable to non-permissive residues not fully accounted for in these models.