A photoaffinity scan maps regions of the p85 SH2 domain involved in phosphoprotein binding.
Src homology 2 (SH2) domains are modular phosphotyrosine binding pockets found within a wide variety of cytoplasmic signaling molecules. Here we develop a new approach to analyzing protein-protein interfaces termed photoaffinity scanning, and apply the method to map regions of the phosphatidylinositol 3-kinase p85 SH2 domain that participate in phospho-protein binding. Each residue except phosphotyrosine (pY) within a tightly binding, IRS-1-derived phosphopeptide (GNGDpYMPMSPKS) was substituted with the photoactive amino acid, benzoylphenylalanine (Bpa). Whereas most substitutions had little effect on binding affinity, Bpa substitution of either Met (+1 and +3 with respect to pY) reduced affinity 50-100-fold to confirm their importance in the pYMXM recognition motif. In three cases photolysis of SH2 domain/Bpa phosphopeptide complexes led to cross-linking of > 50% of the SH2 domain; cross-link positions were identified by microsequence, amino acid composition, and electrospray mass spectrometric analyses. Bpa-1 cross-links within alpha-helix I, whereas Bpa+1 and Bpa+4 cross-link the SH2 domain within the flexible loop C-terminal to alpha-helix II. Moreover, cross-linking at any position prevents SH2 domain cleavage at a trypsin-sensitive site within the flexible loop between beta-strands 1 and 2. Therefore, at least three distinct SH2 regions in addition to the beta-sheet participate in phosphoprotein binding; the loop cross-linked by phosphopeptide residues C-terminal to pY appears to confer specificity to the phosphoprotein/SH2 domain interaction.
Williams
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KP
Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02215.
Shoelson
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SE
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010770:Phosphotransferases / Q000737:chemistry / Q000378:metabolism*; D010777:Photochemistry; D011485:Protein Binding; D017510:Protein Folding; D016392:Proto-Oncogene Proteins pp60(c-src)
D003432:Cross-Linking Reagents; D010750:Phosphoproteins; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D016392:Proto-Oncogene Proteins pp60(c-src)