Thermodynamic studies of tyrosyl-phosphopeptide binding to the SH2 domain of p56lck.
The interaction between SH2 domains and tyrosine-phosphorylated proteins is essential in several cytosolic signal transduction pathways. Here we report thermodynamic studies of the interaction of the p56lck (lck) SH2 domain with several phosphopeptides, using the technique of isothermal titration calorimetry (ITC). This is the first report of the use of ITC to study SH2 domain binding reactions. The free energy of binding of the SH2 domain of lck to a phosphopeptide corresponding to the autoregulatory C-terminus of the protein (pY505) was found to be similar to that measured for a phosphopeptide modeled on the C-terminus of the epidermal growth-factor receptor (delta G degrees approximately -7.0 kcal mol-1 at pH 6.8), although significant differences in the enthalpy and entropy were observed. Binding of a phosphopeptide modeled on the C-terminus of p185neu was weaker (delta G degrees approximately -5.4 kcal mol-1 at pH 6.8). Lowering the pH to 5.5 reduced the binding affinity of pY505 by approximately 1 order of magnitude. We ascribe this to the protonation of a histidine side chain in the SH2 domain (H180), which is involved in a hydrogen-bonding network that optimizes the binding site geometry. No difference in affinity was observed between portions of lck corresponding to the SH3-SH2 (residues 63-228) and SH2 (residues 123-228) domains for the pY505 peptide. We also studied the effect upon pY505 peptide binding of mutations at two highly conserved arginine residues in the lck SH2 domain (R134 and R154).(ABSTRACT TRUNCATED AT 250 WORDS)
Lemmon
M A
MA
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
Ladbury
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D003001:Cloning, Molecular; D017124:Conserved Sequence; D066246:ErbB Receptors / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008214:Lymphocytes / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry* / Q000378:metabolism; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000031:analogs & derivatives*
D010446:Peptide Fragments; D010748:Phosphopeptides; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)