Calorimetric examination of high-affinity Src SH2 domain-tyrosyl phosphopeptide binding: Dissection of the phosphopeptide sequence specificity and coupling energetics

被引:74
作者
Bradshaw, JM [1 ]
Waksman, G [1 ]
机构
[1] Washington Univ, Sch Med, Dept Biochem & Mol Biophys, St Louis, MO 63110 USA
关键词
D O I
10.1021/bi982974y
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
SH2 domains are protein modules which interact with specific tyrosine phosphorylated sequences in target proteins. The SH2 domain of the Src kinase binds with high affinity to a tyrosine phosphorylated peptide containing the amino acids Glu, Glu, and lie (EEI) at the positions +1, +2, and +3 C-terminal to the phosphotyrosine, respectively. To investigate the degree, of selectivity of the Src SH2 domain for each amino acid of the EEI motif, the binding thermodynamics of a panel of substitutions at the +1 (Gln, Asp, Ala, Cry), +2 (Gln, Asp, Ala, Gly:), and +3 (Leu, Val, Ala, Gly) positions were examined using titration microcalorimetry. It was revealed that the Src SH2 domain is insensitive (Delta Delta G degrees less than or equal to 0.6 kcal/mol) to conservative substitutions at all three peptide positions. However, mutation to Ala resulted in moderate reductions in Delta G degrees, with the substitution at the +3 position showing the largest loss in affinity (Delta Delta G degrees = 1.4 kcal/mol), followed by the +2 (Delta Delta G degrees = 1.0 kcal/mol) and +1 (Delta Delta G degrees = 0.5 kcal/mol) positions. This hierarchy of binding was not reflected in the values of the heat capacity change, since only the peptide substituted to Ala at the +3 position showed a Delta C(p)degrees that was reduced in magnitude compared to wild-type. To assess the degree of cooperation upon binding (or coupling) between the amino acids of the EEI sequence, the binding of a series of singly, doubly, and triply Ala substituted phosphopeptides was examined and analyzed using double mutant cycles. It was revealed that the effects of the Ala substitutions on Delta G degrees were additive. However, nonadditive binding enthalpies were observed between the +1 Glu and +3 Ile, as well as the +2 Glu and +3 Ile, suggesting that communication occurs between residues of the EEI motif upon binding.
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页码:5147 / 5154
页数:8
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