J.M. Bradshaw and G. Waksman.
Calorimetric examination of high-affinity Src SH2 domain-tyrosyl
phosphopeptide binding: dissection of the phosphopeptide sequence
specificity and coupling energetics
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 Ile (EEI) at the positions +1, +2, and
+3 C-terminal to the phosphotyrosine, respectively. In order 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, Gly), +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 < 0.6 kcal/mol) to conservative
substitutions at all three peptide positions. However, mutation to Ala
resulted in moderate reductions in delta G, with the substitution at the +3
position showing the largest loss in affinity (delta-deltaG = 1.4 kcal/mol), followed by
the +2 (delta-delta G = 1.0 kcal/mol) and +1 (delta-delta G = 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 Cp that was reduced in magnitude compared to wild type. In order 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 deltaG
were additive. However, non-additive 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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