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The Src homology-2 (SH2) domains are modules of about 100 amino-acid residues
that are found in many intracellular signal-transduction proteins. They bind
phosphotyrosine-containing sequences with high affinity and specificity,
recognizing phosphotyrosine in the context of the immediately adjacent
polypeptide sequence. The protein p56lck (Lck) is a Src-like,
lymphocyte-specific tyrosine kinase. A phosphopeptide library screen has
recently been used to deduce an 'optimal' binding sequence for the Lck SH2
domain. There is selectivity for the residues Glu, Glu and Ile in the three
positions C-terminal to the phosphotyrosine. An 11-residue phosphopeptide
derived from the hamster polyoma middle-T antigen, EPQpYEEIPIYL, binds with an
approximately 1 nM dissociation constant to the Lck SH2 (ref. 17), an affinity
equivalent to that of the tightest known SH2-phosphopeptide complex. We report
here the high-resolution crystallographic analysis of the Lck SH2 domain in
complex with this phosphopeptide. Recent crystallographically derived structures
of the Src SH2 domain in complex with low-affinity peptides, which do not
contain the EEI consensus, and NMR-derived structures of unliganded Abl (ref.
19) and p85 (ref. 20) SH2 domains have revealed the conserved fold of the SH2
domain and the properties of a phosphotyrosine binding pocket. Our high-affinity
complex shows the presence of a second pocket for the residue (pY + 3) three
positions C-terminal to the phosphotyrosine (pY). The peptide is anchored by
insertion of the pY and pY + 3 side chains into their pockets and by a network
of hydrogen bonds to the peptide main chain. In the low-affinity
phosphopeptide/Src complexes, the pY + 3 residues do not insert into the
homologous binding pocket and the peptide main chain remains displaced from the
surface of the domain.
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