1kc2
structure of the triple (Lys(beta)D3Ala, Asp(beta)C8Ala, AspCD2Ala) mutant of the Src SH2 domain bound to the PQpYEEIPI peptide
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OverviewOverview
Src Homology (SH2) domains play critical roles in signaling pathways by, binding to phosphotyrosine (pTyr)-containing sequences, thereby recruiting, SH2 domain-containing proteins to tyrosine-phosphorylated sites on, receptor molecules. Investigations of the peptide binding specificity of, the SH2 domain of the Src kinase (Src SH2 domain) have defined the EEI, motif C-terminal to the phosphotyrosine as the preferential binding, sequence. A subsequent study that probed the importance of eight, specificity-determining residues of the Src SH2 domain found two residues, which when mutated to Ala had significant effects on binding: Tyr beta D5, and Lys beta D3. The mutation of Lys beta D3 to Ala was particularly, intriguing, since a Glu to Ala mutation at the first (+1) position of the, EEI motif (the residue interacting with Lys beta D3) did not significantly, affect binding. Hence, the interaction between Lys beta D3 and +1 Glu is, energetically coupled. This study is focused on the dissection of the, energetic coupling observed across the SH2 domain-phosphopeptide interface, at and around the +1 position of the peptide. It was found that three, residues of the SH2 domain, Lys beta D3, Asp beta C8 and AspCD2, (altogether forming the so-called +1 binding region) contribute to the, selection of Glu at the +1 position of the ligand. A double (Asp beta, C8Ala, AspCD2Ala) mutant does not exhibit energetic coupling between Lys, beta D3 and +1 Glu, and binds to the pYEEI sequence 0.3 kcal/mol tighter, than the wild-type Src SH2 domain. These results suggest that Lys beta D3, in the double mutant is now free to interact with the +1 Glu and that the, role of Lys beta D3 in the wild-type is to neutralize the acidic patch, formed by Asp beta C8 and AspCD2 rather than specifically select for a Glu, at the +1 position as it had been hypothesized previously. A triple mutant, (Lys beta D3Ala, Asp beta C8Ala, AspCD2Ala) has reduced binding affinity, compared to the double (Asp beta C8Ala, AspCD2Ala) mutant, yet binds the, pYEEI peptide as well as the wild-type Src SH2 domain. The structural, basis for such high affinity interaction was investigated, crystallographically by determining the structure of the triple (Lys beta, D3Ala, Asp beta C8Ala, AspCD2Ala) mutant bound to the octapeptide, PQpYEEIPI (where pY indicates a phosphotyrosine). This structure reveals, for the first time contacts between the SH2 domain and the -1 and -2, positions of the peptide (i.e. the two residues N-terminal to pY). Thus, unexpectedly, mutations in the +1 binding region affect binding of other, regions of the peptide. Such additional contacts may account for the high, affinity interaction of the triple mutant for the pYEEI-containing, peptide.
About this StructureAbout this Structure
1KC2 is a Single protein structure of sequence from Rous sarcoma virus with CO as ligand. Active as Transferase, with EC number and 2.7.10.2 2.7.10.1 and 2.7.10.2 Full crystallographic information is available from OCA.
ReferenceReference
Dissection of the energetic coupling across the Src SH2 domain-tyrosyl phosphopeptide interface., Lubman OY, Waksman G, J Mol Biol. 2002 Feb 15;316(2):291-304. PMID:11851339
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