Simultaneous Binding of Two Peptidyl Ligands by a Src Homology 2 Domain

Abstract

Src homology 2 (SH2) domains mediate protein-protein interactions by recognizing phosphotyrosine (pY)-containing sequences of target proteins. In all of the SH2 domain-pY peptide interactions described to date, the SH2 domain binds to a single pY peptide. Here, determination of the co-crystal structure of the N-terminal SH2 domain of phosphatase SHP-2 bound to a class IV peptide (VIpYFVP) revealed a noncanonical 1:2 (protein/peptide) complex. The first peptide binds in the canonical manner with its pY side chain inserted in the usual binding pocket, while the second pairs up with the first to form two anti-parallel β-strands that extend the central β sheet of the SH2 domain. This unprecedented binding mode was confirmed in solution phase by NMR experiments and shown to be adopted by pY peptides derived from cellular proteins. Site-directed mutagenesis and surface plasmon resonance studies revealed that the binding of the first peptide is pY dependent, but phosphorylation is not required for the second peptide. Our findings suggest a potential new function for the SH2 domain as a molecular clamp to promote dimerization of signaling proteins.

New structural and functional insight into the regulation of Ras

“…PTP catalysis is activated when the N-SH2 domain is bound by a tyrosyl-phosphorylated protein ligand [72,73]. Co-crystal structures of ligand-bound Shp2 N-SH2 domains [74] illustrate that the tyrosyl-phosphorylated protein ligand sits in an extended molecular groove that spans the length of the N-SH2 domain. This domain can accommodate four classes of tyrosyl-phosphorylated peptides for effective N-SH2 interaction [75].…”

Phosphotyrosine recognition domains: the typical, the atypical and the versatile

“…A recent model proposed that activation of the SHP-1 phosphatase requires binding of a pTyr ligand to the SH2 domains and a subsequent large structural rearrangement of the C-terminal SH2 domain to allow dissociation of the N-terminal SH2 domain from the catalytic pocket [89]. A further structural study illustrated that the N-terminal SH2 domain of SHP-2 possesses the ability to act either in the single-peptide or in the double-peptide binding mode, depending on the peptide sequence [90]. The single-peptide binding mode follows a canonical ligand binding mechanism, i.e., binding to an open cleft between the EF and BG loops (Figure 2B).…”

“…In this conformation, the BG loop contacts the EF loop and inhibits ligand binding. ( B ) The 1:1 binding mode (PDB ID: 3TL0) [90]. The bound LNpYAQLW peptide is coloured yellow.…”

“…The C-terminal region of the single peptide binds to a cleft between the EF and BG loops. ( C ) The 1:2 binding mode, in which the two identical peptides, with a sequence VIpYFVPL, form a short, antiparallel β-sheet and bind to a single SH2 domain (PDB ID: 3TKZ) [90]. The BG loop is positioned to accommodate the two peptides.…”

Differential recognition of syk‐binding sites by each of the two phosphotyrosine‐binding pockets of the Vav SH2 domain

“…This pocket is the site associated with specific recognition of the SH2 domain for its binding partner and interacts with amino acids C‐terminal to phosphotyrosine. Recognition determinants beyond these canonical binding interactions include binding contacts that extend N‐terminal to phosphotyrosine, phosphorylation‐independent binding, and simultaneous binding of two peptides …”