These structural studies, combined with biochemical evidence [5,6], imply that pY-peptide binding and disruption of the intramolecular N-SH2 C PTP interface, and hence activation of phosphatase activity, are normally coupled. helical and extended regions of the Tyr66 / map. Changing the Tyr66 backbone conformation from extended to left-handed helical induces a closed-to-open transition in the cleft, and the reverse change in backbone conformation induces the reverse, open-to-closed transition. In the open-cleft state, weak Vinburnine solvent-exposed interactions involving the sidechains of Tyr66, Asp40, Lys55, and Gln57 serve to anchor the Mouse monoclonal to Myostatin Tyr66 sidechain to the surface of the protein and away from the binding cleft entrance, thereby facilitating pY-peptide access to the binding cleft. Conclusion The simulations point to a regulatory role for Tyr66 and surrounding residues in SHP-2 function: mutations at Tyr66, Asp40, Lys55, and/or Gln57 are predicted to break the switching mechanism and negatively impact pY-peptide binding. This in turn would Vinburnine interfere with cellular localization and the coupled SHP-2 phosphatase activity. The structurally well-defined binding cleft conformations resulting from the switch-like transition suggest the possibility of applying structure-based methods to develop inhibitors of N-SH2 pY-peptide binding to serve as research tools for signal transduction and precursors to therapeutics for SHP-2-related diseases. Background The ubiquitously expressed vertebrate non-transmembrane protein tyrosine phosphatase SHP-2 takes part in intracellular signal transduction induced by a variety of environmental cues and plays an important role in diverse cellular processes [1-3]. The SHP-2 protein consists of 593 residues, with the first 213 residues comprising two SRC homology 2 domains (SH2) and the remainder a protein tyrosine phosphatase domain (PTP) and the C-terminal tail. The 2 2.0 ? X-ray crystal structure of SHP-2 [4] reveals that the PTP catalytic site is blocked by the formation of an intramolecular protein C protein interface between PTP and the N-terminal SH2 domain (N-SH2), thereby providing a structural explanation for the low baseline SHP-2 tyrosine phosphatase activity [5,6]. In addition to self-inhibiting catalysis, N-SH2, like the second (C-terminal) SH2 domain (C-SH2), has the capacity to selectively bind phosphotyrosine (pY) peptides of a particular sequence [7,8]. Thus, SHP-2 can be recruited to different regions of the cell via the interaction of its N-SH2 or C-SH2 domains with particular pY-peptides localized in these different regions. Crystal structures of N-SH2 alone, both with and without bound pY-peptides [9,10], show an open pY-peptide binding cleft between the EF loop (Tyr66-Gly67-Gly68) and the BG loop (Lys89-Glu90-Lys91-Asn92). This is in contrast to the crystal structure of the complete self-inhibited protein wherein the PTP-bound N-SH2’s peptide-binding cleft is closed due to EF-loop motion and therefore unable to accommodate a pY-peptide (Figure ?(Figure1).1). These structural studies, combined with biochemical evidence [5,6], imply that pY-peptide binding and disruption of the intramolecular N-SH2 C PTP interface, and hence activation of phosphatase activity, are normally coupled. Mutations at the protein C protein interface that disrupt the interface leading to the active form of the protein are associated with the congenital disease Noonan syndrome as well as childhood leukemias [11-13]. Accordingly, it may be anticipated that small-molecule inhibitors of either SHP-2 SH2 pY-peptide binding or PTP activity have the Vinburnine potential to serve as novel research tools and as potential precursors to therapeutics. To better understand the biochemical properties of the N-SH2 domain with the aim of developing N-SH2-specific inhibitors, we have used molecular dynamics (MD) simulations to investigate the closed-to-open transition of the N-SH2 pY-peptide binding cleft. Our data suggest that Tyr66 plays an important role in this conformational switching. Open in a separate window Figure 1 SHP-2 N-SH2 crystal structures. A) Crystal structure of isolated N-SH2 [PDB:1AYD] [9] showing the open pY-peptide binding cleft formed by the EF (red) and BG loops. B) Crystal structures of isolated N-SH2 [PDB:1AYD] (red), and N-SH2 in the full SHP-2 protein [PDB:2SHP] [4] (yellow). Dashed lines connect the C atoms of Gly67 and Asn92. Also shown are pY-peptides from N-SH2 C pY-peptide.
