Structural basis for the binding of high affinity phosphopeptides to Stat3

Abstract

Signal transducer and activator of transcription 3 (Stat3) is constitutively active in a number of cancers where it participates in aberrant transcription of prosurvival, cell cycling, and angiogenesis genes. Since Stat3 initiates its signaling activity through binding of its SH2 domain to phosphotyrosine residues on cell surface receptors, inhibitors targeting this region of the protein are potential chemotherapeutic agents. To date, no NMR or X-ray crystallographic structures of high-affinity phosphopeptides complexed with the Stat3 SH2 domain are available to aid in the development of peptidomimetic antagonists. Examination of the crystal structures of several STAT proteins and the complex of Stat1 with Ac-pTyr-Asp-Lys-Pro-His-NH 2 led to a hypothesis that the specificity determinant for Stat3, glutamine at position pY+3 in pTyr-Xxx-Xxx-Gln sequences, resides in a unique pocket on the protein surface at the juncture of the third strand of the central β-sheet and a unique, STAT specific α-helix. Docking of Ac-pTyr-Leu-Pro-Gln-NHBu to the SH2 domain of Stat3 using molecular modeling showed that the Gln binds tightly in this pocket and participates in a network of hydrogen bonds. Novel interactions between the peptide main chain and the protein were also discovered. Phosphopeptide structure-affinity studies using unnatural amino acids and glutamine derivatives provide evidence for the peptide-protein interactions revealed by the model and lend support to the binding hypothesis. © 2007 Wiley Periodicals, Inc.