MOLECULAR BASIS FOR SYK SIGNALING REVEALED BY ITS TANDEM SH2 DOMAIN/ITAM BOUND STRUCTURE.

Klaus Fütterer1, Richard A. Grucza¹, Andrew C. Chan² and Gabriel Waksman<sup>1

1</sup>Department of Biochemistry and Molecular Biophysics
²Department of Pathology, Washington University School of Medicine, Campus Box 8231, 660 South Euclid Av., Saint Louis, MO 63110, USA.

Syk and the homologous ZAP-70 kinases belong to a separate family of protein tyrosine kinases that play essential roles in antigen and antibody receptor function in several hematopoietic cell lines. The absence of either ZAP-70 or Syk causes severe deficiencies in signaling through immune receptors. Syk and ZAP-70 share many structural and functional features, notably two adjacent N-terminal SH2 domains, which mediate localization of the kinases to the membrane through receptor encoded dually tyrosine phosphorylated motifs, known as ITAMs. Yet the more ubiquitous nature of expression of Syk versus ZAP-70 has suggested that Syk may accommodate a greater variety of peptide targets to mediate its function, potentially including activation through SH2 domain binding to an individual pYxxL/I motif.

The crystal structure of the tandem-SH2 domain of Syk tyrosine kinase bound to a dually phosphorylated ITAM-ligand, was solved by single isomorphous replacement. Diffraction quality crystals have been obtained by the hanging drop vapour diffusion method and were in space group P2₁, comprising 6 molecules per asymmetric unit. X-ray diffraction data complete to 3.0 Å were recorded at the synchrotron and initial phases were determined on the basis of a single platinum derivative to 4 Å resolution, exploiting both the isomorphous and the anomalous signal. Phases could be improved to the level of an interpretable electron density map by non-crystallographic symmetry averaging and phase extension to 3.0 Å.

With 6 molecules present in the asymmetric unit, this structure reveals a previously unkown flexibility in the orientation of the two SH2 domains with respect to each other. Moreover, the C-terminal phosphotyrosine-binding site of Syk is shown to differ significantly from that of ZAP-70, suggesting two independently functional SH2 domains for Syk. The conformational flexibility and structural independence of the SH2 modules of Syk likely provides the molecular basis for the more ubiquitous involvement of Syk in a variety of immune or non-immune signal transduction pathways. New insight is also gained into ITAM recognition by Syk-family kinases.