Structural insights into the tyrosine phosphorylation–mediated inhibition of SH3 domain–ligand interactions

Balázs Merő,László Radnai,Gergő Gógl,Orsolya Tőke,Ibolya Leveles,Kitti Koprivanacz,Bálint Szeder,Metta Dülk,Gyöngyi Kudlik,Virág Vas,Anna Cserkaszky,Szabolcs Sipeki,László Nyitray,Beáta G Vértessy,László Buday

doi:10.1074/jbc.ra118.004732

Abstract

Src homology 3 (SH3) domains bind proline-rich linear motifs in eukaryotes. By mediating inter- and intramolecular interactions, they regulate the functions of many proteins involved in a wide variety of signal transduction pathways. Phosphorylation at different tyrosine residues in SH3 domains has been reported previously. In several cases, the functional consequences have also been investigated. However, a full understanding of the effects of tyrosine phosphorylation on the ligand interactions and cellular functions of SH3 domains requires detailed structural, atomic-resolution studies along with biochemical and biophysical analyses. Here, we present the first crystal structures of tyrosine-phosphorylated human SH3 domains derived from the Abelson-family kinases ABL1 and ABL2 at 1.6 and 1.4 Å resolutions, respectively. The structures revealed that simultaneous phosphorylation of Tyr89 and Tyr134 in ABL1 or the homologous residues Tyr116 and Tyr161 in ABL2 induces only minor structural perturbations. Instead, the phosphate groups sterically blocked the ligand-binding grooves, thereby strongly inhibiting the interaction with proline-rich peptide ligands. Although some crystal contact surfaces involving phosphotyrosines suggested the possibility of tyrosine phosphorylation-induced dimerization, we excluded this possibility by using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and NMR relaxation analyses. Extensive analysis of relevant databases and literature revealed not only that the residues phosphorylated in our model systems are well-conserved in other human SH3 domains, but that the corresponding tyrosines are known phosphorylation sites in vivo in many cases. We conclude that tyrosine phosphorylation might be a mechanism involved in the regulation of the human SH3 interactome.

Highlights

WT and mutant SH3 domains of ABL kinases and the kinase domain of EphB1 receptor were expressed in E. coli Rosetta pLysS (Novagen) and Arctic Express (Agilent Technologies) cells, respectively
We recognized novel major tyrosine phosphorylation sites in the binding groove of SH3 domains and demonstrated that SH3 domain tyrosine phosphorylation strongly inhibits the interaction with proline-rich ligands due to the steric blocking of the ligand-binding groove by the phosphate groups
Based on the occurrence of phosphotyrosine residues in homologous positions of many human SH3 domains, this seems to be a widespread mechanism regulating a significant part of the SH3 interactome in cells

Summary

Introduction

WT and mutant SH3 domains of ABL kinases and the kinase domain of EphB1 receptor were expressed in E. coli Rosetta pLysS (Novagen) and Arctic Express (Agilent Technologies) cells, respectively. Cells were grown in 2YT medium up to A600 ϭ 0.7 at 37 °C and induced overnight with 0.5 mM isopropyl 1-thio-␤-D-galactopyranoside at 16 °C. Cells were harvested by centrifugation and resuspended in 1 liter of minimal medium containing 1 g/liter [15N]NH4Cl and induced by 0.5 mM isopropyl 1-thio-␤-D-galactopyranoside at 37 °C for 4 h. Cells were resuspended in lysis buffer (50 mM Na2HPO4, 500 mM NaCl, 0.25 mM TCEP, pH 8.0) and subsequently lysed by lysozyme treatment, one freeze-thaw cycle, and sonication. Fractions containing the protein of interest were pooled and subsequently dialyzed against lysis buffer. His tags were cleaved by TEV protease digestion. Protein samples were dialyzed against Tris-buffered saline (TBS) supplemented with TCEP (20 mM Tris, 150 mM NaCl, 0.5 mM TCEP, pH 7.6), concentrated by ultrafiltration, aliquoted, and stored at Ϫ80 °

Results

Discussion

Conclusion