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

Balázs Mero, László Radnai, Gergo Gógl, Orsolya To ke, Ibolya Leveles, Kitti Koprivanacz, Bálint Szeder, Metta Dülk, Gyöngyi Kudlik, X. Virág Vas, Anna Cserkaszky, Szabolcs Sipeki, L. Nyitray, B. Vértessy, L. Buday

Research output: Contribution to journalArticle

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 Tyr 89 and Tyr 134 in ABL1 or the homologous residues Tyr 116 and Tyr 161 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 phosphoty-rosines 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.

Original languageEnglish
Pages (from-to)4608-4620
Number of pages13
JournalJournal of Biological Chemistry
Volume294
Issue number12
DOIs
Publication statusPublished - Jan 1 2019

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Phosphorylation
src Homology Domains
Tyrosine
Ligands
Proline
Signal transduction
Dimerization
Dynamic light scattering
Eukaryota
X ray scattering
Signal Transduction
Phosphotransferases
Crystal structure
Phosphates
Nuclear magnetic resonance
X-Rays
Databases
Peptides
Crystals
Proteins

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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Structural insights into the tyrosine phosphorylation–mediated inhibition of SH3 domain–ligand interactions. / Mero, Balázs; Radnai, László; Gógl, Gergo; To ke, Orsolya; Leveles, Ibolya; Koprivanacz, Kitti; Szeder, Bálint; Dülk, Metta; Kudlik, Gyöngyi; Virág Vas, X.; Cserkaszky, Anna; Sipeki, Szabolcs; Nyitray, L.; Vértessy, B.; Buday, L.

In: Journal of Biological Chemistry, Vol. 294, No. 12, 01.01.2019, p. 4608-4620.

Research output: Contribution to journalArticle

Mero, B, Radnai, L, Gógl, G, To ke, O, Leveles, I, Koprivanacz, K, Szeder, B, Dülk, M, Kudlik, G, Virág Vas, X, Cserkaszky, A, Sipeki, S, Nyitray, L, Vértessy, B & Buday, L 2019, 'Structural insights into the tyrosine phosphorylation–mediated inhibition of SH3 domain–ligand interactions', Journal of Biological Chemistry, vol. 294, no. 12, pp. 4608-4620. https://doi.org/10.1074/jbc.RA118.004732
Mero, Balázs ; Radnai, László ; Gógl, Gergo ; To ke, Orsolya ; Leveles, Ibolya ; Koprivanacz, Kitti ; Szeder, Bálint ; Dülk, Metta ; Kudlik, Gyöngyi ; Virág Vas, X. ; Cserkaszky, Anna ; Sipeki, Szabolcs ; Nyitray, L. ; Vértessy, B. ; Buday, L. / Structural insights into the tyrosine phosphorylation–mediated inhibition of SH3 domain–ligand interactions. In: Journal of Biological Chemistry. 2019 ; Vol. 294, No. 12. pp. 4608-4620.
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AU - Mero, Balázs

AU - Radnai, László

AU - Gógl, Gergo

AU - To ke, Orsolya

AU - Leveles, Ibolya

AU - Koprivanacz, Kitti

AU - Szeder, Bálint

AU - Dülk, Metta

AU - Kudlik, Gyöngyi

AU - Virág Vas, X.

AU - Cserkaszky, Anna

AU - Sipeki, Szabolcs

AU - Nyitray, L.

AU - Vértessy, B.

AU - Buday, L.

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N2 - 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 Tyr 89 and Tyr 134 in ABL1 or the homologous residues Tyr 116 and Tyr 161 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 phosphoty-rosines 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.

AB - 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 Tyr 89 and Tyr 134 in ABL1 or the homologous residues Tyr 116 and Tyr 161 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 phosphoty-rosines 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.

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