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

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.

Keywords: 3BP-2; ABI2; ABL tyrosine kinase; Src homology 3 domain (SH3 domain); X-ray crystallography; ligand binding; nuclear magnetic resonance (NMR); phosphotyrosine signaling; post translational modification (PTM); protein phosphorylation; small-angle X-ray scattering (SAXS).

Figure 1.

A, structure and ligand-binding groove of SH3 domains. The SH3 domain of the tyrosine kinase Src in complex with a bound ligand peptide is shown as an example (PDB entry 1QWE). The ligand-binding interface can be divided into three pockets that are mainly flanked by aromatic residues (blue and orange) and a conserved proline (yellow). Binding groove tyrosines starting from the N terminus are labeled as N, M1, M2, and C. These positions are referred to similarly throughout this work. One or more negatively charged residues of the RT loop (Asp in Src) orient the ligand (red) by interacting with a positively charged residue on either side of the PXXP motif (Arg in this example). B, secondary structural elements and sequence alignment of ABL1 SH3 and ABL2 SH3. Tyrosine phosphorylation sites (based on PhosphoSitePlus) are highlighted in red. Conserved residues of the binding groove are shown with backgrounds corresponding to the color-coding in A. Arrows, differences between ABL1 and ABL2. Sequence numbering is based on isoform 1B of both ABL1 (UniProt identifier P00519-2) and ABL2 (UniProt identifier P42684-1). Note that in ABL1 SH3 and ABL2 SH3, Phe and Trp residues occupy positions M1 and M2, respectively. C, protein sequences of Tyr-to-Phe mutant constructs used in this work.

Figure 2.

Sequence logo representation of the tyrosine-phosphorylated human SH3 domains. Key conserved residues of the ligand-binding groove are highlighted in gray background. The four most prominent tyrosine phosphorylation sites (positions 10, 12, 50, and 55, with 32, 13, 20, and 35 occurrences in PhosphoSitePlus, respectively) are indicated in red. (See also the full alignment in Table S2. Note that only positions homologous to ABL1 SH3 are shown here for simplicity.)

Figure 3.

Tryptophan fluorescence–based titrations of ABL1 SH3 and ABL2 SH3. A, tryptophan fluorescence emission spectra of free and 3BP-2 peptide–bound ABL1 SH3 and ABL2 SH3. Ligand binding was associated with a remarkable blue shift. The highest intensity difference was observed at 318 nm. B, complex formation between ABL1 and ABL2 SH3 variants and 3BP-2 followed by fluorescence intensity changes at 318 nm. Both the doubly phosphorylated (ABL1 SH3^pYn/pYc and ABL2 SH3^pYn/pYc) and Tyr-C–phosphorylated (ABL1 SH3^YnF/pYc and ABL2 SH3^YnF/pYc) proteins showed total loss of ligand binding. The inhibitory effect of TyrN phosphorylation (ABL1 SH3^pYn/YcF and ABL2 SH3^pYn/YcF) was weaker but still substantial. C, complex formation between ABL1 and ABL2 SH3 variants and ABI2 followed by fluorescence intensity changes at 318 nm. Although the ABI2-derived ligand showed much weaker binding compared with 3BP-2 (dissociation constants around 100 μm), the effects of phosphorylation were similar. Error bars, S.D. of three independent measurements.

Figure 4.

Comparison of the nonphosphorylated and phosphorylated SH3 domains. Structural alignment of ABL1 SH3 (light blue; PDB code 4JJC) with ABL1 SH3^pYn/pYc (dark blue, PDB code 5NP2) (A) and ABL2 SH3 (yellow; PDB code 5NP3) with ABL2 SH3^pYn/pYc (orange; PDB code 5NP5) (B). Key residues of the binding grooves are labeled and shown as sticks.

Figure 5.

Alignment of the crystal structure of ABL1 SH3^pYn/pYc and ABL1 SH3 in complex with a peptide ligand derived from 3BP-1 (PDB code 1ABO (14)). A, for simplicity, only the ligand (salmon) and the phosphorylated domain are shown. Surfaces of the three neighboring hydrophobic pockets within the binding groove are shown in different colors. B, the phosphate group of pTyrC (pTyr134) occupies the binding groove, making it totally inaccessible for the ligand. (Both Pocket I and Pocket II are affected.) C, although the phosphate group of pTyrN (pTyr89) is located at the edge of the binding groove and this side chain seems to be more flexible, steric clashes with the ligand can be expected. (Mainly Pocket I is affected.)