NMR-guided structural and dynamic characterization of β-PIX SH3 domain for the design of peptide inhibitors targeting oncogenic protein-protein interactions

Srijon Sen (IIT Kharagpur, India)

Abstract: Protein–protein interactions (PPIs) mediated by Src homology 3 (SH3) domains are central to oncogenic signaling and represent challenging therapeutic targets. In breast cancer, the β-PIX SH3 domain interacts with the proline-rich region of Cbl, sequestering it and impairing EGFR downregulation, thereby promoting aberrant signaling. Here, we employ solution-state NMR spectroscopy to structurally and dynamically characterize the β-PIX SH3 domain and to elucidate its interaction with designed peptide inhibitors.

Uniformly ¹⁵N-labeled β-PIX SH3 domain was expressed and purified, and ¹⁵N–¹H HSQC spectra exhibited well-dispersed resonances indicative of a folded protein. Backbone resonance assignments were achieved with high completeness. Secondary structure propensity analysis confirmed a canonical SH3 fold comprising five β-strands and a short 3₁₀ helix. ¹⁵N relaxation measurements (R₁, R₂, and heteronuclear NOE) revealed a structurally stable core with localized flexibility in the RT loop, a key determinant of ligand binding.

Peptide inhibitors were designed using computational approaches and molecular dynamics simulations. NMR titration experiments showed significant chemical shift perturbations upon peptide binding, enabling mapping of the interaction interface on the SH3 domain. Cyclic peptides exhibited enhanced binding compared to linear counterparts, as evidenced by larger perturbations and improved binding affinities. These observations were further supported by isothermal titration calorimetry and fluorescence anisotropy measurements.

Overall, this study demonstrates the power of NMR in resolving structural, dynamic, and interaction features of the β-PIX SH3 domain and provides a framework for the rational design of peptide-based inhibitors targeting SH3-mediated PPIs in cancer.