What makes a good partner? - Two Regulatory Complexes of the Cortactin SH3 Domain with Diverse Affinities Determined by NMR

The SH3 domain of cortactin interacts with proline-rich domains in WASp-Interacting Protein (WIP) and protein tyrosine kinase Pyk2 in the regulation of cytoskeleton assembly. The cortactin-WIP interaction and the phosphorylation of cortactin by Pyk2 are essential for invadopodia formation and extracellular matrix degradation, both processes of tumor cell invasion. Thus, it is clear that the question of how cortactin interacts with these two binding partners is of clinical relevance.

Chemical shift perturbations of SH3 with WIP and Pyk2 as ligands show variations in profile, a sign of molecular recognition. The two complexes differ significantly in affinity: while the strong Pyk2 complex exhibits sub-micromolar affinity, the WIP complex is in fast-to-intermediate exchange on the NMR timescale, as shown by titration experiments, isothermal titration calorimetry (ITC), and backbone ¹⁵N R₂ relaxation rates, all indicating a complex that is weaker by two orders of magnitude.

We employed a suite of edited-filtered NOESY experiments in combination with selective isotopic labeling to provide intra- and inter-molecular distance constraints for these two complexes. Based on these, we determined high-resolution structures of both complexes, whose affinity differences result from the degree of interaction with hydrophobic residues and the charge distribution profile. A comparison of the two structures and mutagenesis analysis of both ligands and the SH3 domain define the determinants of affinity and explain the mechanism behind peptide inhibition of metastasis formation observed in in vivo studies in mice. This can also greatly contribute to fine-tuning of new SH3-inhibitors to optimize their effectiveness.