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Unveiling Unexpected Complexity in Protein Interactions: Insights from NMR Studies of SH3 Domains and Proline-Rich Ligands

• 0:08 Introduction to Project Focus: The research group utilizes biomolecular NMR to investigate the structure, dynamics, and function of proteins, including their interactions with other proteins and ligands.

• 1:19 Core Research Area: The presentation centers on the interaction between SH3 domains (well-known protein-binding structures) and proline-rich ligands.

• 1:39 SH3 Binding Types: SH3 domains typically bind proline-rich ligands in two ways (Class 1 and Class 2), determined by the location of a key basic residue and its interaction with the SH3 domain's selectivity loop.

• 2:12 Protein of Interest: Whip: The study focuses on the intrinsically disordered protein "Whip" (500 amino acids), specifically its central proline-rich region, which was previously thought to interact with the C-terminal SH3 domain of cortactin.

• 2:55 Cortactin's Role: Cortactin is a crucial player in cytoskeletal modification.

• 3:00 Initial Interaction Studies: Early yeast two-hybrid studies suggested an interaction between Whip and cortactin's SH3 domain, but no structural data existed.

• 3:27 Experimental Approach: Chemical Shift Perturbation: NMR, specifically chemical shift perturbation, was employed to study the interaction. This involved observing changes in the NMR spectrum of Whip upon adding the SH3 domain.

• 4:10 Key Finding: Single Binding Motif: Despite multiple potential binding motifs (PXXP) within Whip, only one primary binding site (residues 166-183) was identified, with some minor binding.

• 4:32 Confirmation of Canonical Binding: The labeling was reversed (labeled SH3, unlabeled Whip) to confirm that the interaction occurred within the canonical binding groove of the SH3 domain, as expected.

• 5:03 Unexpected Results from NOSY: The crucial turning point arose from analyzing NOESY spectra, which reveal inter-molecular contacts. The data showed an unexpectedly extensive network of contacts. The binding motif was longer and did not conform to any expected structure.

• 5:50 Embracing Unexpected Results: The researchers acknowledged the impossibility of fitting the data to a single structure and considered alternative explanations.

• 6:06 Multiple Binding Modes: The NOESY data was categorized to identify the minimum number of structures that could explain the observed interactions.

• 6:25 Three Structures Identified: Three distinct binding modes were identified: Class 1, "pseudo" Class 2 (lacking a typical basic residue), and a non-canonical mode utilizing additional surface of interaction.

• 7:42 Extended Binding Surface: An extended binding surface (Surface Two) on the cortactin domain, adjacent to the canonical site, plays a vital role in the non-canonical binding.

• 8:13 Revised Model of SH3 Binding: The findings led to a revised model of SH3 binding, incorporating the additional interaction surface and its specific residue requirements.

• 8:50 Connection to Cancer Research: This non canonical binding is important for invadopodia formation in tumor cells and, therefore, metastasis.

• 9:07 Peptide Inhibition of Metastasis: Collaborators discovered that a peptide derived from the unrelated protein "Peak 2" could inhibit invadopodia formation by interfering with the cortactin-SH3 interaction.

• 9:35 Structural Basis of Inhibition: The structure of the Peak 2-derived peptide bound to the SH3 domain was solved, revealing the importance of the extended interaction region for strong binding.

• 10:05 Future Directions: Peptide Design: The improved understanding of binding motifs is being utilized to design modified peptides with enhanced affinity and selectivity for the cortactin SH3 domain, potentially leading to new therapeutic strategies.