Native MS and ligand observed NMR uncovers subtle SLiM binding variations that mediate HSP90-Hop PPI modulation.

We have previously demonstrated that a non-natural tetrazole containing peptide (2), which mimics the MEEVD Short Linear Motif (SLiM) found at the Heat Shock Protein 90 (HSP90) C-terminus, disrupts the transient protein-protein interaction (PPI) formed between HSP90 and the HSP70-HSP90 organizing protein (Hop) co-chaperone. However, the native MEEVD SLiM (1) showed negligible inhibitory activity despite similar binding affinity to the interacting HopTPR2A domain. To investigate the origin of this discrepancy, we combined native mass spectrometry (nMS) coupled to ion mobility (IM) with saturation transfer difference (STD) and water ligand observed via gradient spectroscopy (WLOGSY) NMR to interrogate the interaction of peptides 1 and 2 alongside a series of peptide derivatives with HopTPR2A. Collectively, these data revealed that the variation in sequence between peptides 1 and 2 imparts subtle variations in conformational stability and magnetization transfer, indicating that differences in PPI modulation arise from altered binding mode rather than binding affinity. These results not only provide a structural framework for developing peptidomimetic HSP90-Hop PPI inhibitors, but a generalized strategy for exploiting transient PPIs for drug discovery.