Native Mass Spectrometry Dissects the Structural Dynamics of an Allosteric Heterodimer of SARS-CoV-2 Nonstructural Proteins

Structure-based drug design, which relies on precise understanding of the target protein and its interaction with the drug candidate, is dramatically expedited by advances in computational methods for candidate prediction. Yet, the accuracy needs to be improved with more structural data from high throughput experiments, which are challenging to generate, especially for dynamic and weak associations. Herein, we applied native mass spectrometry (native MS) to rapidly characterize ligand binding of an allosteric heterodimeric complex of SARS-CoV-2 two nonstructural proteins (nsp) nsp10 and nsp16 (nsp10/16). Native MS showed that the dimer is in equilibrium with monomeric states in solution. Consistent with literature, well characterized small co-substrate, RNA substrate and product bind with high specificity and affinity to the dimer but not the free monomers. Unsuccessfully designed ligands bind indiscriminately to all forms. Using neutral gas collision, the nsp16 monomer with bound co-substrate can be released from the holo dimer complex, confirming the binding to nsp16 as revealed by the crystal structure. However, an unusual migration of the endogenous zinc ions bound to nsp10 to nsp16 after collisional dissociation was observed, and can be suppressed using an alternative surface collision method at reduced precursor charge states. This highlighted the importance of careful optimization of experimental techniques. Overall, with minimal sample input (~µg), native MS can rapidly detect ligand binding affinities and locations in dynamic multi-subunit protein complexes, demonstrating the potential of an “all-in-one” native MS assay for rapid structural profiling of protein-to-AI-based compound systems to expedite drug discovery.