The spike protein is essential to the SARS-CoV-2 virus life cycle, facilitating virus entry and mediating viral-host membrane fusion. The spike contains a fatty acid (FA) binding site between every two neighbouring receptor-binding domains. This site is coupled to key regions in the protein, but the impact of glycans on these allosteric effects has not been investigated. Using dynamical nonequilibrium molecular dynamics (D-NEMD) simulations, we explore the allosteric effects of the FA site in the fully glycosylated spike of the SARS-CoV-2 ancestral variant. Our results identify the allosteric networks connecting the FA site to functionally important regions in the protein, including the receptor-binding motif, an antigenic supersite in the N-terminal domain, the fusion peptide region, and another allosteric site known to bind heme and biliverdin. The networks identified here highlight the complexity of the allosteric modulation in this protein and reveal a striking and unexpected link between different allosteric sites. Comparison of the FA site connections from D-NEMD in the glycosylated and non-glycosylated spike revealed that glycans do not qualitatively change the internal allosteric pathways but can facilitate the transmission of the structural changes within and between subunits.

Deep Mutational Scanning (DMS) is an emerging method to systematically test the functional consequences of thousands of sequence changes to a protein target in a single experiment. Because of its utility in interpreting both human variant effects and protein structure-function relationships, it holds substantial promise to improve drug discovery and clinical development. However, applications in this domain require improved experimental and analytical methods. To address this need, we report novel DMS methods to precisely and quantitatively interrogate disease-relevant mechanisms, protein-ligand interactions, and assess predicted response to drug treatment. Using these methods, we performed a DMS of the melanocortin-4 receptor (MC4R), a G-protein-coupled receptor (GPCR) implicated in obesity and an active target of drug development efforts. We assessed the effects of >6600 single amino acid substitutions on MC4R’s function across 18 distinct experimental conditions, resulting in >20 million unique measurements. From this, we identified variants that have unique effects on MC4R-mediated Gα_s- and Gα_q-signaling pathways, which could be used to design drugs that selectively bias MC4R’s activity. We also identified pathogenic variants that are likely amenable to a corrector therapy. Finally, we functionally characterized structural relationships that distinguish the binding of peptide versus small molecule ligands, which could guide compound optimization. Collectively, these results demonstrate that DMS is a powerful method to empower drug discovery and development.