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We report a distinct difference in the interactions of the glycans of the host-cell receptor, ACE2, with SARS-CoV-2 and SARS-CoV S-protein receptor-binding domains (RBDs). Our analysis demonstrates that the ACE2 glycan at N90 may offer protection against infections of both coronaviruses, while the ACE2 glycan at N322 enhances interactions with the SARS-CoV-2 RBD. The interactions of the ACE2 glycan at N322 with SARS-CoV RBD are blocked by the presence of the RBD glycan at N357 of the SARS-CoV RBD. The absence of this glycosylation site on SARS-CoV-2 RBD may enhance its binding with ACE2.

Both SARS-CoV and SARS-CoV-2 bind to the human ACE2 receptor. Based on high-resolution structures, the two viruses bind in practically identical conformations, although several residues of the receptor-binding domain (RBD) differ between them. Here we have used molecular dynamics (MD) simulations, machine learning (ML), and free energy perturbation (FEP) calculations to elucidate the differences in RBD binding by the two viruses. Although only subtle differences were observed from the initial MD simulations of the two RBD-ACE2 complexes, ML identified the individual residues with the most distinctive ACE2 interactions, many of which have been highlighted in previous experimental studies. FEP calculations quantified the corresponding differences in binding free energies to ACE2, and examination of MD trajectories provided structural explanations for these differences. Lastly, the energetics of emerging SARS-CoV-2 mutations were studied, showing that the affinity of the RBD for ACE2 is increased by N501Y and E484K mutations but is slightly decreased by K417N.

Protein intrinsically disordered regions (IDRs) are often targets of combinatorial post-translational modifications (PTMs) that serve to regulate protein structure and/or function. Emerging evidence suggests that the N-terminal tails of G protein γ subunits – essential components of heterotrimeric G protein complexes – are intrinsically disordered, highly phosphorylated governors of G protein signaling. Here, we demonstrate that the yeast Gγ Ste18 undergoes combinatorial, multi-site phosphorylation within its N-terminal IDR. Phosphorylation at S7 is responsive to GPCR activation and osmotic stress while phosphorylation at S3 is responsive to glucose stress and is a quantitative indicator of intracellular pH. Each site is phosphorylated by a distinct set of kinases and both are also interactive, such that phosphomimicry at one site affects phosphorylation on the other. Lastly, we show that phosphorylation produces subtle yet clear changes in IDR structure and that different combinations of phosphorylation modulate the activation rate and amplitude of the scaffolded MAPK Fus3. These data place Gγ subunits among the growing list of intrinsically disordered proteins that exploit combinatorial post-translational modification to govern signaling pathway output.