Molecular dynamics and free energy simulations have been carried out to elucidate the structural origin of differential protein–protein interactions between the common receptor protein angiotensin converting enzyme 2 (ACE2) and the receptor binding domains of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [A. E. Gorbalenyaet al.,Nat. Microbiol.5, 536–544 (2020)] that causes coronavirus disease 2019 (COVID-19) [P. Zhouet al.,Nature579, 270–273 (2020)] and the SARS coronavirus in the 2002–2003 (SARS-CoV) [T. Kuikenet al., Lancet 362, 263–270 (2003)] outbreak. Analysis of the dynamic trajectories reveals that the binding interface consists of a primarily hydrophobic region and a delicate hydrogen-bonding network in the 2019 novel coronavirus. A key mutation from a hydrophobic residue in the SARS-CoV sequence to Lys417 in SARS-CoV-2 creates a salt bridge across the central hydrophobic contact region, which along with polar residue mutations results in greater electrostatic complementarity than that of the SARS-CoV complex. Furthermore, both electrostatic effects and enhanced hydrophobic packing due to removal of four out of five proline residues in a short 12-residue loop lead to conformation shift toward a more tilted binding groove in the complex in comparison with the SARS-CoV complex. On the other hand, hydrophobic contacts in the complex of the SARS-CoV–neutralizing antibody 80R are disrupted in the SARS-CoV-2 homology complex model, which is attributed to failure of recognition of SARS-CoV-2 by 80R.

SUMMARY We propose that cell cycle-dependent timing of FEN1 nuclease activity is essential for cell cycle progression and the maintenance of genome stability. After DNA replication is complete at the exit point of the S-phase, removal of excess FEN1 may be crucial. Here, we report a mechanism that controls the programmed degradation of FEN1 via a sequential cascade of post-translational modifications. We found that FEN1 phosphorylation stimulated its SUMOylation, which, in turn, stimulated its ubiquitination and ultimately led to its degradation via the proteasome pathway. Mutations or inhibitors that blocked the modification at any step in this pathway suppressed FEN1 degradation. Critically, the presence of SUMOylation- or ubiquitination- defective, non-degradable FEN1 mutant protein caused accumulation of Cyclin B, delays in the G1 and G2/M phases and polyploidy. These findings may represent a newly identified regulatory mechanism used by cells to ensure precise cell cycle progression and to prevent transformation.

Genetic alterations in the PRKACA gene coding for the catalytic α subunit of the cAMP-dependent protein kinase A (PKA-C) are linked to cortisol-secreting adrenocortical adenomas, resulting in Cushing’s syndrome. Among those, a single mutation (L205R) has been found in up to 67% of patients. Because the x-ray structures of the wild-type and mutant kinases are essentially identical, the mechanism explaining aberrant function of this mutant remains under active debate. Using NMR spectroscopy, thermodynamics, kinetic assays, and molecular dynamics simulations, we found that this single mutation causes global changes in the enzyme, disrupting the intramolecular allosteric network and eliciting losses in nucleotide/pseudo-substrate binding cooperativity. Remarkably, by rewiring its internal allosteric network, PKA-CL205R is able to bind and phosphorylate non-canonical substrates, explaining its changes in substrate specificity. Both the lack of regulation and change in substrate specificity reveal the complex role of this mutated kinase in the formation of cortisol-secreting adrenocortical adenomas.

Enzymes accelerate the rate of chemical transformations by reducing the activation barriers of uncatalyzed reactions. For signaling enzymes, substrate recognition, binding, and product release are often rate-determining steps in which enthalpy-entropy compensation plays a crucial role. While the nature of enthalpic interactions can be inferred from structural data, the molecular origin and role of entropy in enzyme catalysis remains poorly understood. Using thermocalorimetry, NMR, and MD simulations, we studied the conformational landscape of the catalytic subunit of cAMP-dependent protein kinase A, a ubiquitous phosphoryl transferase involved in a myriad of cellular processes. Along the enzymatic cycle, the kinase exhibits positive and negative cooperativity for substrate and nucleotide binding and product release. We found that globally coordinated changes of conformational entropy activated by ligand binding, together with synchronous and asynchronous breathing motions of the enzyme, underlie allosteric cooperativity along the kinase’s cycle.

ATP-competitive inhibitors are currently the largest class of clinically approved drugs for protein kinases. By targeting the ATP-binding pocket, these compounds block the catalytic activity, preventing substrate phosphorylation. A problem with these drugs, however, is that inhibited kinases may still recognize and bind downstream substrates, acting as scaffolds or binding hubs for signaling partners. Here, using protein kinase A as a model system, we show that chemically different ATP-competitive inhibitors modulate the substrate binding cooperativity by tuning the conformational entropy of the kinase and shifting the populations of its conformationally excited states. Since we found that binding cooperativity and conformational entropy of the enzyme are correlated, we propose a new paradigm for the discovery of ATP-competitive inhibitors, which is based on their ability to modulate the allosteric coupling between nucleotide and substrate-binding sites.