Long-range allosteric communication, double mutant cycles, and energetic coupling in SARS-CoV-2 spike protein

SARS-CoV-2 spike protein is continuously evolving, leading to new variants. While mutations in the receptor-binding domain (RBD) enhance binding to the ACE2 receptor and evade neutralizing antibodies, the function of mutations in the N-terminal domain (NTD) remains poorly understood. Using two independent methods, surface plasmon resonance (SPR) and cryo-EM, we show that NTD mutations increase the population of spike protein with the RBD in the “up” conformational state. SPR association and dissociation kinetics of spike binding to ACE2 and antibodies, analyzed using a coupled equilibrium model, determined the relative populations and indicated that the RBD-up-to-down transition is rate-limiting relative to the RBD-down-to-up transition. Advanced model fitting of cryo-EM Coulomb potential maps confirmed the populations. The combined effect of NTD and RBD mutations exceeds the sum of their individual effects, indicating long-range allosteric communication and energetic coupling within the spike protein.