Abstract
Spike (S) protein is the primary antigenic target for neutralization and vaccine development for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It decorates the virus surface and undergoes large motions of its receptor binding domains (RBDs) to enter the host cell. Here, we observe Down, one-Up, one-Open, and two-Up-like structures in enhanced molecular dynamics simulations, and characterize the transition pathways via inter-domain interactions. Transient salt-bridges between RBD(A) and RBD(C) and the interaction with glycan at N343(B) support RBD(A) motions from Down to one-Up. Reduced interactions between RBD(A) and RBD(B) in one-Up induce RBD(B) motions toward two-Up. The simulations overall agree with cryo-electron microscopy structure distributions and FRET experiments and provide hidden functional structures, namely, intermediates along Down-to-one-Up transition with druggable cryptic pockets as well as one-Open with a maximum exposed RBD. The inherent flexibility of S-protein thus provides essential information for antiviral drug rational design or vaccine development.