Dynamical asymmetry exposes 2019-nCoV prefusion spike

Susmita Roy

A major challenge was simulating the gigantic structure of the full-length trimeric spike, as it is associated with the largescale conformational transition. It is indeed a daunting task to explore the full conformational landscape at an atomic length-scale. To overcome this, a structure-based coarse-grained molecular dynamic simulation approach has been adopted (15) . The simulation started with a full-length homo-trimeric spike protein structure generated from homology modeling which involves the alignment of a target sequence and a template structure (pdb: 6vsb) (7, 16) . This also helped to build the missing loops. The domain-specific residuerange for the full-length, trimeric SARS-CoV-2 S is given in Fig. 2A . The S1 head coordination of the trimeric spike is programmed by developing a super-symmetric topology-based modeling framework ( Fig. 2B ) (described in the Method pipeline in the supplementary material). With this, the molecular machine is ready to swing each of its S1 head between its 'up' and 'down' conformations (Movie S1, S2). A number of Cryo-EM structures captured the 'up' and 'down' conformations of the RBD domain of spike proteins of other coronaviruses including SARS-CoV-2 where the S1 subunit undergoes a hinge-like conformational movement prerequisite for receptor binding (Fig. 2C) (7, 8, 10, 17) . Apart from the hinge-responsive RBD-cleft interaction, in this study, a few inter-chain interactions are found to assist the 'RBD-up' and the 'RBD-down' conformations (shown in Fig. 2D and 2E, Movie S3). These few interactions are identified to impact the breathing of RBD of SARS-CoV-2 S. This makes the early referred 'RBD-up/down' conformations slightly different from the 'S1-head-up/down' conformation for trimeric SARS-CoV-2 S as the former is regulated only by intra-chain interactions while the latter is regulated by both intra and inter-chain interactions (Fig. S1 ). After identifying all these unique intra and inter-chain contacts (18, 19) extracted from the corresponding 'S1-head-up' and 'S1-head-down' conformations, a super-symmetric contact map is generated. This follows the development of a structure-based model Hamiltonian (Materials and Methods in Supplementary) which is based on the energy landscape theory of protein folding (20) (21) (22) (23) (24) . This approach not only potentiates the trimeric spike to adopt C3 symmetric '3up' and '3down' states but also to break the symmetry in a thermodynamically governed way (Fig. S1-S4) (25, 26) . Residue-residue native contact map identifying unique intra and inter-chain contact-pairs formed by any single monomer in its S1-head up and S1-head down states. C. Within intra-chain contacts, the unique contacts that drive hinge motion leading to RBD-up and RBD-down states are highlighted in the structure, as well as in the contact map. D. Inter-chain unique contacts between RBD and NTD domains upholding the S1-head-up state. E. Inter-chain unique contacts are responsible for connecting the RBD of ChainA with the S2-stalk of ChainB and the S2 stalk of ChainC.