Exploring Conformational Transition of 2019 Novel Coronavirus Spike Glycoprotein Between Its Closed and Open States Using Molecular Dynamics Simulations

Mert Gur; Elhan Taka; Sema Zeynep Yilmaz; Ceren Kilinc; Umut Aktas; Mert Golcuk

Each S protein protomer consists of two functional subunits ( Fig. 1(c,d) ): S1 subunit is responsible for binding to host receptor and S2 subunit contains the membrane fusion machinery (Walls et al. 2017 , Kirchdoerfer et al. 2018 . S1 subunit comprises two functional domains: N-terminal domain (NTD) and a receptor-binding domain (RBD), both of which are responsible for the binding to the host cell receptor (Li, Moore et al. 2003) . S2 subunit contains three functional domains; fusion peptide (FP), heptad repeat (HR) 1, and HR2 (Jiang et al. 2020) . Based on the RBD position, S protein can be in a receptor inaccessible closed or receptor accessible open state. As shown in Fig. 2 , in the receptor inaccessible closed state, all RBDs are in the down position covering the S2 subunits; whereas in the receptor accessible open state, at least a single RBD is in an up position being rotated outwards from S2, hence exposing its receptor binding surface. Conformational switch of RBD from the down to up conformation is a prerequisite for ACE2 binding of the S protein. There are two cleavage sites in each protomer of the S protein. Among them, the S2' site is located upstream from the FP. S1/S2 site, on the other hand, is located 103 residues downstream from S2' and located at the boundary between the S1 and S2 subunits. Cleavage at S2' sites is a requirement for fusion of host cell and virus membranes as it exposes the FPs at S2 to the environment and also separates the protein into S1-ectodomain and viral anchored S2 subunit. However, there is no consensus whether S1/S2 site cleavage is a requirement for S protein transition from pre to post fusion state. Upon S1/S2 site cleavage, S protein is divided into S1 and S2 subunits, which remain non-covalently bound in this metastable prefusion conformation (Bosch et al. 2003 , Park et al. 2016 CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https: //doi.org/10.1101 .04.17.047324 doi: bioRxiv preprint Wang et al. 2018 . During cell infection, S protein binds to the host cell receptor ACE2 via RBD (Li, Moore et al. 2003 , Wong et al. 2004 . Protein-receptor binding is expected to initiate a set of structural rearrangements in the S protein. Cleavage in S2' promotes rearrangement of HR1 in the S2 subunit into an extended α-helix and insertion of the FP into the host membrane. Subsequently, a six-helix bundle comprising HR2 and HR1 is formed (Bullough et al. 1994 , Bosch, van der Zee et al. 2003 , Walls, Tortorici et al. 2017 ). This completes the transition of the S protein from its metastable pre-fusion state to a highly stable post-fusion state. S protein conformational transition results in the fusion of viral and host membranes, thus allowing the release of the viral genome into host cell (Walls, Tortorici et al. 2017) . The first SARS-CoV-2 S protein structure was resolved in its open state and released on February 26 2020 with the PDB ID 6VSB. This structure had a 3.46 Å resolution and covers 76.8% of the sequence. Shortly after, on March 11 2020, SARS-CoV-2 S protein structures were resolved in their closed and open states having PDB IDs 6VXX and 6VYB. These closed and open structures have resolutions 2.8 Å and 3.2 Å, respectively, and each cover 76.4% of the protein sequence. Based on the crystal structures, the transition between the two states can be predicted to occur through a hing