The spike (S) glycoprotein of SARS-COV-2, the pathogen of the COVID-19 pandemic, is a key target for drug development due to its vital role of the spike in enabling a coronavirus to enter the host cell. The relatively variable S1 subunit of the spike is responsible for receptor recognition and binding, while the much more highly conserved S2 subunit catalyzes viral and host cell membrane fusion by undergoing dramatic conformational changes. It has been hypothesized that the spike-mediated entry mechanism involves a series of steps, including binding of S1 to the ACE2 receptor, shedding of the S1 subunit, and refolding of S2. However, the detailed mechanism remains unknown. A large number of cryo-EM structures (&gt;500) of the S protein have been resolved. The vast majority are highly similar prefusion structures with both S1 and S2 present; the other two show the postfusion structure of S2, which adopts a strikingly different fold in the absence of S1. In this work, we identified an unusual loop region in the S2 subunit, the serine rich loop (SRL), which is highly conserved among all coronaviruses. We speculated that the SRL is crucial to S2 refolding and designed a previously unknown disulfide staple to restrict SRL motion. Simulations showed that adding the disulfide did not influence the overall structure of the spike. Cryo-EM characterization by our experimental collaborators shows that the disulfide bond indeed forms in the intact (S1+S2) spike, with minimal structural perturbation. Importantly, our designed staple prevents S2 refolding, resulting in the first-ever structure of a key intermediate in the coronavirus entry pathway.

Designed disulfide staple leads to the first-ever structure of a key intermediate in coronavirus cell entry

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