Crystallographic and biophysical analysis of the fusion core from SARS‐CoV‐2 spike protein

Abstract Severe acute respiratory syndrome‐coronavirus 2 (SARS‐CoV‐2) is a newly emerging infectious pathogen causing coronavirus disease 2019 (COVID‐19). The virus primarily infects cells via its spike glycoprotein, which is cleaved into S1 and S2 subunits to aid in cell attachment and membrane fusion, respectively. Heptad repeat 1 (HR1) and heptad repeat 2 (HR2) of the S2 subunit are essential for membrane fusion, culminating in an expected six‐helix bundle termed fusion core. To better understand the structural and biophysical features of the SARS‐CoV‐2 fusion core, we designed, constructed, and bacterially produced a recombinant single‐chain HR1‐L6‐HR2 protein and conducted a series of biochemical and biophysical experiments. Our findings demonstrate that the HR1‐L6‐HR2 protein spontaneously assembles into a highly stable trimeric complex, further confirmed by x‐ray crystallographic analysis. The crystal structure of the fusion core reveals a trimeric coiled‐coil structure of HR1 antiparallelly surrounded by three HR2 to form a six‐helical bundle. Additionally, four residues of HR1 that contribute to binding with HR2 through the formation of hydrogen bonds and salt bridges were observed. These results indicate that the SARS‐CoV‐2 fusion core exhibits similar characteristics to other class I viral glycoproteins, suggesting potential for drug repurposing as an alternative strategy to combat COVID‐19.