A pan-coronavirus fusion inhibitor targeting the HR1 domain of human coronavirus spike

Xia, Shuai; Yan, Lei; Xu, Wei; Agrawal, Anurodh Shankar; Algaissi, Abdullah; Tseng, Chien-Te K.; Wang, Qian; Du, Lanying; Tan, Wenjie; Wilson, Ian A.; Jiang, Shibo; Yang, Bei; Lu, Lu

To investigate the structural basis for the pan-CoV inhibitory effect of EK1 peptide, we crystallized EK1 in complex with HR1 peptides from three representative HCoVs, including the most pathogenic SARS-CoV and MERS-CoV in -HCoVs and 229E in -HCoVs, the HR1 of which is 14 amino acids longer than those of -HCoVs. (table S3) . In all three structures, EK1 snugly fits into the hydrophobic groove formed between two adjacent HR1 helices in an oblique and antiparallel manner (Fig. 4 , A to C), producing a 6-HB structure resembling the 3HR1-3HR2 postfusion state of corresponding HCoVs ( fig. S4) . Notably, the binding sites of EK1 coincide with those of native HR2s (fig. S4 ). These results indicate that the presence of EK1 would preclude binding of HR2 onto their corresponding 3HR1 core, thereby blocking formation of the 3HR1-3HR2 6-HB, which is an indispensable step during host-viral membrane fusion. Hence, administration of the EK1 peptide would block cellular entry of those HCoVs (Fig. 2) . In all three structures, the EK1 peptide adopts a mixed secondary structure conformation (Fig. 4 , A to C). The central region of EK1 folds into a five-turn helix, which packs against two neighboring HR1 helices via extensive hydrophobic interactions (3HR1 cores of different HCoVs are illustrated as electrostatic surfaces, while residues on EK1 that are involved in hydrophobic packing are shown as stick models; residues L12, E15, M16, L19, A22, I23, L26, S29, and Y30 on EK1 locate within the five-turn helix of EK1 and form strong hydrophobic interactions with the 3HR1 cores; Fig. 5A ) and a few electrostatic or polar interactions (residues E13, E15, K18, E20, E27, E28, and Y30 locate within the five-turn helix region of EK1 and interact with HR1 residues through side chain-to-side chain hydrophilic interactions; Fig. 6A ). The rest of EK1 adopts an extended conformation, except that one extra turn is formed at the C-terminal end of EK1 (around E35) in the HR1(229E)-L6-EK1 structure (Fig. 6 , right panel). In the extended region of EK1, polar interactions between side chain (HR1 residues) and main chain (EK1 residues) dominate (compare Fig. 6, B and A) . Most of these side chain-to-main chain polar interactions cluster at either end of the EK1 helical region, which interweave into extensive H-bond networks and likely help secure the EK1 helical region in the correct register (Fig. 6B) . Hydrophobic residues in the extended region of EK1 also insert their bulky side chains into hydrophobic pockets on the surface of the 3HR1 cores (residues L2, I5, V7, L10, I31, and L36; Fig. 5A ), further strengthening the adhesion of the EK1 extended region onto the 3HR1 cores.