Fig 1.
Overall structure of MjHKU4r-CoV-1 RBD in complex with human DPP4 (hDPP4) or pangolin DPP4 (MjDPP4).
(A) Schematic diagram of the MjHKU4r-CoV-1 spike (S) protein ectodomain. NTD, N-terminal domain of S1. RBD, receptor binding domain. SD, subdomain. UH, upstream helix. FP, fusion peptide. HR, heptad repeat. TM, transmembrane domain. CT, cytoplasmic tail. (B) Crystal structure of MjHKU4r-CoV-1 RBD bound to hDPP4. MjHKU4r-CoV-1 RBD and hDPP4 are colored in light pink and wheat, respectively. Blades IV and V of hDPP4 are highlighted in lemon and blue, respectively. MjHKU4r-CoV-1 RBD adopts a β barrel structure and contains eleven β-strands. (C) Crystal structure of MjHKU4r-CoV-1 RBD bound to MjDPP4. MjHKU4r-CoV-1 RBD and MjDPP4 are colored in light pink and pale cyan, respectively. Blades IV and V are highlighted in green and blue. (D) Superposition of MjHKU4r-CoV-1 RBD-hDPP4 and MjHKU4r-CoV-1 RBD-MjDPP4 complexes. The two structures exhibit high similarity and the RMSD between them is 0.468 Å.
Fig 2.
Structure based sequence alignment of different DPP4s and merbecoviruses RBDs.
(A) Sequence alignment of hDPP4 (NP_001926.2), MjDPP4 (XM_017664375.2), TpDPP4 (MH345671.1), macaques DPP4 (NM_001039190.2), cat DPP4 (NM_001009838.1), camel DPP4 (XP_006176870.1), pig DPP4 (NM_214257.1), goat DPP4 (KF574265.1) and PpDPP4 (AGF80256.1). Residues on hDPP4 or MjDPP4 that bind to MjHKU4r-CoV-1 RBD, MERS-CoV RBD, and Ty-BatCoV-HKU4 RBD are labelled according to the code of the key below the sequences. (B) Sequence alignment of MjHKU4r-CoV-1 RBD (UVJ46720.1), Ty-BatCoV-HKU4-1 RBD (ABN10848.1), MERS-CoV RBD (JX869059), Ty-BatCoV-HKU4-2 RBD (EF065506.1), Ty-BatCoV-HKU4-SM3A RBD (MW218395.1) and pangolin-HKU4-P251T RBD (OM009282.1). Compared to MjHKU4r-CoV-1 RBM, the non-conserved V544 residue in pangolin-CoV-HKU4-P251T RBM is highlighted in pink. The secondary structure elements are generated with ESPript1. Residues on viral RBDs that bind to hDPP4 are labelled according to the code of the key below the sequences. Genbank accession numbers of corresponding sequences in (A) and (B) are enclosed in brackets.
Fig 3.
MjHKU4r-CoV-1 recognizes conserved residues on human and pangolin DPP4 receptors.
(A-D) Atomic details of the interaction between MjHKU4r-CoV-1 RBD and hDPP4/MjDPP4. MjHKU4r-CoV-1 RBD, hDPP4 and MjDPP4 are colored in light pink, wheat and pale cyan, respectively. Contacting residues on respective proteins are represented as sticks, with nitrogen and oxygen atoms colored in blue and red, respectively. MjHKU4r-CoV-1 RBD and hDPP4/MjDPP4 are colored as in Fig 1B and 1C. (E-G) Footprints of MjHKU4r-CoV-1 RBD (E), MERS-CoV RBD (F) and Ty-BatCoV-HKU4 RBD (G) on hDPP4. hDPP4 is represented as gray surface. Residues on hDPP4 contacting these three RBDs are colored in red, yellow and cyan, respectively. (H) Overlay of MjHKU4r-CoV-1 RBD, MERS-CoV RBD and Ty-BatCoV-HKU4 RBD footprints on hDPP4. The boundaries of these RBD footprints are circled with red, yellow and blue dotted lines, respectively.
Table 1.
The affinities of wild-type (WT) or mutant MjHKU4r-CoV-1 RBD/Ty-BatCoV-HKU4 RBD and hDPP4/MjDPP4 measured by SPR.
Fig 4.
Effect of residue mutation on MjHKU4r-CoV-1 and Ty-BatCoV-HKU4 entry into cells.
(A, B) Expression of WT or mutant MjHKU4r-CoV-1 (A) and Ty-BatCoV-HKU4 (B) spike proteins was detected in HEK293T cells. HEK293T cells were transfected with WT or mutant spike protein expression plasmid containing a C-terminal S-tag. At 48 h post transfection (h.p.t.), the cells were harvested, lysed, and subjected to western blotting analysis using the anti-S-tag antibody. Expression of full-length spike proteins and GAPDH was shown. (C) Comparison of the entry efficiencies between wild-typed (WT) MjHKU4r-CoV-1 and Ty-BatCoV-HKU4 spike protein packaged pseudoviruses in HEK293T-hDPP4 cells. Luciferase activity was determined at 48 h.p.t.. (D, E) Entry efficiency of pseudotyped viruses bearing the WT or mutant MjHKU4r-CoV-1 (D) and Ty-BatCoV-HKU4 (E) spike proteins in HEK293T-hDPP4 cells was determined by measuring luciferase activity at 48 h.p.t.. Data are presented as means and standard errors of the means (SEMs) of triplicate assays. Statistical significance was assessed using a two-tailed Student’s t-test. P < 0.05 was considered statistically significant. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significant difference.