Fig. 2. Molecular basis for CendR binding of SARS-CoV-2 S1 with NRP1.

(A) Binding of NRP1 b1 with native (green line) and mutant (orange line) form of S1 CendR peptide (corresponding to residues 679-685) by ITC at two different pH conditions (N=3). All ITC graphs represents the integrated and normalized data fit with 1-to-1 ratio binding. (B) Left: NRP1 b1 – S1 CendR peptide complex superposed with NRP1 b1 – VEGF-A fusion complex (PDB ID: 4DEQ). Bound peptides are shown in stick representation. RMSD = root mean square deviation. Right: Enlarged view highlighting the binding of S1 CendR peptide b1. Key binding residues on b1 are shown in stick representation. (C). HEK293T cells were co-transfected with combinations of GFP-tagged S1^493-685 and S1^493-685 R685D, and mCherry or mCherry-NRP1 b1, and subjected to mCherry-nanotrap (N=5). Two-tailed unpaired t-test; P <0.0001. (D). HEK293T cells were co-transfected with combinations of GFP-tagged S1^493-685 and mCherry, mCherry-NRP1 b1 or mCherry-NRP1 b1 T316R mutant and subjected to mCherry-nanotrap (N=5). Two-tailed unpaired t-test; P <0.0001. (E) HeLa^NRP1KO + ACE2 cells transfected with GFP, NRP1 wt-GFP or NRP1 T316R-GFP constructs were infected 24 h later with SARS-CoV-2. At 16 hpi the cells were fixed and stained for SARS-CoV-2-N, and viral infection quantified in the GFP-positive subpopulation of cells (N=3). The percentage of infection was normalized to that of GFP-transfected cells. Two-tailed unpaired t-test; p = 0.002. The bars, error bars and circles represent the mean, SEM (C-D) and SD (E), individual data points, respectively. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001.