Crystal Structure of MERS-CoV N-NTD Complexed with Potent Compounds
We attempted to obtain crystals of MERS-CoV N-NTD in complex with compounds P1, P2, and P3 by cocrystallization or ligand-soaking. With the exception of P2, the complex structures of N-NTD with P1 and P3 were solved at resolutions of 3.09 and 2.77 Å, respectively (Table S1). The overall structures of the complexes resembled that of apo-MERS-CoV. Both complexes revealed well-defined unbiased densities in the dimer interface and permitted detailed analysis of the interactions between the compounds and MERS-CoV N-NTD (Figure 5). The interactions between the N protein and each compound were calculated with the Discovery Studio Client (v19.1.0.18287). Most interactions were hydrophobic contacts, which were consistent with our selection rationale. In the P1 complex, N68, F135, and D143 on monomer 1 and V41, G106, P107, and T137 on monomer 2 packed against P1 to create a dimer (Figure 5A). In addition, two nonbonding interactions were detected between P1 and the monomers. There was a π-anion interaction between the benzene ring of the P1 indoline moiety and D143 of monomer 1. There was also a π-donor hydrogen bond between the other P1 benzene ring and the T137 side chain of monomer 2 (Figure 5B). Relative to P1, P3 bound more deeply into the dimer interface and interacted with a larger number of residues on both N-NTD monomers. The amino acid composition of this binding region was W43, N66, N68, S69, T70, N73, and F135 on monomer 1 and V41, G104, T105, G106, A109, and T137 on monomer 2. These residues along with P3 generated a massive hydrophobic driving force allowing the proteins and ligands to pack against each other and stabilize the dimeric conformation of the N protein (Figure 5C). Several nonbonding interactions were also observed at the P3-binding site. These included the interaction between the P3 benzene ring and N68 of monomer 1 and A109 of monomer 2 via π-lone pair and π-alkyl interactions. The dimethylaminomethyl moiety of P3 was a major source of nonbonding interactions. Three π-cation interactions formed between this moiety and the aromatic groups of W43 and F135 in monomer 1. This moiety also formed a π-lone pair interaction with N66 and a π-sigma interaction with W43 of monomer 1 (Figure 5D). The structural analyses explain the comparatively stronger binding of P3 to N-NTD (Figure 2B) and corroborated the thermal stabilization effects (Figure 2C) and antiviral activities (Table 1) of the compounds.
Figure 5  Structures of MERS-CoV N-NTD complexed with potent compounds. The structures were solved using HCoV-OC43 N-NTD (PDB:4J3K) as the search model.24 Left panel: (Upper) structural superimposition of the MERS-CoV N-NTD:P1 complex (monomers 1 and 2 are in purple and pink, respectively) and the MERS-CoV N-NTD:P3 complex (monomers 1 and 2 are in brown and green, respectively) with compounds depicted as stick structures. (Lower) Interactions involving vector-fusion residues in the non-native dimer of the apoprotein shown for comparison with (A) and (B). Color is the same as in Figure 1A. Right panel: detailed interactions among MERS-CoV N-NTD and P1 (A, B) and P3 (C, D). Different Fo–Fc maps were contoured at ∼2.5 σ. (A) Detailed stereoview of interactions at the P1-binding site. The color of each monomer is the same as in the left panel. Residues constructing the P1-binding pocket are labeled and showed as sticks. (B) Schematic of P1 bound to MERS-CoV N-NTD. Hydrophobic contacts between P1 and each monomer are displayed as dashed lines. Nonbonding interactions are indicated by cyan arrows. (C) Detailed stereoview of interactions at the P3-binding site. The color of each monomer is the same as in the left panel. Residues belonging to the P3-binding pocket are labeled and shown as sticks. (D) Schematic of P3 bound to MERS-CoV N-NTD. Hydrophobic contacts between P3 and each monomer are displayed as dashed lines. Nonbonding interactions are indicated by red arrows.