Crystal structure-based exploration of the important role of Arg106 in the RNA-binding domain of human coronavirus OC43 nucleocapsid protein

Abstract

Human coronavirus OC43 (HCoV-OC43) is a causative agent of the common cold. The nucleocapsid (N) protein, which is a major structural protein of CoVs, binds to the viral RNA genome to form the virion core and results in the formation of the ribonucleoprotein (RNP) complex. We have solved the crystal structure of the N-terminal domain of HCoV-OC43 N protein (N-NTD) (residues 58 to 195) to a resolution of 2.0Å. The HCoV-OC43 N-NTD is a single domain protein composed of a five-stranded β-sheet core and a long extended loop, similar to that observed in the structures of N-NTDs from other coronaviruses. The positively charged loop of the HCoV-OC43 N-NTD contains a structurally well-conserved positively charged residue, R106. To assess the role of R106 in RNA binding, we undertook a series of site-directed mutagenesis experiments and docking simulations to characterize the interaction between R106 and RNA. The results show that R106 plays an important role in the interaction between the N protein and RNA. In addition, we showed that, in cells transfected with plasmids that encoded the mutant (R106A) N protein and infected with virus, the level of the matrix protein gene was decreased by 7-fold compared to cells that were transfected with the wild-type N protein. This finding suggests that R106, by enhancing binding of the N protein to viral RNA plays a critical role in the viral replication. The results also indicate that the strength of N protein/RNA interactions is critical for HCoV-OC43 replication.

Structure-Based Stabilization of Non-native Protein–Protein Interactions of Coronavirus Nucleocapsid Proteins in Antiviral Drug Design

“…We determined the crystal structure of MERS-CoV N-NTD by molecular replacement (MR) using the structure of HCoV-OC43 N-NTD (PDB ID: 4J3K) as the search model. 24 The final structure was refined to R-factor and R-free values of 0.26 and 0.29, respectively, at a resolution of 2.6 Å (Table S1). Each asymmetric unit contained four N-NTD molecules assembled into two identical dimers with an overall RMSD of 0.28 Å between the dimers ( Figure S1A,B).…”

Structural Characterization of Human Coronavirus NL63 N Protein

“…However, the nucleic acid interaction surface within SARS-CoV NTD was roughly delineated experimentally by monitoring nuclear magnetic resonance (NMR) chemical shift perturbation upon RNA titration (36). Abundant mutagenesis data from homologous NTDs corroborate the extent of the binding surface and highlight particular residues involved in the interaction (35)(36)(37)(38)(39). The crystal structure of the NL63 NTD revealed a network of sulfate ions, mostly coordinated by positively charged residues.…”

“…The results described above are consistent with prior mutagenesis data obtained for beta-and gammacoronavirus NTDs. In IBV, residues equivalent to positions 61, 63, and 77 in NL63 were experimentally demonstrated to participate in the nucleic acid interaction (37), and the residue equivalent to position 61 is involved in nucleic acid binding by OC43 (38). Further, residues at positions equivalent to positions 59 and 75 (39), and 77 of NL63 (33), are important for nucleic acid binding by MHV.…”

Recent insights into the development of therapeutics against coronavirus diseases by targeting N protein

“…Previous studies have reported that the N terminus of the N protein provides a scaffold for RNA binding. Structural analysis revealed that the fold and the distribution of the secondary structures of the N-terminal domain of the N protein are essentially conserved across the various CoVs [41,55]. The core of the structure of CoV N-NTD adopts a unique five-stranded antiparallel b-sheet sandwiched between loops (or short 3 10 helix) with the topology of b4-b2-b3-b1-b5; and the whole structure presents a righthanded fist-shaped structure in which palm and finger are rich in basic residues and the flexible loops are organized around the bsheet core of the N-terminal domain ( Fig.…”

“…All CoV N-NTDs possess positively charged amino acids on or near the protruding loop; however, in the SARS-CoV, mouse hepatitis virus (MHV) and avian infectious bronchitis virus (IBV) N-NTDs the protruding segment comprises a b-hairpin, whereas a flexible loop predominates in the HCoV-OC43 N-NTD. Based on the surface charge distribution, the protruding loop of the CoV N-NTD containing several positively charged residues, for example, Arg 106, Arg 107 and Arg 117 in HCoV-OC43, were identified to provide a site for binding of the phosphate backbone of RNA through electrostatic interactions [55]. In the central core of MHV N-NTDs, there are two crucial conserved Tyr residues, Tyr 127 and Tyr 129, that are involved in RNA binding and mutations of these residues to alanine can lead to loss of virus replication [56].…”