title: Solution structure of mouse hepatitis virus (MHV) nsp3a and determinants of the interaction with MHV nucleocapsid (N) protein.
abstract: Coronaviruses (CoVs) are positive-sense, single-stranded, enveloped RNA viruses that infect a variety of vertebrate hosts. The CoV nucleocapsid (N) protein contains two structurally independent RNA binding domains, designated the N-terminal domain (NTD) and the dimeric C-terminal domain (CTD), joined by a charged linker region rich in serine and arginine residues (SR-rich linker). An important goal in unraveling N function is to molecularly characterize N-protein interactions. Recent genetic evidence suggests that N interacts with nsp3a, a component of the viral replicase. Here we present the solution nuclear magnetic resonance (NMR) structure of mouse hepatitis virus (MHV) nsp3a and show, using isothermal titration calorimetry, that MHV N219, an N construct that extends into the SR-rich linker (residues 60 to 219), binds cognate nsp3a with high affinity (equilibrium association constant [K(a)], [1.4 ± 0.3] × 10(6) M(-1)). In contrast, neither N197, an N construct containing only the folded NTD (residues 60 to 197), nor the CTD dimer (residues 260 to 380) binds nsp3a with detectable affinity. This indicates that the key nsp3a binding determinants localize to the SR-rich linker, a finding consistent with those of reverse genetics studies. NMR chemical shift perturbation analysis reveals that the N-terminal region of an MHV N SR-rich linker peptide (residues 198 to 230) binds to the acidic face of MHV nsp3a containing the acidic α2 helix with an affinity (expressed as K(a)) of 8.1 × 10(3) M(-1). These studies reveal that the SR-rich linker of MHV N is necessary but not sufficient to maintain this high-affinity binding to N.
authors.affiliation: Department of Chemistry, Indiana University, Bloomington, IN, USA.
authors.forename: Sarah C
authors.initials: SC
authors.lastname: Keane
authors.forename: David P
authors.initials: DP
authors.lastname: Giedroc
mesh_terms: D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D006517:Murine hepatitis virus / Q000737:chemistry* / Q000502:physiology*; D019590:Nucleocapsid Proteins / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D019590:Nucleocapsid Proteins; D017361:Viral Nonstructural Proteins; C099600:nucleocapsid protein, Hepatitis virus