Abstract
Coronaviruses are a family of positive‐stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C‐terminal domain (∼100 amino‐acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library‐based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet‐substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P43. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS‐derived composite model. The structure consists of a single domain with a predominantly α‐helical content displaying a unique fold that could be engaged in protein–protein interactions.
Highlights
The Coronaviridae family, which is comprised of the genera Coronavirus and Torovirus, and the more distantly related Arteriviridae and Roniviridae families together form the order Nidovirales (Gorbalenya et al, 2006)
The incorporation of SeMet was verified by matrix-assisted laser desorption ionization (MALDI) mass spectrometry
The high conservation of the C-terminal domain of nsp4 suggests that it plays a ubiquitous role in the coronavirus life cycle, and that nsp4 proteins from different subgroups are structurally similar and have similar modes of operation
Summary
The Coronaviridae family, which is comprised of the genera Coronavirus and Torovirus, and the more distantly related Arteriviridae and Roniviridae families together form the order Nidovirales (Gorbalenya et al, 2006). Coronaviruses are positive-stranded RNA viruses that are frequently associated with enteric or respiratory diseases in humans, livestock and companion animals (Dye & Siddell, 2005) At present, they are formally classified into three genetic groups (1–3), with the first two groups further divided into two subgroups (1a/b and 2a/b; Gorbalenya et al, 2004; Lai & Holmes, 2001), but as our understanding of natural coronavirus diversity progresses novel subgroups continue to be recognized (Woo et al, 2009). Tertiary structures, solved using X-ray or/and NMR analyses, have been reported for a substantial number of nsps from at least one coronavirus, typically SARS-CoV (reviewed in Bartlam et al, 2005; Mesters et al, 2006) These structures represent a variety of separate domains, entire proteins and even multiprotein complexes. A family-wide comparative sequence analysis of the nsp C-terminal domain was performed in order to identify residues/regions that might be important for function rather than for structural integrity
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