Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein

Abstract

The multi-domain non-structural protein 3 (Nsp3) is the largest protein encoded by the coronavirus (CoV) genome, with an average molecular mass of about 200 kD. Nsp3 is an essential component of the replication/transcription complex. It comprises various domains, the organization of which differs between CoV genera, due to duplication or absence of some domains. However, eight domains of Nsp3 exist in all known CoVs: the ubiquitin-like domain 1 (Ubl1), the Glu-rich acidic domain (also called "hypervariable region"), a macrodomain (also named "X domain"), the ubiquitin-like domain 2 (Ubl2), the papain-like protease 2 (PL2pro), the Nsp3 ectodomain (3Ecto, also called "zinc-finger domain"), as well as the domains Y1 and CoV-Y of unknown functions. In addition, the two transmembrane regions, TM1 and TM2, exist in all CoVs. The three-dimensional structures of domains in the N-terminal two thirds of Nsp3 have been investigated by X-ray crystallography and/or nuclear magnetic resonance (NMR) spectroscopy since the outbreaks of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) in 2003 as well as Middle-East Respiratory Syndrome coronavirus (MERS-CoV) in 2012. In this review, the structures and functions of these domains of Nsp3 are discussed in depth.

Keywords: Innate immunity; Macrodomain; Nucleic-acid binding domain; Papain-like protease; Structural biology; Ubiquitin-like domain.

Copyright © 2017 Elsevier B.V. All rights reserved.

Figures

Fig. 1

Genome organization of coronaviruses; Nsp3 domains and their functions. (A) The 5′-terminal two thirds of the CoV genome comprise ORF1a and ORF1b. ORF1a encodes the polyprotein 1a (Nsp1-11) while ORF1a plus ORF1b produce the polyprotein 1ab (Nsp1-16) through a ribosomal frameshift overreading the stop codon of ORF1a (indicated by a black arrow). The 3′-proximal third encodes the structural proteins S, E, M, and N as well as accessory proteins. The polyproteins pp1a and pp1ab are processed by the viral proteases PL1pro, PL2pro (both domains of Nsp3), and Mpro (3CLpro, Nsp5). The domain organization of Nsp3 is different in different CoV genera. The Nsp3 of HCoV NL63 as a representative of alpha-CoVs, and of SARS-CoV in clade B of the genus beta-CoV, are zoomed out. The question mark within HCoV-NL63 Nsp3 indicates a region of unknown function and structure. (B) Summary of the functions and domain organization of SARS-CoV Nsp3. Nsp3 is bound to double-membrane vesicles recruited from the endoplasmic reticulum (ER) membrane. The protein passes through this membrane twice, via the two transmembrane regions TM1 and TM2. AH1 is possibly an amphipathic helix attached to the ER membrane, next to TM2. Except for the 3Ecto domain, all other Nsp3 domains are located in the cytosol. All domains with known three-dimensional structures are indicated in light green (X-ray structures) or orange (NMR structures), whereas parts with unknown structure are in red. The best characterized functions of each domain of Nsp3 are shown.*: glycosylation sites in the 3Ecto domain (Asn1431 and Asn1434; Harcourt et al., 2004).

Fig. 2

Structures (in cartoon view) of the ubiquitin-like domain 1 (Ubl1) and Ubl2 in SARS-CoV, Ubl1 in MHV, as well as their structural homologues. (A) Ubl1 (residues 20–108) of SARS-CoV (PDB entry: 2IDY; Serrano et al., 2007). (B) Ubl1 (19–114) of MHV (PDB entry: 2M0A; Keane and Giedroc, 2013). (C) Ubl2 (residues 1–60) of SARS-CoV (PDB entry: 2FE8; Ratia et al., 2006). (D) human ubiquitin (PDB entry: 1UBQ; Vijay-Kumar et al., 1987). (E) human interferon-stimulated gene 15 (hISG15; PDB entry: 1Z2M; Narasimhan et al., 2005). hISG15 contains two linked ubiquitin-like domains; here, the N-terminal Ubl domain is shown. (F) the Ras-interacting domain of RalGDS (PDB entry: 1LFD; Huang et al., 1998). The N and C termini of all structures are marked. All α and 310 (η) helices are labeled and shown in cyan. β strands are in purple and loops are in brown. This figure and Fig. 3, Fig. 5, as well as 8 were generated by using Chimera (Pettersen et al., 2004).

Fig. 3

Crystal structure of the papain-like protease domain 1 (PL1pro) of TGEV. Cartoon view of the overall structure (PDB entry: 3MP2; Wojdyla et al., 2010). The thumb, fingers, and palm subdomains are shown in blue, brown, and green, respectively. The Cα atoms of the catalytic triad residues (Cys32−His183−Asp196) are displayed as yellow, blue, and red spheres. Residue Gln27 contributing to the oxyanion hole is shown in ball & stick style. Ile155, Thr209, and Tyr175 forming the S4 pocket are labeled; Ile155 is in black and the latter two are in red. The N and C termini of the PL1pro are indicated.

Fig. 4

Structure of the MERS-CoV macrodomain I (Mac1, X domain) in complex with ADP-ribose (ADPr) (PDB entry: 5HOL). The protein features an α/β/α sandwich fold. The central β sheet with the strand order β1−β2−β7−β6−β3−β5−β4 is shown in purple, β1 and β4 are labeled. An Fo-Fc omit difference map of ADPr is shown in black (contoured at 4.0 σ). The ADPr itself is displayed as brown sticks. The five regions (blue) relating to ADPr binding are marked by Roman numbers I – V. Fixing the two ends of the ADPr, Asp21 and Asn39 are displayed by thicker red sticks. The O2′ of ADPr forms a hydrogen bond with a water molecule (H2O 308; green sphere) being stabilized by the side-chain of Asn155. The “GGG” triple-glycine motif is displayed in black. H2O 310 (green sphere) corresponds to a water molecule that has been proposed to mediate a nucleophilic attack onto the C1″ atom of the ADPr in the de-MARylation reaction catalyzed by the VEEV X domain (Li et al., 2016a). The N and C termini of the X domain are marked. This figure and Fig. 6 were prepared using Pymol (Schrödinger; http://www.pymol.org/).

Fig. 5

Structures (in cartoon style) of the macrodomains II (Mac2) and III (Mac3), of the Domain Preceding Ubl2 and PL2pro (DPUP) of SARS-CoV and MHV, as well as of the frataxin-like fold protein Yfh1. (A) and (B) Mac2 and Mac3 (PDB entry: 2W2G; Tan et al., 2009). Both domains possess the α/β/α sandwich fold. The central six β strands in the order β1−β6−β5−β2−β4−β3 are displayed in purple. A predominantly positively charged surface patch (Lys563+Lys565+Lys568+Glu571; Nsp3 numbering) of Mac3 being involved in binding oligo(G) (Kusov et al., 2015) is labeled. (C) The SARS-CoV DPUP NMR structure (PDB entry: 2KQW; Johnson et al., 2010). (D) The MHV DPUP X-ray crystal structure (PDB entry: 4YPT; Chen et al., 2015). (E) Structure of the yeast frataxin-like protein Yfh1, as determined by NMR spectroscopy (PDB entry: 2GA5; He et al., 2004). All structures shown in (C), (D), and (E) display the typical frataxin-like fold. Two α helices located at the N- and C- terminal of each structure form one plane and the β sheet forms the other plane. The negatively charged residues (Asp or Glu) in the first α helix (α1) are shown in red (in (C), (D), and (E)); they are possibly involved in binding metal ions. The N and C termini of all structures are marked.

Fig. 6

Structure of the SARS-CoV papain-like protease 2 (PL2pro) in complex with Lys48-linked diubiquitin (PDB entry: 5E6J; Békés et al., 2016). The Ubl2 is shown as a grey cartoon. The catalytic domain (PL2pro) is displayed in surface view. The thumb, fingers, and palm subdomains are shown in blue, light brown, and green, respectively. The blocking loop 2 (BL2) is depicted in red. The Lys48-linked diubiquitin is displayed as a light-blue cartoon. Lys48 of Ub1 is linked to the C-terminal Gly75 of Ub2 (black sticks) via a triazole (red sticks). The N and C termini of Ub1 (N1, C1) as well as the N terminus of Ub2 (N2) are marked. The conserved hydrophobic patches (Ile44, Ala46, Gly47) of Ub1 and Ub2 are indicated by purple and orange dots, respectively. The residue Phe70 (yellow) interacting with the hydrophobic patch of Ub2 is labeled. The C-terminal Arg−Leu−Arg−Gly−Gly residues (RLRGG) of Ub1 are shown in ball & stick style (purple). P3-Arg and P5-Arg are marked.

Fig. 7

Recently described inhibitors of the CoV PL2pro. (A) Structural formula of the purine derivative 8-(trifluoromethyl)-9H-purin-6-amine (compound 4). This compound is a competitive MERS-CoV PL2pro inhibitor (Lee et al., 2015). It is also active against SARS-CoV PL2pro but acts as an allosteric inhibitor in this case. (B) A natural-product chalcone, compound 6 from the perennial plant Angelica keiskei, inhibits the SARS-CoV Mpro (3CLpro) and PL2proin vitro (Park et al., 2016).

Fig. 8

NMR structure of the nucleic acid-binding (NAB) domain in SARS-CoV (cartoon style; PDB entry: 2K87; Serrano et al., 2009). The order of secondary-structure elements is β1−β2−β3−α1−β4−β5−η1−η2−β6−β7−α2−β8. The overall structure of NAB represents a unique fold. The residues involved in RNA binding (Lys75, Lys76, Lys99, and Arg106) are displayed in blue. The N and C termini of the NAB domain are labeled.

Fig. 9

Multiple sequence alignment of the 3Ecto and the N-terminal portion of the Y1 domain containing two potential zinc fingers. The conserved cysteines in 3Ecto as well as cysteines and histidines in the N-terminal portion of Y1 are marked by triangles. Two glycosylation sites in the 3Ecto domain of SARS-CoV (Asn1431 and Asn1434; Harcourt et al., 2004) are indicated by asterisks. The corresponding sequence accession numbers are: SARS-CoV, Genbank: AY274119.3; MERS-CoV, Genbank: JX869059.2; MHV, Genbank: AY700211.1; HCoV NL63, Genbank: AY567487.2; IBV, Genbank: M95169.1. The figure was generated using the program ESPript (Gouet et al., 1999).