Crystal structures of the X-domains of a Group-1 and a Group-3 coronavirus reveal that ADP-ribose-binding may not be a conserved property

Abstract

The polyproteins of coronaviruses are cleaved by viral proteases into at least 15 nonstructural proteins (Nsps). Consisting of five domains, Nsp3 is the largest of these (180-210 kDa). Among these domains, the so-called X-domain is believed to act as ADP-ribose-1''-phosphate phosphatase or to bind poly(ADP-ribose). However, here we show that the X-domain of Infectious Bronchitis Virus (strain Beaudette), a Group-3 coronavirus, fails to bind ADP-ribose. This is explained on the basis of the crystal structure of the protein, determined at two different pH values. For comparison, we also describe the crystal structure of the homologous X-domain from Human Coronavirus 229E, a Group-1 coronavirus, which does bind ADP-ribose.

Figure 1

Isothermal titration calorimetry profile for the binding of ADP-ribose to HCoV-229E X-domain.

Figure 2

Structure-based sequence alignment of the X-domains of HCoV 229E and IBV (strain Beaudette) with the homologue in SARS-CoV. Secondary structure elements of the HCoV-229E X-domain and the IBV X-domain are represented above and below the alignment, respectively. Amino acid residues labeled in cyan have been included in the calculation of the r.m.s.d. value. Asterisks above and below the alignment indicate aligned and conserved amino acid residues.

Figure 3

Ribbon-representation of the superimposed X-domains of HCoV 229E (blue) and IBV (strain Beaudette) (green, pH 8.5; red, pH 5.6). N- and C-termini are indicated; the symbol “N” is framed for IBV-X.

Figure 4

A: Stereo representation showing the binding of ADP-ribose to the X-domain of SARS-CoV, compared with the equivalent site in IBV-X8.5. The ADP-ribose molecule and the amino acid residues involved in the binding are represented as sticks. Carbon atoms of the ADP-ribose are colored white, carbon atoms of the protein are colored yellow and green, for SARS-X and IBV-X, respectively. Nitrogens and oxygens are colored blue and red, respectively. B: Residues ₄₂GGG₄₄ and ₁₂₂SCGIFG₁₂₇ (L11) of 229E-X (sticks, blue carbon atoms), likely involved in binding ADP-ribose, superimposed onto the corresponding site (₄₆GSG₄₈ and ₁₂₈SLGIFG₁₃₃) in IBV-X8.5 (transparent surface colored according to electrostatic potential, green sticks). Residue labels for 229E-X are framed. Note that Ile 131, and with it, the entire loop L11, in IBV-X is “pushed away” (red arrows) due to steric hindrance from Ser 47, thereby destroying the binding site for ADP-ribose.