Without its N-finger, the main protease of severe acute respiratory syndrome coronavirus can form a novel dimer through its C-terminal domain

Abstract

The main protease (M(pro)) of severe acute respiratory syndrome coronavirus (SARS-CoV) plays an essential role in the extensive proteolytic processing of the viral polyproteins (pp1a and pp1ab), and it is an important target for anti-SARS drug development. It was found that SARS-CoV M(pro) exists in solution as an equilibrium of both monomeric and dimeric forms, and the dimeric form is the enzymatically active form. However, the mechanism of SARS-CoV M(pro) dimerization, especially the roles of its N-terminal seven residues (N-finger) and its unique C-terminal domain in the dimerization, remain unclear. Here we report that the SARS-CoV M(pro) C-terminal domain alone (residues 187 to 306; M(pro)-C) is produced in Escherichia coli in both monomeric and dimeric forms, and no exchange could be observed between them at room temperature. The M(pro)-C dimer has a novel dimerization interface. Meanwhile, the N-finger deletion mutant of SARS-CoV M(pro) also exists as both a stable monomer and a stable dimer, and the dimer is formed through the same C-terminal-domain interaction as that in the M(pro)-C dimer. However, no C-terminal domain-mediated dimerization form can be detected for wild-type SARS-CoV M(pro). Our study results help to clarify previously published controversial claims about the role of the N-finger in SARS-CoV M(pro) dimerization. Apparently, without the N-finger, SARS-CoV M(pro) can no longer retain the active dimer structure; instead, it can form a new type of dimer which is inactive. Therefore, the N-finger of SARS-CoV M(pro) is not only critical for its dimerization but also essential for the enzyme to form the enzymatically active dimer.

FIG. 1.

(A) Elution profile of M^pro-C from gel filtration analysis. The solid line is for the purification of the M^pro-C protein, and the elution peaks for the monomeric (M) and dimeric (D) forms are indicated. The broken line and dotted line are for the purified M^pro-C monomeric and dimeric protein samples after 3 days at room temperature, respectively. (B) SDS-PAGE analysis of M^pro-C. Lanes M2 and M3 are the M^pro-C monomer with/without 10 mM DTT, respectively; lanes D2 and D3 are the M^pro-C dimer with/without 10 mM DTT, respectively; lanes M1 and D1 are the M^pro-C monomeric and dimeric forms treated with cross-linking agent EGS; and the center lane is the molecular mass marker.

FIG. 2.

(A) An overlay of the 2D ¹H-¹⁵N HSQC spectra of the monomeric (blue) and dimeric (red) forms of M^pro-C. The peaks with combined NH chemical shift difference larger than 0.05 ppm are labeled with the one-letter amino acid code and residue number; “sc” is used to indicate the side chain signals. (B) Plot of combined NH chemical shift difference versus residue number. The combined chemical shift difference was calculated using the empirical equation Δδ_comb = [Δδ_HN² + (Δδ_N/6.5)²]^1/2, where Δδ_HN and Δδ_N represent the chemical shift differences of ¹H and ¹⁵N, respectively (18). (C) Plot of ¹³C^α chemical shift difference versus residue number. Residues without assignment are indicated by short red bars.

FIG. 3.

(A) The dimerization interface of the M^pro-C dimer mapped on the crystal structure of SARS-CoV M^pro. A ribbon diagram of the crystal structure 1UK3 is shown. The C-terminal domain of one protomer is colored in light blue, and the residues with a Δδ_comb value of >0.05 ppm are colored in blue. The C-terminal domain of the other protomer is colored in pink, and the residues with a Δδ_comb value of >0.05 ppm are colored in red. (B) Ribbon diagram of a model structure for the M^pro-C dimer. The model structure was calculated using the software program Haddock. The dimer interfaces are colored in red and blue in two protomers, respectively. The side chains of residues W218, F219, F223, and L271, which may be important for dimer formation due to hydrophobic interactions at the dimer interface, are shown.

FIG. 4.

(A) Gel filtration analysis of WT SARS-CoV M^pro and M^pro-Δ7. The broken lines represent WT SARS-CoV M^pro at the indicated concentrations. The peak heights have been adjusted arbitrarily to make the figure clearer. The solid line is the purification profile for M^pro-Δ7 with the elution peaks for the monomeric form (M) and dimeric form (D) marked. (B) Gel filtration analysis of M^pro-Δ7 stability. The solid line is the purification profile for M^pro-Δ7, with the peaks of the monomeric form (M) and dimeric form (D) indicated. The lines of purified M^pro-Δ7 after being placed at room temperature for 1 day and 3 days are indicated. The broken and dotted lines are for the monomeric and dimeric forms, respectively. The peak heights have been adjusted arbitrarily to make the figure clearer. (C) Enzymatic activity of WT SARS-CoV M^pro and M^pro-Δ7. The solid line is for WT SARS-CoV M^pro; the broken line is for the M^pro-Δ7 dimer; and the dotted line is for the M^pro-Δ7 monomer.

FIG. 5.

An overlay of 2D ¹H-¹⁵N HSQC spectra of monomeric and dimeric M^pro-Δ7 and M^pro-C. The black peaks belong to the M^pro-Δ7 dimer, the green peaks belong to the M^pro-Δ7 monomer, the red peaks are from the M^pro-C dimer, and the blue peaks are from the M^pro-C monomer. The signature NH peaks of the M^pro-C dimer are indicated by orange squares, which are labeled with a one-letter amino acid code and a residue number. Six areas of the spectra are enlarged and displayed for clarity.

FIG. 6.

Cartoon diagrams illustrating the dimerization pattern of WT SARS-CoV M^pro (A) or M^pro-Δ7 (B). The N-terminal and C-terminal domains are labeled “N” and “C”, respectively. The N-finger is illustrated as a thick black line and is indicated in the figure. The novel dimer interface of the C-terminal domain is represented by hatched bars.