Recognition of Lys48-Linked Di-ubiquitin and Deubiquitinating Activities of the SARS Coronavirus Papain-like Protease

Abstract

Deubiquitinating enzymes (DUBs) recognize and cleave linkage-specific polyubiquitin (polyUb) chains, but mechanisms underlying specificity remain elusive in many cases. The severe acute respiratory syndrome (SARS) coronavirus papain-like protease (PLpro) is a DUB that cleaves ISG15, a two-domain Ub-like protein, and Lys48-linked polyUb chains, releasing diUb(Lys48) products. To elucidate this specificity, we report the 2.85 Å crystal structure of SARS PLpro bound to a diUb(Lys48) activity-based probe. SARS PLpro binds diUb(Lys48) in an extended conformation via two contact sites, S1 and S2, which are proximal and distal to the active site, respectively. We show that specificity for polyUb(Lys48) chains is predicated on contacts in the S2 site and enhanced by an S1-S1' preference for a Lys48 linkage across the active site. In contrast, ISG15 specificity is dominated by contacts in the S1 site. Determinants revealed for polyUb(Lys48) specificity should prove useful in understanding PLpro deubiquitinating activities in coronavirus infections.

Graphical abstract

Figure 1

Distal diUb^Lys48 ABP Labels SARS PLpro (A) Ub-conjugate cleavage in TNF-α-treated HeLa cell lysates by SARS and MERS PLpro. Dotted lines added for clarity. (B and C) Schematics of (B) Ub chain recognition by SARS PLpro and (C) recognition and cleavage of biotin-tagged triUb^Lys48. (D) Cleavage of biotin-triUb^Lys48 by SARS PLpro. Cleavage intermediates detected by avidin-HRP reveal biotin on the diUb product. (E) Qualitative labeling of SARS PLpro by Ub-ABPs (cartoons at bottom with red stars indicate warhead positions). (F) Quantitative labeling of SARS PLpro by Ub-ABPs indicating percent of SARS PLpro labeled as derived from gels in Figure S2C. Error bars represent ±SEM. See also Figures S1 and S2.

Figure 2

Crystal Structure of SARS PLpro Bound to a diUb^Lys48-ABP (A) Cartoon and surface representations of SARS PLpro covalently bound to diUb^Lys48-ABP. SARS PLpro shown in surface representation with the USP-family DUB domain colored blue-white, the N-terminal Ubl domain in gray, the ^S2Ub binding motif in green, and the active site cysteine (C112) in yellow. Cartoon representation of Lys48-linked diUb with proximal Ub (^S1Ub) in salmon and distal Ub (^S2Ub) in raspberry. (B–E) Cartoon representation of interaction surfaces between SARS PLpro and diUb^Lys48; color as in (A). (B) Structure-based alignment of SARS-PLpro-diUb^Lys48 and a closed conformation of diUb^Lys48 (PDB 1AAR). Ub Ile44 (hydrophobic patch) that contact SARS PLpro shown as spheres. (C) View of the covalent triazole linkage between ^S1Ub-Lys48 and ^S2Ub-Gly75 indicating minimal contacts to SARS PLpro. (D) View of contacts between the SARS PLpro palm domain (M209 and Arg167/Asp168) and the ^S1Ub, highlighting both hydrophobic and polar interactions (^S1Ub-Ile44 and -Gln49/Arg42), respectively. (E) View of contacts between the SARS PLpro ^S2Ub binding motif (Phe70, Glu71, and His74) and the ^S2Ub-Ile44 hydrophobic patch (^S2Ub-Ile44, -Leu8, and -His68). See also Figure S3.

Figure 3

Contribution of SARS PLpro ^S1Ub and ^S2Ub Sites to polyUb^Lys48 Cleavage (A) Ub chain cleavage activities of SARS PLpro WT (black) and its mutants (^S2Ub, top, green; ^S1Ub, bottom, blue) on pentaUb^Lys48. Representative gels used to derive graphs shown in Figure S3B. Error bars represent ±SEM. (B) Schematics of tetraUb^Lys48 and diUb^Lys48 recognition by SARS PLpro. (C) Gel-based cleavage assays of SARS PLpro WT and mutants on tetraUb^Lys48 (top) and diUb^Lys48 (bottom) indicating differential effects of diUb ^Lys48 and tetraUb ^Lys48 cleaving activities for ^S1Ub and ^S2Ub mutants of SARS PLpro. Additional mutants analyzed in Figure S4C. (D) Cartoon and surface representation of SARS-PLpro∼diUb^Lys48 indicating the location of the ^S1Ub and ^S2Ub mutants. (E) Michaelis-Menten kinetics of WT (black) and selected SARS PLpro mutants (M209S, hydrophobic S1 mutant, light blue; R167S/E168R, polar S1 mutant, dark blue; F70S/E71K/H74G, S2 mutant, green) on diUb^Lys48-AMC substrates and comparison to SARS PLpro WT on monoUb-AMC (in red). Extracted kinetic parameters (k_cat and K_M) are in Table 2. See also Figure S4.

Figure 4

SARS PLpro Activity Is Restricted to Lys48-Linked Ub Chains with Specificity Dominated by S2-S1 Interactions (A and B) S2-S1 linkage specificity of SARS PLpro probed by (A) SDS-PAGE analysis of cross-linking to linkage-specific diUb-ABPs (SYPRO-stained) and (B) release of AMC using diUb-AMC fluorogenic substrates with initial linear cleavage rates (V_i) plotted as percent of diUb^Lys48-AMC cleavage rate by WT SARS PLpro as derived from curves in Figure S4B. (C) View of SARS-PLpro∼diUb^Lys48-ABP highlighting the location of ^S1Ub-Lys48 (dark green, sticks) and its proximity to Phe70 (green) in the SARS PLpro ^S2Ub binding site. Other chain-forming ^S1Ub residues (Lys-6, -11, -27, -29, -33, and -63 and Met1) shown as sticks (red). (D) Linkage specificity of SARS PLpro assayed using homotypic tetraUb chains. Representative gel shown and bar graph indicating ±SEM from duplicate experiments. (E) Schematics of triUb^Lys48 chain recognition by SARS PLpro via S2-S1 (green) and S1′ (gray). (F) Cartoon of tetraUb chains, indicating SARS PLpro preferred sites of recognition (curved line) and cleavage (dotted line). (G) Time-course for cleavage of tetraUb chains by SARS PLpro and its S2 mutant. (H) Quantification of V_i as determined from duplicate experiments in Figure 4G. Error bars represent ±SEM. See also Figure S5.

Figure 5

Recognition of DiUb^Lys48 and ISG15 by SARS PLpro Appears Distinct (A) Michaelis-Menten kinetics of WT (black) and selected SARS PLpro mutants (M209S, S1 mutant, light blue; R167S/E168R, S1 mutant, dark blue; F70S/E71K/H74G, S2 mutant, green) using ISG15-AMC. Extracted kinetic parameters (k_cat and K_M) in Table 2. (B) Cleavage of HMW-Ub^Lys48 (top, WB anti-K48) and ISG15-conjugates (bottom, WB ISG15) in lysates prepared from IFNβ/MG132-treated cells by SARS PLpro WT and S2 and S1 mutants. (C) Quantification of loss of HMW-Ub^Lys48 (left) and appearance of free ISG15 (right) from duplicate experiments shown in Figure 5B. Error bars represent ±SEM. (D) Schematic representation of SARS PLpro substrate specificity. Dashed lines can indicate the -AMC substrate, a non-Lys48-linked Ub unit, or a protein substrate. See also Figure S6.