Figure 1.
The PLpro-ubiquitin interface involves both polar and hydrophobic interactions.
PLpro is shown in blue and ubiquitin is shown in orange. Areas of interest are labeled. The ‘hydrophobic patch’ of ubiquitin is colored in magenta. Ubiquitin residues are italicized. A. Surface representation of PLpro bound to ubiquitin aldehyde. B. Overall schematic of the interface. C. A detailed view of the polar area of contacts designated in (B). Hydrogen bonds directly between PLpro and ubiquitin and those between intervening water molecules (red spheres) are shown as dashed lines. D. A detailed view of the hydrophobic area of contacts designated in (B).
Figure 2.
The crystal structure of the PLpro-Ubal complex reveals a dense hydrogen-bonding pattern between the active site of PLpro and the C-terminus of ubiquitin.
A. Stereoview of PLpro active-site interactions with ubiquitin-aldehyde. PLpro residues are shown in blue and labeled in black, and ubiquitin residues are shown and labeled in orange. Hydrogen bonds between PLpro and ubiquitin are shown as dashed lines. B. Electron density associated with the region surrounding the C-terminal residues of ubiquitin (orange density) and their interactions with the PLpro active site in the region of the mobile loop (blue density). The residues shown and the view depicted are similar to those in panels A and C. The electron density maps were calculated by omitting ubiquitin from the structure factor calculations. The Fo-Fc map for ubiquitin (orange) is contoured at 3σ and the 2Fo-Fc map for PLpro (blue density) is contoured at 1.5σ. The figure was generated using the program Pymol. C. Comparison of the PLpro active site loop in bound (blue with white surface) and unbound (yellow) conformations. The C-terminus of ubiquitin is shown in orange. The orientation of the structure is similar to that shown in panels A and B.
Figure 3.
PLpro prefers K48-linked ubiquitin to K63-linked ubiquitin and ISG15 to mono-ubiquitin.
A. SDS-PAGE analysis of PLpro isopeptidase activity with K48-Ub5 (top gel) and K63-Ub5 (bottom gel) chains. Reactions times (in minutes) are indicated above each well, and the different ubiquitin species are marked to the right of the gels. Note the inability of PLpro to process K48-Ub2 to mono-ubiquitin. B. Comparison of PLpro activity with Ub-AMC (gray circles) and ISG15-AMC (white squares) as a function of substrate concentration. C. The inhibition of PLpro activity by free mono-ubiquitin (gray circles), free ISG15 (white squares), K63-Ub2 (black diamonds) and K48-Ub2 (black circles) is plotted as a function of inhibitor concentration.
Figure 4.
Proposed recognition models of K48-Ub2 and ISG15 by PLpro.
PLpro is shown as a surface representation and colored grey. The ubiquitin binding subsites distal to the isopeptide bond are indicated in red for SUb1 and blue for SUb2. Ubiquitin molecules in the chain are indicated as circles in yellow and numbering follows conventional protease substrates numbering with ubiquitins distal to the isopeptide bond as Ub2, Ub1 and those proximal as Ub1′ etc. Ubiquitin lysines are labeled as K48 or K63. The greater than symbols (>>) designate the relative affinity of one complex over the other from data presented in Figure 3.
Figure 5.
Modeling and mutational analysis suggest that PLpro binds to ISG15 and K48-Ub2, but not K63-Ub2, in a bidentate manner.
Initial model of PLpro (blue surface) bound to ISG15 (yellow) (A), K48-Ub2 (orange) (B), and K63-Ub2 (orange) (C). The multiple conformations of the distal ubiquitin depicted as transparent cartoons in (B) represent the range of pre-experimental binding configurations for K48-Ub2. D. A detailed view of mutated PLpro residues designed to locate the second binding site for K48-Ub2 and ISG15. These residues are shaded in cyan in A–D. Residues marked with an asterisk designate follow-up mutations.
Figure 6.
K48-Ub2 and ISG15 interact with a “ridge” region on the thumb domain of PLpro.
PLpro mutants were purified and tested for activity with Ub-AMC (white bars) and ISG15-AMC (blue bars) and for their ability to be inhibited by 5 µM K48-Ub2 (gray bars). All measurements were performed in triplicate and are reported in % activity or % inhibition relative to wild-type PLpro. The ribbon diagram below the bar graph illustrates the position of each mutant on PLpro. Mutants marked with an asterisk represent follow-up mutants.
Figure 7.
Updated models of (A) ISG15 (yellow) and (B) K48-Ub2 (orange) and bound to PLpro (blue).
The ubiquitin hydrophobic patch residues are shown in magenta. PLpro residues identified through site-directed mutagenesis as important for K48-Ub2 and ISG15 binding are highlighted in a yellow circle. The two distal regions involved in binding Ub2 or ISG15 are labeled as distal-1 (closest to active site, location of single ubiquitin binding) and distal-2 (binding of second ubiquitin-like domain).
Figure 8.
Deubiquitinating activity and NF-κB antagonism are reduced by mutation of SARS-CoV PLpro residue F70.
(A) HEK293T cells were transfected with expression plasmids encoding FLAG-Ub and the indicated PLpro. At 18 hours post transfection, cells were lysed and immunoblotted for FLAG-Ub, calnexin, and PLpro-V5. (B) HEK293T cells were transfected with constructs expressing nsp2-3-GFP and SARS-CoV PLpro-V5. Lysates were immunoblotted with anti-GFP, anti-calnexin, and anti-V5. (C) HEK293 cells were transfected with IkBα-HA and the indicated PLpro. After 16 hours incubation, cells were stimulated with TNFα (20 ng/ml). Lysates were analyzed by 10% SDS-PAGE and immunoblotted for anti-HA, anti-calnexin, and anti-V5. (D) 293HEK cells were transfected with NFkB-reporter and Renilla luciferase control constructs and the indicated PLpro. After 12 hours, TNFα was added to a final concentration of 10 ng/mL and the cells were incubated for an additional 4 hours. Results are normalized to induction of NFkB reporter activity by TNFα. Panels below are western blots of the lysates using anti-V5 for detection of PLpro and anti-actin as a protein loading control. Experiments were performed in triplicate and repeated twice. * = p<.05 statistical difference from mock transfected cells by student t-test.