Turnip yellow mosaic virus protease binds ubiquitin suboptimally to fine-tune its deubiquitinase activity

Abstract

Single-stranded, positive-sense RNA viruses assemble their replication complexes in infected cells from a multidomain replication polyprotein. This polyprotein usually contains at least one protease, the primary function of which is to process the polyprotein into mature proteins. Such proteases also may have other functions in the replication cycle. For instance, cysteine proteases (PRO) frequently double up as ubiquitin hydrolases (DUB), thus interfering with cellular processes critical for virus replication. We previously reported the crystal structures of such a PRO/DUB from Turnip yellow mosaic virus (TYMV) and of its complex with one of its PRO substrates. Here we report the crystal structure of TYMV PRO/DUB in complex with ubiquitin. We find that PRO/DUB recognizes ubiquitin in an unorthodox way: It interacts with the body of ubiquitin through a split recognition motif engaging both the major and the secondary recognition patches of ubiquitin (Ile⁴⁴ patch and Ile³⁶ patch, respectively, including Leu⁸, which is part of the two patches). However, the contacts are suboptimal on both sides. Introducing a single-point mutation in TYMV PRO/DUB aimed at improving ubiquitin-binding led to a much more active DUB. Comparison with other PRO/DUBs from other viral families, particularly coronaviruses, suggests that low DUB activities of viral PRO/DUBs may generally be fine-tuned features of interaction with host factors.

Keywords: crystal structure; deubiquitylation (deubiquitination); plant virus; plus-stranded RNA virus; protein complex; ubiquitin; viral protease.

Figure 1.

Overall structure of the covalent TYMV PRO/DUB–Ub complex. A, crystal structure of the covalent TYMV PRO/DUB–Ub complex. TYMV PRO/DUB is represented as molecular surface, with the N-terminal (N-ter) lobe in yellow, the central lobe in magenta, and the C-terminal (C-ter) lobe in green. The enzyme's catalytic dyad, composed of Cys⁷⁸³ and His⁸⁶⁹, is indicated in red. HA–Ub-VME is shown in ribbon diagram and colored in orange. Residues Ile³⁶ and Ile⁴⁴ are displayed in ball-and-stick format and colored in cyan. Ubiquitin residues are labeled in italics and underlined. B, sequence alignment of polyprotein processing endopeptidases belonging to the Tymoviridae family. The sequence of TYMV PRO/DUB was aligned with enzymes encoded by Chayote mosaic virus (ChMV), Physalis mottle virus (PhMV), Eggplant mosaic virus (EMV), Dulcamara mottle virus (DuMV), Okra mosaic virus (OkMV), and Kennedya yellow mosaic virus (KYMV) as in the work of Lombardi et al. (34). Alignment was performed by CLUSTALW (82), edited, and displayed with ESPript3 (83). White characters in red boxes indicate identity, and red characters in white boxes indicate homologous residues. Secondary structures of TYMV PRO/DUB (PDB code 4A5U (34)) are indicated on top. Black stars indicate the residues of enzyme interacting with Ub Ile⁴⁴ hydrophobic patch, black circles indicate the residues interacting with Ub Ile³⁶ hydrophobic patch, and black triangles indicate the residues interacting with the C-ter extremity of Ub.

Figure 2.

Interactions between TYMV PRO/DUB and Ub. A, close-up view of the Ub Ile⁴⁴ patch. Both proteins are shown in cartoon, and residues involved in the interaction are shown in stick. Proteins are colored as in Fig. 1, with oxygen and nitrogen atoms in red and blue, respectively. The hydrogen bond between Gln⁴⁹ from Ub and Thr⁷⁶³ from TYMV PRO/DUB is shows as a dotted line. B and C, analysis of interactions around the Ub Ile⁴⁴ patch by molecular dynamics simulations. The distances between three pairs of residues were measured during 90 ns of production time in two simulations, and their frequency was plotted. Gray, hydrogen bonds and electrostatic interactions; black, hydrophobic contact. D, close-up view of the Ub Ile³⁶ patch. Proteins and residues are represented and colored as in A. E and F, analysis of interactions around the Ub Ile³⁶ patch by molecular dynamics simulations. The distances between two pairs of residues were measured during 90 ns of production time in two simulations, and their frequency was plotted, as in B and C. G, close-up view around Leu⁸ of Ub. Both proteins are displayed in cartoon loop with some side chains shown in stick. The cavity of TYMV PRO/DUB that fits Ub is highlighted by the gray molecular surface of the enzyme. Three crystal structures of Ub were superimposed to compare the position of the loop encompassing Leu⁸: purple, loop-out conformation (PDB code 1UBQ (73)); orange, intermediate conformation (this work); blue, loop-in conformation (PDB code 2G45 (84)). H, overall view of the two polar loops of TYMV PRO/DUB that bind the two hydrophobic patches of Ub.

Figure 3.

In vitro DUB activity of structure-guided mutants of TYMV PRO/DUB. A, DUB activity of recombinant TYMV PRO/DUB (WT and structure-guided mutants) was measured by a fluorescence assay using Ub-AMC as substrate. K_app was determined according to the equation V/[E] = K_app [S], where V is the initial velocity calculated from the kinetic data, and [E] and [S] are the corresponding enzyme and substrate concentrations. The values are expressed as the percentages of that of WT protein. B and C, behavior of residue 844 side chain (B) and of the catalytic dyad (Cys⁷⁸³ and His⁸⁶⁹) (C) was investigated by performing molecular dynamics simulations of the product state complex, using WT TYMV PRO/DUB or R844A mutant. The R844A mutant was generated by truncating the Arg side chain at Cβ to mimic an alanine. The distances were measured along the same 90 ns in two simulations as in Fig. 2 (WT, red histograms) and along 90 ns in two simulations for R844A (black histograms). B, distance between the side chains of TYMV PRO/DUB residue 844 (Cβ atom) and Ub Ile³⁶ (Cγ1 atom). C, distance between TYMV PRO/DUB Cys⁷⁸³ (Sγ atom) and His⁸⁶⁹ (Nδ1 atom). The minor peak at 3.5 Å signals alignment of the catalytic dyad.

Figure 4.

Interactions network at the C-terminal tail of Ub. A, detailed hydrogen bonding between the last five residues of Ub and TYMV PRO/DUB. The C-terminal extremity backbone of Ub (including Arg⁷² to Gly-VME76) is represented as sticks. The residues of TYMV PRO/DUB involved in the interaction with Ub are displayed as sticks. Hydrogen bonds are shown as dotted lines. B, electrostatic interactions between three Arg of Ub (Arg⁴², Arg⁷², and Arg⁷⁴) with the acidic pocket of TYMV PRO/DUB constituted by Glu⁸¹⁶ and Glu⁸²⁵, and Asp³⁹.C, global hydrophobic interactions network between the last five residues of Ub and TYMV PRO/DUB. Both proteins are shown in cartoon, with residues involved in interaction depicted in sticks. The overall coloring scheme is the same as that in Figs. 1 and 2.

Figure 5.

Comparison of the binding interfaces in the PRO/DUB–Ub and PRO/DUB·PRO complexes. A and C, covalent and noncovalent complexes between TYMV PRO/DUB and Ub (this work) (A) or TYMV PRO/DUB from the PRO↓HEL cleavage site (PDB code 4A5U (34)) (C), respectively, are shown in surface representation. The enzyme TYMV PRO/DUB is colored in gray, and the substrates Ub and TYMV PRO/DUB are colored in orange and pink, respectively. B and D, the interacting surfaces used by the enzyme to bind its substrates (B, Ub; D, PRO/DUB) are colored in cyan and are shown after rotation of the protein.

Figure 6.

Ub-binding mode with TYMV PRO/DUB and other viral and cellular DUBs. Comparison of overall Ub-binding mode for viral PRO/DUBs (black lettering), viral OTU DUBs (blue lettering), and cellular OTU DUBs (magenta lettering). Whether each enzyme belongs to the OTU or USP family is indicated. Crystal structures of Ub in complex with viral or cellular PRO/DUBs or DUBs were aligned with PyMOL. In each case, Ub is displayed as molecular surface and colored in orange, whereas the enzyme is shown in green cartoon. The two hydrophobic patches of Ub are colored in cyan. Ile⁴⁴ and Ile³⁶ of Ub, at the center of the two patches, are shown in blue, and Leu⁸, located in a loop between the two patches, is highlighted in red. The conformation of the Ub Leu⁸ loop in each complex is compared with the classical loop-out and loop-in conformations and to the intermediate conformation found in TYMV PRO/DUB–Ub complex (inset, see also Fig. 2G). We chose for comparison PRO/DUBs encoded by SARS-CoV (PDB code 4MM3 (41)), MERS-CoV (PDB code 4RF1 (59)), MHV (PDB code 5WFI, unpublished structure), and EAV (PDB code 4IUM (25)) (black) and DUBs encoded by CCHFV (PDB code 3PHW (55)) and DUGV (PDB code 4HXD (56)) (blue). We also compared DUBs from yeast (PDB code 3BY4 (60)) and human (PDB code 4BOS (61)) (magenta).