Polyclonal antisera raised against the TYMV 66K protein, the PRR domain of 98K protein (hereafter anti-98K antiserum) and the TYMV capsid were described previously [54, 89], and were used at dilutions of 1/2,000, 1/8,000, and 1/50,000, respectively. Detection of 66K-Ub conjugates following 66K IP was performed as described [28, 32]. (3.4M) GUID:?60DEA158-808F-468E-AC10-5E2F8F3D9DEA S4 Fig: Overlay of the asymmetric units of crystals of C5 and I847A. Color code as in Fig 2. No fitting was performed.(TIF) ppat.1006714.s004.tif (4.9M) GUID:?CE207857-559B-45AA-A4AB-B729A57D58F5 S5 Fig: Layouts of the two sides of the active site clefts in the Lobeline hydrochloride coronavirus USP-like PRO/DUBs in the same representation as for the OTU DUBs of Fig 8. (A) The SARS CoV PRO/DUB in free (PDB 2FE8) and diubiquitin-bound (PDB 5E6J) forms. Note the large size and displacement of the glycine-hinged loop bearing Lobeline hydrochloride C271 between the two forms. (B) The MERS CoV PRO/DUB in free (PDB 4REZ) and ubiquitin-bound (PDB 4RF0) forms. Top left panel: Here the glycine-hinged loop is disordered in the free form. The asterisks denote G1752 and G1758 on either side of the disordered loop.(TIF) ppat.1006714.s005.tif (5.5M) GUID:?386E6800-BA1F-47F8-8579-90DB3C4BE72D S6 Fig: Layouts of the two sides of the active site cleft Lobeline hydrochloride in the USP7 cellular DUB in a similar representation as for the OTU DUBs of Fig 8. The switching loop is located by residue Q293 and labeled. In the bottom panels, the distances between the sulfur of C223 and the N1 of H464 are indicated as in Fig 1B. Left, free USP7 (PDB 1NB8). Right, ubiquitin-bound USP7 (PDB 1NBF).(TIF) ppat.1006714.s006.tif (9.2M) GUID:?5FCA7E52-0677-4605-BAC9-81806C2B21DE S1 Dataset: RT-qPCR raw data, quantification and normalization, and relative RNA accumulation of mutant transcripts in Arabidopsis protoplasts and Arabidopsis plants. (XLSX) ppat.1006714.s007.xlsx (178K) GUID:?5C16DF6D-BEE1-4D01-89BC-368B28B227DD S2 Dataset: Original dataset for Fig 4. (TIF) ppat.1006714.s008.tif (1.1M) GUID:?D69BC4BE-953D-430A-A351-F149F73AD33F S3 Dataset: Original dataset for Fig 5. (TIF) ppat.1006714.s009.tif (3.7M) GUID:?2A46EFD8-D63C-496E-A260-3A00CB9E9CC1 S4 Dataset: Original dataset for Fig 6. (TIF) ppat.1006714.s010.tif (8.7M) GUID:?1FF64080-1303-45D4-8085-331C412FBCB6 Data Availability StatementCoordinates and diffraction data Cdc14A1 have been deposited in the Protein Data Bank as entries 5LW5 and 5LWA. All other relevant data are within the paper and its Supporting Information files. Abstract The positive-strand RNA virus (TYMV) encodes an ovarian tumor (OTU)-like protease/deubiquitinase (PRO/DUB) protein domain involved both in proteolytic processing of the viral polyprotein through its PRO activity, and in removal of ubiquitin chains from ubiquitylated substrates through its DUB activity. Here, the crystal structures of TYMV PRO/DUB mutants and molecular dynamics simulations reveal that an idiosyncratic mobile loop participates in reversibly constricting its unusual catalytic site by adopting “open”, “intermediate” or “closed” conformations. The two and (TYMV), the type member of the genus and [26C28]. We also reported that the TYMV papain-like cysteine PRO domain involved in the proteolytic processing of the viral replication precursor protein [29C31], also displays a DUB activity that was able to rescue the viral polymerase from degradation [32]. The structure of the recombinant TYMV PRO/DUB domain we solved previously (residues 728C879 of the TYMV replication precursor protein) [21] highlighted its homology with members of the ovarian tumor domain-containing (OTU) superfamily of DUBs [33]. However, TYMV PRO/DUB appeared to be a very peculiar OTU DUB, both Lobeline hydrochloride functionally and structurally. Indeed, in contrast to other OTU DUBs, TYMV PRO/DUB does not possess a general deubiquitylating activity, but instead displays a specificity towards particular ubiquitylated substrates, among them TYMV RNA-dependent RNA polymerase [32]. In addition, the TYMV PRO/DUB structure displayed a surprisingly exposed and minimal catalytic site, with a Cys783-His869 catalytic dyad [21] instead of the conserved OTU DUB Cys-His-Asp/Asn catalytic triad [6, 34, 35]. In TYMV polyprotein processing, one endopeptidase site lies at the C-terminus of PRO/DUB itself [31] (S1 Fig), and, in.