Rhodanese-Like Domain Protein UbaC and Its Role in Ubiquitin-Like Protein Modification and Sulfur Mobilization in Archaea

RHD protein fusions and stand-alone proteins are widespread in archaea but poorly understood. Of the seven RHD proteins of H. volcanii, two are tandem RHDs (HVO_0025 and HVO_0024), three are RHDs fused to a C-terminal metallo-β-lactamase domain, and two are stand-alone RHDs (UbaB and UbaC) (Fig. 1). Of these RHDs, UbaB is in genome synteny with the E1-like UbaA, suggesting that it may be associated with the Ubl systems; however, UbaB is not required for Ubl modification or sulfur mobilization to form Moco or thiolated wobble uridine tRNA, based on a previous study (8).

To further examine the relationship of RHDs to Ubl systems, two sequence similarity networks (SSNs) were generated. First, archaeal E1s (including those with C-terminal RHDs) were analyzed for their similarity in amino acid sequence and protein domain architecture (Fig. 2). The most common domain fused to the archaeal E1s was the RHD, followed by domains related to JAB/MPN metalloproteases that cleave Ub/Ubl isopeptide linkages (3, 27, 29) and β-grasp fold proteins (Ub and ThiS) (Fig. 2). Most archaeal E1s, however, were stand-alone proteins, including H. volcanii UbaA, which shared close relationship to the E1s fused to RHDs. To further understand the E1-RHD relationship, a second SNN was generated by comparing the RHDs extracted from the eukaryotic E1s (MOCS3/Cnx5/Uba4) and archaeal arCOG02021 (Fig. 3A). When examining the H. volcanii RHDs in this SSN configuration, UbaB/C and the metallo-β-lactamase-fused RHDs were found in distinct clusters connected by edges to the central cluster of stand-alone and E1-fused RHDs (Fig. 3A). The H. volcanii tandem RHDs (HVO_0024 and HVO_0025) were distinct and did not appear to be related to the E1-RHDs (Fig. 3A). Alignment of UbaB/C to the C-terminal domain of the eukaryotic E1-RHD fusions MOCS3/Uba4/Cnx5 revealed the key active-site cysteine that carries the persulfidic sulfur is conserved (Fig. 3B). UbaB was also found to share 36% identity with UbaC (Fig. 3C, left), with both proteins having predicted three-dimensional (3D) structures related to the human MOCS3 RHD X-ray crystal structure (Fig. 3C, middle and right). Thus, UbaB/C are related to each other and to E1 RHDs and have a conserved cysteine predicted to be modified by a persulfide group (30–32).

To determine the archaeal RHD required for Moco biosynthesis, a series of H. volcanii RHD mutants (ΔubaB, ΔubaC, Δhvo_0024, and Δhvo_0025) was generated and compared to parent strain H26 cells in terms of DMSO respiration. H. volcanii does not grow anaerobically if DMSO is excluded from the medium, and it requires MPT synthase subunit homologs (MoaE and SAMP1) and the E1-like UbaA for DMSO reductase activity (4). After 3 days of incubation at 42°C, the ΔubaB and the tandem RHD (Δhvo_0024 and Δhvo_0025) mutants displayed growth comparable to that of the parent (H26) when supplied with DMSO as the terminal electron acceptor (Fig. 4A). In contrast, little to no growth was observed for the ΔubaC mutant under these conditions (Fig. 4A). The Δsamp1 and ΔubaA mutants were also unable to grow on DMSO (Fig. 4A), as observed in a previous study (8). When ubaC was expressed in trans, the ΔubaC mutant was restored for anaerobic respiration on DMSO, revealing that the effect was specific to ubaC. The conserved active-site cysteine (C64) of the UbaC RHD loop was required, as the UbaC C64S variant did not complement the ΔubaC mutation (Fig. 4A). While UbaC shares amino acid sequence identity with UbaB, presumed overexpression of UbaB from a constitutive rRNA P2 promoter did not restore the ability of the ΔubaC mutant to respire on DMSO (Fig. 4A). Inclusion of tungstate, a known antagonist of molybdate insertion into the pterin ring during Moco maturation in E. coli (33), prevented all H. volcanii strains from respiring DMSO (Fig. 4A). This effect could be reversed for the H. volcanii parent (H26) and ΔubaC mutant trans-expressing ubaC strains by including molybdate in the medium (Fig. 4A). Thus, tungstate had an antagonistic effect on DMSO respiration that could be overcome by molybdate supplementation. The ΔubaC mutant and its ubaC C64S trans-expression strain did not respire on DMSO under all conditions examined (Fig. 4A). Furthermore, all strains grew in the presence of oxygen even when supplemented with tungstate (Fig. 4A), revealing that the shift in terminal electron acceptor from oxygen to DMSO was important for the phenotypes observed. Collectively, these results reveal that UbaC and its active-site cysteine (C64) are required for DMSO respiration and do not share redundant functions with UbaB in this pathway.

To further understand the role of UbaC in DMSO respiration, the H. volcanii strains were analyzed for DMSO reductase activity, which requires Moco (34, 35). To test for this activity, clarified lysates were assayed for the oxidization of reduced methyl viologen in the presence of DMSO. By this approach, DMSO reductase activity was detected in the parent (H26) but not in the ΔubaC mutant (Fig. 4B). Furthermore, ectopic expression of UbaC restored the DMSO reductase activity of the ΔubaC mutant, while the empty-vector control and the UbaC C64S mutant did not (Fig. 4B). Thus, ubaC was important for DMSO reductase activity, consistent with its putative role in Moco biosynthesis.

Hypothiolation of wobble uridine tRNA is correlated with reduced translational fidelity and impaired growth at elevated temperature (8, 28, 36, 37). To indirectly address the role of the archaeal RHDs in tRNA thiolation, the H. volcanii RHD mutants were examined for growth at 50°C compared to the 42 to 45°C optimum. The ΔubaB, Δhvo_0024, and Δhvo_0025 RHD mutants were found to grow similarly to the parent H26 at all temperatures examined (Fig. 4C). In contrast, the ΔubaC mutant was impaired for growth at 50°C (but not at 42°C) (Fig. 4C), having a growth phenotype that corresponded to those of ΔubaA and ΔncsA mutants deficient in the 2-thiolation of wobble uridine tRNA (8, 28). Growth of the ΔubaC mutant at 50°C was restored by ectopic expression of ubaC but not ubaC C64S (Fig. 4C). Thus, UbaC and its conserved cysteine (C64) were found to be important for growth at elevated temperature, which may be correlated with tRNA thiolation.

The observed impact of UbaC on cellular functions associated with sulfur mobilization suggested that this RHD may physically associate with UbaA in H. volcanii. To examine this potential interaction, a pulldown assay was performed using UbaC–Strep-tag II as the bait. Untagged UbaC was included as a negative control to rule out any nonspecific proteins that may bind the affinity column. UbaA (encoded from the H. volcanii genome) was found to copurify with UbaC–Strep-tag II in a specific manner (Fig. 5). This pulldown experiment provided evidence that UbaA and UbaC interact in the cell.

The physical association of UbaC with UbaA suggested that UbaC may be required for Ubl modification of protein targets, similarly to the RHD of the Uba4 E1-RHD fusion in urmylation. Thus, the ΔubaC mutation was analyzed for its impact on (i) the covalent attachment of the Ubl SAMP1 to MoaE (3, 5, 8) and (ii) the accumulation of Ubl modifications after exposure of cells to the oxidizing agents DMSO, methionine sulfoxide (MetO), and NaOCl and the proteasome inhibitor bortezomib (5, 11, 38). Ubl modification by SAMP1, SAMP2, and SAMP3 was monitored by individually Flag tagging the SAMPs. The parent H26 and the ΔubaA (E1-like) mutant were included for comparison, with the latter impaired in all Ubl modifications examined (7, 8). By this approach, ubaC was found to be important for the covalent modification of MPT synthase (SAMP1 linked to MoaE). The covalently modified MPT synthase that migrated at 50 kDa was not apparent in the ΔubaC mutant (Fig. 6A and B, lanes 1 versus 4), which instead accumulated Ubl (SAMP1) linkages at 37 and 60 kDa (Fig. 6A, lane 4) that migrated similarly to the Ubl linkages detected when UbaA–Strep-tag II was ectopically expressed (Fig. 6A, lane 5). These alternative Ubl linkages were speculated to be UbaA modified by SAMP1, based on this close relationship in SDS-PAGE migration (Fig. 6A, lane 4 versus 5) and our previous finding by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis that UbaA is autosampylated at K87 and K157 (7). UbaC was not needed for formation of the SAMP1 Ubl linkages that were induced by DMSO (Fig. 6A, lane 10) or MetO (Fig. 6B, lane 9). Contrastingly, most of the SAMP2 Ubl linkages that accumulated in the presence of an oxidizing agent and/or proteasome inhibitor were impaired in the ΔubaC mutant (Fig. 7A, lanes 3, 11, 19, and 25). Note that of the three H. volcanii SAMPs, only SAMP2 appears to function like eukaryotic Ub in targeting proteins for proteasome-mediated proteolysis (1, 5). In addition to SAMP2, the oxidant-induced SAMP3 Ubl linkages were also abolished in the ΔubaC mutant (Fig. 7B, lanes 3 and 10). Ectopic expression of ubaC was found to restore the Ubl SAMP2/3 modifications in the ΔubaC mutant (Fig. 7A, lanes 4, 12, 20, and 26, and B, lanes 4 and 11); UbaC64S was not functional in this restoration (Fig. 7B, lane 8). Consistent with a role in stress-induced Ubl modifications, UbaC and its active-site C64 were important for cell survival after exposure to NaOCl similarly to UbaA (Fig. 8). Thus, UbaC and its conserved active-site cysteine (C64) not only are important for the Ubl SAMP2/3 linkages that accumulate after exposure to oxidant and proteasome inhibitor but also are needed for cell survival during stress, such as exposure to the potent oxidant hypochlorite generated during NaOCl treatment.

Biochemicals and analytical-grade inorganic chemicals were purchased from Fisher Scientific (Atlanta, GA), Bio-Rad (Hercules, CA), and Sigma-Aldrich (St. Louis, MO). Desalted oligonucleotides were from Integrated DNA Technologies (Coralville, IA). DNA polymerases and restriction enzymes were from New England Biolabs (Ipswich, MA). Hi-Lo DNA standards were from Minnesota Molecular, Inc. (Minneapolis, MN). Hybond-P polyvinylidene fluoride (PVDF) membranes for Western blots were from GE Healthcare.

The genotypic profiles of bacterial and haloarchaeal strains used in this study are detailed in Table S1 in the supplemental material. Nutrient broth cultures were grown with rotary agitation at 200 rpm unless otherwise noted. Bacterial cultures were grown at 37°C in Luria-Bertani (LB) medium supplemented with ampicillin (Amp) (100 μg·ml⁻¹) as needed. H. volcanii strains were grown at 42°C in ATCC 974 complex medium and yeast-peptone-Casamino Acids (YPC) (50). For H. volcanii strains carrying the expression plasmids, media were supplemented with novobiocin (Nov, 0.2 μg·ml⁻¹). All strains were stored in 20% glycerol stocks at −80°C and freshly streaked for isolated colonies for analysis.

DNA was separated by electrophoresis using 0.8% or 2% (wt/vol) agarose gels in 1× TAE electrophoresis buffer (40 mM Tris acetate, 2 mM EDTA, pH 8.5). Gels were stained with ethidium bromide at 0.5 μg · ml⁻¹ and photographed with a Mini visionary imaging system (Fotodyne, Hartland, WI). Sizes of the DNA fragments were estimated using Hi-Lo DNA molecular weight markers (Minnesota Molecular, Inc., Minneapolis, MN). Plasmid DNA was isolated from E. coli strains using a QIAprep spin miniprep kit (Qiagen, Valencia, CA). PCRs were carried out using primers detailed in Table S2 in the supplemental material and Phusion and Taq polymerase for cloning and colony-based confirmatory tests, respectively. PCR products were purified by MinElute (Qiagen).

Amino acid exchange of UbaC Cys64 (conserved at the RHD active-site loop) to Ser was performed by PCR-based site-directed mutagenesis (Stratagene, La Jolla, CA). UbaC–Strep-tag II (pJAM983; for H. volcanii expression) was used as a template to generate pJAM2705 (UbaC C64S–Strep-tag II).

The open reading frame hvo_1947 (encoding the putative RHD protein, UbaC) was targeted for markerless deletion from the chromosome of H. volcanii H26 using the pyrE2-based “pop-in/pop-out” method as previously described (51) using suicide plasmid pJAM1900. Transformants were plated on Hv-minimal salts-Casamino Acids-agar medium (50), and direct colony PCR screening was used to check for pop-ins. Four pop-in colonies were pooled in 4 ml ATCC 974 with 5-fluoroorotic acid (5-FOA) (50 μg · ml⁻¹) and plated on the same medium with agar. Null mutants were screened for the absence of 370-bp PCR product using primers that amplify 0.5 kb upstream/downstream flanking regions of hvo_1947 (ubaC). Plasmids pJAM983 (UbaC–Strep-tag II), pJAM2705 (UbaC C64S–Strep-tag II), pJAM2716 (UbaB–Strep-tag II), and pJAM202c (empty vector) were transformed into H. volcanii ΔubaC. A similar approach was used to generate the other H. volcanii mutant strains listed in Table S1.

H. volcanii cells from freshly streaked ATCC 974 plates were grown overnight in 4 ml YPC medium to log phase (optical density at 600 nm [OD₆₀₀] of 0.4 to 0.6) and subcultured to 4 ml fresh YPC medium using a 0.25% (vol/vol) inoculum. Cells at log phase (OD₆₀₀ of 0.4 to 0.6) were used as an inoculum to fresh YPC medium with DMSO (to a final concentration of 100 mM) in screw-cap tubes (9 ml, 13 by 100 mm) from a starting OD₆₀₀ of 0.02. Tubes were filled completely with medium and capped with phenolic caps. Cells were incubated at 42°C without agitation, and the cell density was monitored as OD₆₀₀ using a Spectronic20+ spectrophotometer (Thermo Scientific, Madison, WI). To examine tungstate as a molybdate antagonist, the medium was supplemented with tungstate (10 mM) and/or molybdate (1 mM) as indicated.

H. volcanii cells were cultured in triplicate in 50 ml YPC medium with DMSO (100 mM) for 36 h at 42°C with agitation at 200 rpm. Cells were harvested by centrifugation (6,000 × g, 4°C), resuspended in 15 ml high-salt Tris buffer (2 M NaCl, 1 mM EDTA, 50 mM Tris, pH 7.5), and lysed by French press thrice (2,000 lb/in²). Whole-cell lysate was clarified by centrifugation (17,000 × g, 4°C) and filtration (0.2 μm), and the protein concentration of the soluble fraction was measured using a bicinchoninic acid (BCA)-based protein assay kit. DMSO reductase activity was monitored by A₆₀₀ (15-s intervals for 3.5 min) with nitrogen as the headspace. Assay mixtures (4 ml) included cell lysate (1 to 1.5 mg protein) and 0.3 mM methyl viologen in buffer A. The mixture was titrated with fresh 20 mM sodium dithionite (Na₂S₂O₄) in 20 mM sodium bicarbonate (NaHCO₃) until an A₆₀₀ of 1 to 1.2 units was reached prior to addition of 10 mM DMSO. One unit of enzyme activity is defined as 1 μmol substrate consumed per min at room temperature with an extinction coefficient A₆₀₀ of 13.6 mM⁻¹cm⁻¹ for methyl viologen. All assays were performed in biological triplicate, with the means ± standard deviations (SD) calculated.

H. volcanii cells from freshly streaked ATCC 974 plates were grown overnight in 4 ml ATCC 974 medium to log phase (OD₆₀₀ of 0.4 to 0.6) and subcultured to fresh 4 ml ATCC 974 medium using a 0.25% (vol/vol) inoculum. Cells at log phase (OD₆₀₀ of 0.4 to 0.6) were serially diluted onto ATCC 974 plates and grown at 42°C and 50°C. Alternatively, the log-phase cells were exposed to NaOCl (20 mM) or a mock control for 10 to 60 min, serially diluted, spotted onto ATCC 974 plates, and then incubated for 3 days at 42°C.

Flag-SAMPs, alone or with MoaE–Strep-tag II, UbaC–Strep-tag II, UbaA–Strep-tag II, or variant proteins, were expressed in H. volcanii. Novobiocin (0.2 μg · ml⁻¹) was included in the medium to maintain the expression plasmids. Cells were grown overnight in 4 ml ATCC 974 and subcultured to 4 ml fresh ATCC 974 with DMSO (100 mM), methionine sulfoxide (MetO) (25 mM), or a mock control using a 0.25% (vol/vol) inoculum. Cells were grown to stationary phase (OD₆₀₀ of 2.0 to 3.0) with agitation (200 rpm) at 42°C and harvested by centrifugation. Alternatively, cultures were supplemented with a proteasome inhibitor (100 μM bortezomib) or NaOCl (20 mM) at log phase and incubated overnight at 42°C (200 rpm). Cells were harvested and analyzed for sampylation (Ubl modifications) by immunoblotting.

H. volcanii H26-pJAM983, which expresses UbaC–Strep-tag II, and H26-pJAM986, which expresses UbaC (untagged), were used for the pulldown assays. Cells were grown to log phase (OD₆₀₀ of 0.8) in 50 ml ATCC 974 medium with novobiocin (0.2 μg · ml⁻¹) in 250-ml flasks at 42°C with agitation (200-rpm rotary shaking). Harvested cells were lysed in 20 ml high-salt Tris buffer using a French press, and whole-cell lysate was clarified by centrifugation (20,000 × g for 10 min, 4°C) and filtration (0.45 μm). The protein concentration was determined using a BCA protein assay kit (Pierce). Clarified cell extract (1.5 mg protein in 1 ml lysate) was incubated with 50 μl Streptactin-Sepharose resin slurry (GE Healthcare) for 1 h at 4°C with gentle rocking to allow binding. The resin was washed thrice with 1 ml high-salt buffer. UbaC–Strep-tag II and its associated protein partners were eluted from the resin by the addition of 100 μl Tris-salt buffer (50 mM Tris, 150 mM NaCl, pH 7.5) supplemented with d-desthiobiotin (5 mM). Eluted proteins were separated by reducing SDS-PAGE (12%) and examined for protein constituents by immunoblotting using anti-UbaA and anti-Strep-tag II antibodies.

Cell pellets were resuspended and boiled for 20 min in 2× reducing SDS-PAGE loading buffer (62.5 mM Tris-Cl buffer at pH 6.8 with 2% [wt/vol] SDS, 25% [vol/vol] glycerol, 0.1 mg · ml⁻¹ bromophenol blue with 5% [vol/vol] β-mercaptoethanol). Proteins were separated by 12% SDS-PAGE. Loading amounts were normalized to 0.065 OD₆₀₀ unit per lane, with equal loading confirmed by staining parallel gels with Coomassie brilliant blue for total protein (with regions of the gels stained for total protein displayed alongside the immunoblots for ease of comparison). Proteins were electroblotted from the SDS-polyacrylamide gels onto Hybond-P polyvinylidene fluoride (PVDF) membranes (GE Healthcare Bio-Sciences, Piscataway, NJ) for 3 h at 90 V. Flag-tagged proteins were detected using alkaline phosphatase-linked anti-Flag M2 monoclonal antibodies (Sigma). Unconjugated rabbit anti-Strep-tag II polyclonal antibodies (GenScript USA, Piscataway, NJ) were used to detect Strep-tag II-tagged proteins. Polyclonal antibodies raised in rabbit against UbaA (Cocalico Biologicals, Reamstown, PA), found to be specific for UbaA by comparison of parent and ΔubaA mutant strains (28), were used to detect UbaA. The secondary antibodies used with the rabbit polyclonal antibodies were alkaline phosphatase-linked goat anti-rabbit IgG(H+L) antibodies (SouthernBiotech, Birmingham, AL). CDP-Star (Applied Biosystems, Carlsbad, CA) was used as the substrate to detect the alkaline phosphatase-conjugated antibodies. Chemiluminescent products were visualized with X-ray film (Hyperfilm; GE Healthcare Bio-Sciences).

Protein domain architectures, amino acid positions of the domains, and IPR superfamilies were according to InterPro (52). Archaeal clusters of orthologous genes (arCOGs) were according to Makarova et al. (53). Sequence similarity networks (SSNs) were generated using the Enzyme Function Initiative Tools-Enzyme Similarity Tool (EIF-EST) according to Gerlt et al. (54). The amino acid sequences of the archaeal E1 homologs of the Ub-activating enzyme superfamily (IPR035985) were input as a FASTA file into EIF-EST; the SSN generated at an alignment score of 46 at 40% amino acid sequence identity with a minimum length of 150 amino acids (aa) was used to assess the relationships of these archaeal E1s. The amino acid sequences of the RHDs from the MOCS3/Uba4 E1s (human, yeast, and plant) and from arCOG02021 were extracted and input as a FASTA file into EIF-EST; the SSN with an alignment score of 13, which represents 36% amino acid sequence identity, was used for the RHD comparison. The 3D structures of UbaC and UbaB were modeled using Phyre2 (55) and compared to the X-ray crystal structure of the MOCS3 RHD (PDB no. 3I2V) using Chimera (56).