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Depupylase Dop Requires Inorganic Phosphate in the Active Site for Catalysis*

  • Marcel Bolten
    Affiliations
    ETH Zurich, Institute of Molecular Biology & Biophysics, 8093 Zurich, Switzerland
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  • Christian Vahlensieck
    Affiliations
    ETH Zurich, Institute of Molecular Biology & Biophysics, 8093 Zurich, Switzerland
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  • Colette Lipp
    Affiliations
    ETH Zurich, Institute of Molecular Biology & Biophysics, 8093 Zurich, Switzerland
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  • Marc Leibundgut
    Affiliations
    ETH Zurich, Institute of Molecular Biology & Biophysics, 8093 Zurich, Switzerland
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  • Nenad Ban
    Affiliations
    ETH Zurich, Institute of Molecular Biology & Biophysics, 8093 Zurich, Switzerland
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  • Eilika Weber-Ban
    Correspondence
    To whom correspondence should be addressed: Otto-Stern-Weg 5, 8093 Zurich, Switzerland. Tel.: 41-44-6333678; .
    Affiliations
    ETH Zurich, Institute of Molecular Biology & Biophysics, 8093 Zurich, Switzerland
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  • Author Footnotes
    * This work was supported by Swiss National Science Foundation (SNF) Grants 31003A_141236 and 31003A_163314 and the National Center for Excellence in Research (NCCR) Structural Biology program of the SNF. The authors declare that they have no conflict of interest with the contents of this article.
    2 The abbreviations used are: Pupprokaryotic ubiquitin-like proteinPafAproteasome accessory factor AGSglutamine synthetaseDopdeamidase of PupPanBketopantoate hydroxymethyltransferaseScGCLS. cerevisiae glutamate cysteine ligaseTSAtransition state analog5-FAM Lysfluorescein-5-carboxamide lysineAp5Abisadenosine pentaphosphateATPγSadenosine 5′-O-(thiotriphosphate)AMP-PCPadenosine 5′-(β,γ-methylenetriphosphate)DON6-diazo-5-oxo-l-norleucineTEVtobacco etch virusBistris propane1,3-bis[tris(hydroxymethyl)methylamino]propanePDBProtein Data Bank.
Open AccessPublished:January 24, 2017DOI:https://doi.org/10.1074/jbc.M116.755645
      Analogous to eukaryotic ubiquitination, proteins in actinobacteria can be post-translationally modified in a process referred to as pupylation, the covalent attachment of prokaryotic ubiquitin-like protein Pup to lysine side chains of the target protein via an isopeptide bond. As in eukaryotes, an opposing activity counteracts the modification by specific cleavage of the isopeptide bond formed with Pup. However, the enzymes involved in pupylation and depupylation have evolved independently of ubiquitination and are related to the family of ATP-binding and hydrolyzing carboxylate-amine ligases of the glutamine synthetase type. Furthermore, the Pup ligase PafA and the depupylase Dop share close structural and sequence homology and have a common evolutionary history despite catalyzing opposing reactions. Here, we investigate the role played by the nucleotide in the active site of the depupylase Dop using a combination of biochemical experiments and X-ray crystallographic studies. We show that, although Dop does not turn over ATP stoichiometrically with substrate, the active site nucleotide species in Dop is ADP and inorganic phosphate rather than ATP, and that non-hydrolyzable analogs of ATP cannot support the enzymatic reaction. This finding suggests that the catalytic mechanism is more similar to the mechanism of the ligase PafA than previously thought and likely involves the transient formation of a phosphorylated Pup-intermediate. Evidence is presented for a mechanism where the inorganic phosphate acts as the nucleophilic species in amide bond cleavage and implications for Dop function are discussed.

      Introduction

      In pupylation, proteins are marked by the post-translational modification of lysine side chains with the small, monomeric prokaryotic ubiquitin-like protein (Pup)
      The abbreviations used are: Pup
      prokaryotic ubiquitin-like protein
      PafA
      proteasome accessory factor A
      GS
      glutamine synthetase
      Dop
      deamidase of Pup
      PanB
      ketopantoate hydroxymethyltransferase
      ScGCL
      S. cerevisiae glutamate cysteine ligase
      TSA
      transition state analog
      5-FAM Lys
      fluorescein-5-carboxamide lysine
      Ap5A
      bisadenosine pentaphosphate
      ATPγS
      adenosine 5′-O-(thiotriphosphate)
      AMP-PCP
      adenosine 5′-(β,γ-methylenetriphosphate)
      DON
      6-diazo-5-oxo-l-norleucine
      TEV
      tobacco etch virus
      Bistris propane
      1,3-bis[tris(hydroxymethyl)methylamino]propane
      PDB
      Protein Data Bank.
      (
      • Samanovic M.I.
      • Li H.
      • Darwin K.H.
      The pup-proteasome system of Mycobacterium tuberculosis.
      ,
      • Striebel F.
      • Imkamp F.
      • Özcelik D.
      • Weber-Ban E.
      Pupylation as a signal for proteasomal degradation in bacteria.
      ,
      • Burns K.E.
      • Liu W.T.
      • Boshoff H.I.
      • Dorrestein P.C.
      • Barry 3rd., C.E.
      Proteasomal protein degradation in Mycobacteria is dependent upon a prokaryotic ubiquitin-like protein.
      ,
      • Pearce M.J.
      • Mintseris J.
      • Ferreyra J.
      • Gygi S.P.
      • Darwin K.H.
      Ubiquitin-like protein involved in the proteasome pathway of Mycobacterium tuberculosis.
      ). Bacterial pupylation shows many functional parallels to eukaryotic ubiquitination, for example, the employment of a macromolecular tag, the nature of the generated covalent linkage, and the role played as an important recognition element in a protein degradation pathway involving a proteasome complex. However, bacteria have evolved this functionally analogous system independently, and the enzymes involved in pupylation and depupylation are not related to ubiquitination or deubiquitination systems but rather belong to the superfamily of carboxylate-amine/ammonia ligases (
      • Striebel F.
      • Imkamp F.
      • Sutter M.
      • Steiner M.
      • Mamedov A.
      • Weber-Ban E.
      Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes.
      ,
      • Iyer L.M.
      • Burroughs A.M.
      • Aravind L.
      Unraveling the biochemistry and provenance of pupylation: a prokaryotic analog of ubiquitination.
      ,
      • Abbott J.J.
      • Pei J.
      • Ford J.L.
      • Qi Y.
      • Grishin V.N.
      • Pitcher L.A.
      • Phillips M.A.
      • Grishin N.V.
      Structure prediction and active site analysis of the metal binding determinants in γ-glutamylcysteine synthetase.
      ).
      Ligation of Pup to target proteins is catalyzed by the enzyme PafA (proteasome accessory factor A) and results in the formation of an isopeptide bond between the side chain carboxylate of the C-terminal glutamate of Pup and the ϵ-amino group of a lysine residue in the target protein (
      • Striebel F.
      • Imkamp F.
      • Sutter M.
      • Steiner M.
      • Mamedov A.
      • Weber-Ban E.
      Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes.
      ,
      • Sutter M.
      • Damberger F.F.
      • Imkamp F.
      • Allain F.H.
      • Weber-Ban E.
      Prokaryotic ubiquitin-like protein (Pup) is coupled to substrates via the side chain of its C-terminal glutamate.
      ). In accordance with the energy requirement of isopeptide bond formation the ligation process requires the turnover of ATP. The structure of PafA shows that it has a similar active site arrangement as glutamine synthetase (GS), consisting of a curved anti-parallel β-sheet with ATP bound at one end of the β-sheet cradle and the triphosphate chain running along the strands toward the opposite side of the sheet, where the glutamate residue of Pup is bound (
      • Barandun J.
      • Delley C.L.
      • Ban N.
      • Weber-Ban E.
      Crystal structure of the complex between prokaryotic ubiquitin-like protein and its ligase PafA.
      ,
      • Özcelik D.
      • Barandun J.
      • Schmitz N.
      • Sutter M.
      • Guth E.
      • Damberger F.F.
      • Allain F.H.
      • Ban N.
      • Weber-Ban E.
      Structures of Pup ligase PafA and depupylase Dop from the prokaryotic ubiquitin-like modification pathway.
      ). The γ-carboxylate of the C-terminal glutamate of Pup binds in close proximity to the γ-phosphate, allowing an attack of the glutamyl γ-carboxylate oxygen on the γ-phosphate of ATP to cleave off ADP and generate the γ-glutamyl phosphate-mixed anhydride intermediate of Pup (
      • Guth E.
      • Thommen M.
      • Weber-Ban E.
      Mycobacterial ubiquitin-like protein ligase PafA follows a two-step reaction pathway with a phosphorylated pup intermediate.
      ). This phospho-Pup intermediate is activated for the nucleophilic attack of the ϵ-amino group of the target lysine in the next step, which then leads to formation of the isopeptide bond. This reaction is chemically similar to the activation of the glutamate side chain for the attack of ammonia in GS. However, whereas bacterial GS is an oligomeric assembly (a double ring of hexamers) with the active sites buried in deep pockets at the intra-ring subunit interfaces (
      • Eisenberg D.
      • Gill H.S.
      • Pfluegl G.M.
      • Rotstein S.H.
      Structure-function relationships of glutamine synthetases.
      ), the Pup ligase PafA is active as a monomer and features a broad, easily accessible active site (
      • Barandun J.
      • Delley C.L.
      • Ban N.
      • Weber-Ban E.
      Crystal structure of the complex between prokaryotic ubiquitin-like protein and its ligase PafA.
      ,
      • Özcelik D.
      • Barandun J.
      • Schmitz N.
      • Sutter M.
      • Guth E.
      • Damberger F.F.
      • Allain F.H.
      • Ban N.
      • Weber-Ban E.
      Structures of Pup ligase PafA and depupylase Dop from the prokaryotic ubiquitin-like modification pathway.
      ).
      In mycobacteria and several other actinobacteria, Pup is encoded with a C-terminal glutamine instead of glutamate, necessitating deamidation of the C-terminal glutamine to glutamate before ligation to a target is possible. This activity is carried out by Dop (deamidase of Pup), which is structurally highly similar to the ligase PafA and is also encoded in the pupylation gene locus (
      • Striebel F.
      • Imkamp F.
      • Sutter M.
      • Steiner M.
      • Mamedov A.
      • Weber-Ban E.
      Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes.
      ,
      • Özcelik D.
      • Barandun J.
      • Schmitz N.
      • Sutter M.
      • Guth E.
      • Damberger F.F.
      • Allain F.H.
      • Ban N.
      • Weber-Ban E.
      Structures of Pup ligase PafA and depupylase Dop from the prokaryotic ubiquitin-like modification pathway.
      ,
      • Cerda-Maira F.A.
      • Pearce M.J.
      • Fuortes M.
      • Bishai W.R.
      • Hubbard S.R.
      • Darwin K.H.
      Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis.
      ). Intriguingly, Dop also opposes the ligase activity by catalyzing the specific cleavage of the isopeptide bond formed between Pup and the protein (
      • Burns K.E.
      • Cerda-Maira F.A.
      • Wang T.
      • Li H.
      • Bishai W.R.
      • Darwin K.H.
      “Depupylation” of prokaryotic ubiquitin-like protein from mycobacterial proteasome substrates.
      ,
      • Imkamp F.
      • Striebel F.
      • Sutter M.
      • Ozcelik D.
      • Zimmermann N.
      • Sander P.
      • Weber-Ban E.
      Dop functions as a depupylase in the prokaryotic ubiquitin-like modification pathway.
      ). Thus, at least in mycobacteria, Dop is involved in both the pupylation and depupylation of protein substrates, suggesting an intricate network of regulation. Furthermore, pupylated target proteins can be recruited to a bacterial proteasome complex consisting of the 20S proteasome core and the AAA-ATPase ring Mpa (mycobacterial proteasomal ATPase), where recognition of Pup takes place (
      • Burns K.E.
      • Pearce M.J.
      • Darwin K.H.
      Prokaryotic ubiquitin-like protein provides a two-part degron to Mycobacterium proteasome substrates.
      ,
      • Striebel F.
      • Hunkeler M.
      • Summer H.
      • Weber-Ban E.
      The mycobacterial Mpa-proteasome unfolds and degrades pupylated substrates by engaging Pup's N-terminus.
      ,
      • Sutter M.
      • Striebel F.
      • Damberger F.F.
      • Allain F.H.
      • Weber-Ban E.
      A distinct structural region of the prokaryotic ubiquitin-like protein (Pup) is recognized by the N-terminal domain of the proteasomal ATPase Mpa.
      ). Pupylated substrates can escape this fate, however, when Pup is cleaved off by the depupylase Dop. The fate of a pupylation target is therefore tightly controlled by all four activities.
      In agreement with its structural similarity to PafA, Dop also features an active site nucleotide (
      • Özcelik D.
      • Barandun J.
      • Schmitz N.
      • Sutter M.
      • Guth E.
      • Damberger F.F.
      • Allain F.H.
      • Ban N.
      • Weber-Ban E.
      Structures of Pup ligase PafA and depupylase Dop from the prokaryotic ubiquitin-like modification pathway.
      ). Yet, cleavage of the isopeptide bond does not require energy and it has been shown that Dop does not turn over ATP stoichiometrically with substrate deamidated or Pup cleaved off (
      • Striebel F.
      • Imkamp F.
      • Sutter M.
      • Steiner M.
      • Mamedov A.
      • Weber-Ban E.
      Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes.
      ,
      • Imkamp F.
      • Striebel F.
      • Sutter M.
      • Ozcelik D.
      • Zimmermann N.
      • Sander P.
      • Weber-Ban E.
      Dop functions as a depupylase in the prokaryotic ubiquitin-like modification pathway.
      ). It is, therefore, an intriguing question what role the nucleotide plays in the catalytic mechanism of the enzyme.
      To investigate the role of the nucleotide in the active site of Dop we used a combination of biochemical experiments and X-ray crystallographic analysis. Our results show that the active site of Dop requires ADP and inorganic phosphate to support both the deamidation and the depupylation activities. This has implications for the catalytic mechanism and suggests that Dop is mechanistically more closely related to the ligase PafA than previously thought.

      Results

      Non-hydrolyzable ATP Analogs Cannot Support Dop Activity

      It was shown earlier that Dop does not hydrolyze ATP stoichiometrically during the reaction progress of deamidation (
      • Striebel F.
      • Imkamp F.
      • Sutter M.
      • Steiner M.
      • Mamedov A.
      • Weber-Ban E.
      Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes.
      ) and depupylation (
      • Imkamp F.
      • Striebel F.
      • Sutter M.
      • Ozcelik D.
      • Zimmermann N.
      • Sander P.
      • Weber-Ban E.
      Dop functions as a depupylase in the prokaryotic ubiquitin-like modification pathway.
      ). In addition it was found that Dop shows only marginal deamidase activity in the presence of the non-hydrolyzable ATP analog ATPγS (
      • Striebel F.
      • Imkamp F.
      • Sutter M.
      • Steiner M.
      • Mamedov A.
      • Weber-Ban E.
      Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes.
      ). To better understand the role of the nucleotide for the catalytic activity of Dop we analyzed the depupylation reaction of the known pupylation substrate PanB (ketopantoate hydroxymethyltransferase) in the presence of different nucleotides. The enzymatic removal of Pup from PanB-Pup was probed by pulling aliquots along the depupylation time course and quenching them with SDS-loading dye to stop the reaction. Aliquots from the respective time points were subjected to SDS-PAGE followed by Coomassie staining (Fig. 1A). In addition, the gels were densitometrically evaluated for a more quantitative assessment (Fig. 1B). In the presence of ATP, all PanB-Pup is converted to PanB within 3 min under the conditions used, whereas in the presence of the non-hydrolyzable analog AMP-PCP even after 30 min no PanB-Pup has been depupylated. This is a curious observation, considering that ATP is not turned over stoichiometrically during the reaction.
      Figure thumbnail gr1
      FIGURE 1Depupylation of PanB-Pup is catalyzed by Dop in the presence of ADP and Pi. 3 μm CgluDop was incubated with 3 μm PanB-Pup, 0.5 mm nucleotides, and 10 mm phosphate at 30 °C. The adenylate kinase inhibitor Ap5A, where present, was used at a concentration of 0.3 mm. A, SDS-polyacrylamide gels showing depupylation time courses of PanB-Pup in the presence of different nucleotides. B, densitometric analysis of the PanB band in relationship to the total amount of PanB used in the reaction. PupE is poorly stained by Coomassie Blue and was not expected to contribute to the density of the PanB-Pup band at the concentrations under which the assay was performed.
      One possible explanation could be that ATP cleavage, although not accompanying substrate turnover, is nevertheless required to produce the correct arrangement of the active site. A consequence of this would be that hydrolyzed ATP, i.e. ADP and phosphate (Pi) in the active site should be able to support the reaction, although no stoichiometric turnover is taking place during the reaction. Indeed, testing this hypothesis, we found that depupylation can be catalyzed in the presence of ADP and Pi (Fig. 1). After 12 s ∼40% of PanB-Pup are depupylated and complete turnover of PanB-Pup in the presence of ADP and Pi is reached after 2 min compared with 3 min with ATP. Interestingly, ADP alone shows no activity on the time scale where in the presence of ADP and Pi or ATP complete conversion occurs. However, at later time points depupylation activity slowly starts up. This activity of Dop in the presence of ADP alone can be traced to minute amounts of adenylate kinase impurities in the recombinantly produced proteins, which results in very slow turnover of ADP to ATP and AMP. It was shown that due to the high catalytic rate of the enzyme, contaminating Escherichia coli adenylate kinase present at 0.01% in highly pure protein preparations can perturb experiments under ADP-only conditions (
      • Chen B.
      • Sysoeva T.A.
      • Chowdhury S.
      • Guo L.
      • Nixon B.T.
      ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli.
      ). Bisadenosine pentaphosphate (Ap5A), a competitive inhibitor of adenylate kinase, was shown to suppress activity of the E. coli enzyme at a ratio of Ap5A to nucleotide of 2:1. Unfortunately, the inhibitor carries about 1% ATP impurity, preventing us from using it in the ADP-only time trace at that ratio. However, the addition of 0.3 mm Ap5A to the ADP time trace results in a decrease of the observed activity, whereas activity in the presence of ATP remains unchanged, indicating that activity in the presence of ADP stems from the adenylate kinase impurity and not from Dop.

      Co-crystal Structure of Dop and ATP Reveals ADP and Phosphate as the Active Site Species

      One of our previously solved crystal structures of Dop contained ATP (
      • Özcelik D.
      • Barandun J.
      • Schmitz N.
      • Sutter M.
      • Guth E.
      • Damberger F.F.
      • Allain F.H.
      • Ban N.
      • Weber-Ban E.
      Structures of Pup ligase PafA and depupylase Dop from the prokaryotic ubiquitin-like modification pathway.
      ). However, as the nucleotide was not co-crystallized but soaked into the crystal, the occupancy was poor. To investigate whether the active site species in Dop might be ADP and Pi rather than ATP, we now co-crystallized ATP and Dop to obtain full occupancy.
      The co-crystallization attempts with Dop from Acidothermus cellulolyticus (AcelDop) and ATP yielded well diffracting crystals. The structure was solved by molecular replacement using the previously solved Dop structure (Protein Data Bank code 4b0r (
      • Özcelik D.
      • Barandun J.
      • Schmitz N.
      • Sutter M.
      • Guth E.
      • Damberger F.F.
      • Allain F.H.
      • Ban N.
      • Weber-Ban E.
      Structures of Pup ligase PafA and depupylase Dop from the prokaryotic ubiquitin-like modification pathway.
      )) and was refined to 1.9 Å. With the exception of a disordered region between residues 42 and 79 in the so-called Dop loop, the electron density was continuous. The structure clearly shows that ADP and Pi are bound in the active site with high occupancy as indicated by the density (Figs. 2A and 3). In contrast to the earlier, ATP-soaked structure, all magnesium binding sites characteristic for members of the carboxylate-amine/ammonia ligase superfamily (n1–n3) are occupied and their importance for mediating contacts between the amino acid residues and the phosphate groups becomes obvious (Fig. 2, A and B). An additional Mg2+ binding site (n5) was observed that contributes to the binding of the β-phosphate. All Mg2+ ions are coordinated in almost perfect octahedral symmetry. Notably, Asp-94, a residue previously shown to be important for activity (
      • Özcelik D.
      • Barandun J.
      • Schmitz N.
      • Sutter M.
      • Guth E.
      • Damberger F.F.
      • Allain F.H.
      • Ban N.
      • Weber-Ban E.
      Structures of Pup ligase PafA and depupylase Dop from the prokaryotic ubiquitin-like modification pathway.
      ,
      • Cerda-Maira F.A.
      • Pearce M.J.
      • Fuortes M.
      • Bishai W.R.
      • Hubbard S.R.
      • Darwin K.H.
      Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis.
      ) and proposed to form a mixed anhydride intermediate during catalysis (
      • Burns K.E.
      • McAllister F.E.
      • Schwerdtfeger C.
      • Mintseris J.
      • Cerda-Maira F.
      • Noens E.E.
      • Wilmanns M.
      • Hubbard S.R.
      • Melandri F.
      • Ovaa H.
      • Gygi S.P.
      • Darwin K.H.
      Mycobacterium tuberculosis prokaryotic ubiquitin-like protein-deconjugating enzyme is an unusual aspartate amidase.
      ), is coordinating Mg2+ at the n1 position.
      Figure thumbnail gr2
      FIGURE 2Crystal structure of Dop reveals ADP and Pi in the active site. A, AcelDop active site with bound ADP, phosphate, magnesium, and sodium ions. The unbiased mFO − DFC Fourier-simulated annealing omit map was calculated with a model in which all but the protein atoms and waters were omitted and is contoured at 5 σ levels. B, schematic representation of polar interactions between Dop active site residues, ADP, phosphate, magnesium ions, sodium ion, and water. Residues labeled in blue are located in the C-terminal domain of Dop. C, comparison of active sites of AcelDop (green) and S. cerevisiae glutamate cysteine ligase (PDB code 3lvv, gray, residue numbers in parentheses). The phosphate group of the transition state mimic buthionine sulfoximine phosphate overlays with the inorganic phosphate bound by Dop.
      Figure thumbnail gr3
      FIGURE 3ADP and Pi are bound in the active site of Dop. A, unbiased mFO − DFC Fourier map at 4.2 σ contour level (gray) and 8 σ contour level (green) after molecular replacement with PDB code 4b0r (which did not include any ligands) and one round of refinement in PHENIX without any further model building. The final model is shown for orientation. B, final model of Dop with ADP and Pi in the active site. The Pi is separated 4.2 Å from the ADP β-phosphate. The anomalous difference Fourier map (gray, 3.5 σ contour level) shows the location of the phosphorus and sulfur atoms.
      A comparison of the active site of Dop with the one of Saccharomyces cerevisiae glutamate cysteine ligase (ScGCL) in complex with ADP and the transition state analog (TSA) inhibitor buthionine sulfoximine phosphate (PDB 3lvv (
      • Biterova E.I.
      • Barycki J.J.
      Structural basis for feedback and pharmacological inhibition of Saccharomyces cerevisiae glutamate cysteine ligase.
      )) (Fig. 2C) shows that the phosphate complexed by Dop superimposes almost perfectly with the phosphate group of the TSA, indicating the general relevance of the bound phosphate and in particular the conservation of the phosphate location within the carboxylate-amine/ammonia ligase superfamily. Arg-472 of ScGCL is positioned to form hydrogen bonds with the sulfoximine oxygen and an oxygen of the phosphate group. The equivalent residue in Dop (Arg-227), while offset from Arg-472 in the alignment by 3.4 Å, nevertheless, is kept at the same distance to the phosphate. Although in the absence of an additional hydrogen bond partner the guanidyl group only coordinates the Pi, the conformational freedom of Arg-227 would allow forming a similar stabilizing interaction with Pup as Arg-472 of ScGCL with the TSA. Judging by the position of the carboxylate end of the TSA it is likely that Arg-205 in Dop coordinates the free C terminus of Pup.

      ATP Hydrolysis Depends on the C-terminal Residue of Pup

      Our structural analysis suggests that ATP is cleaved into ADP and phosphate in the active site of Dop. In contrast to the ligase, this reaction is not coupled to the turnover of substrate. Nevertheless, it is possible that the presence of Pup in the active site might influence the cleavage. To investigate a potential role of Pup in the hydrolysis of ATP we therefore followed the conversion of [α-32P]ATP to [α-32P]ADP in the active site of Dop by TLC and phosphorimaging in the absence and presence of Pup. As the ATP cleavage presumably occurs to configure the active site rather than to turn over the substrate, a high concentration of Dop (30 μm) had to be employed to detect the cleavage. When Pup was absent, Dop showed only a very low basal ATPase activity (Fig. 4, upper left panel). However, when either PupE or PupQ were included in the reaction mixture, significant amounts of ADP were produced in the measured time frame of 50 min (Fig. 4, two upper middle panels). This increased Dop activity is due to a stimulation of the ATP cleavage by Pup and not due to any possible impurity in the Pup preparations, because PupQ or PupE alone did not show turnover in the same time period (Fig. 4, two lower middle panels). To test whether hydrolysis of ATP is stimulated by a conformational change of Dop upon binding of Pup or rather by the presence of the very C-terminal residue in the active site, a Pup variant lacking the last residue (PupGG) was used. Strikingly, PupGG was not able to accelerate ATP hydrolysis over the background level that Dop exhibits on its own, indicating that indeed the C-terminal residue of Pup in the active site stimulates the hydrolysis of ATP.
      Figure thumbnail gr4
      FIGURE 4ATP hydrolysis mediated by Dop is dependent on the C-terminal residue of Pup. The C-terminal residues glutamine or glutamate of Pup accelerate the ATP hydrolysis mediated by Dop. A Pup variant lacking the C-terminal residue (PupGG) shows the same ATPase activity as Dop alone. ATP and the Pup variants alone show negligible ATPase activity over the assay time. 30 μm CgluDop was incubated with 190 μm PupE, 250 μm PupQ, or 250 μm PupGG and 100 μm ATP (100 mCi/mmol of [α-32P]ATP) at 23 °C.

      ATP Hydrolysis Is Necessary for Dop Activity

      We next analyzed whether ATP hydrolysis is required for the active site to carry out catalysis of the depupylation reaction. To obtain a continuous record of the depupylation reaction we used the fluorescent model substrate Pup-Fl (
      • Hecht N.
      • Gur E.
      Development of a fluorescence anisotropy-based assay for Dop, the first enzyme in the pupylation pathway.
      ), which can be monitored by fluorescence anisotropy. In parallel, we followed the ATP hydrolysis radiochemically (Fig. 5A). The depupylation reaction shows an initial lag phase, during which Pup-Fl is turned over only very slowly (Fig. 5B). Maximal Dop activity is reached after ∼12 min under the used conditions, at which point an ATP amount has been cleaved that is equivalent to 1.4 times the Dop active sites. This is rather close to stoichiometric with active sites, considering that ADP and Pi also dissociate off during this time frame, and suggests that Pi is indeed required for catalysis. To further demonstrate that the lag phase in the presence of ATP is due to ATP first needing to be cleaved to ADP and Pi, we measured turnover time traces of Pup-Fl providing Dop with either ATP or ADP and Pi (Fig. 6). In agreement with the notion that Pi is required in the active site for catalysis, when ADP and Pi instead of ATP is provided, no lag phase is observed.
      Figure thumbnail gr5
      FIGURE 5Initial ATP hydrolysis is necessary for depupylation of model substrate Pup-Fl. A, ATP hydrolysis catalyzed by Dop in the absence and presence of model substrate Pup-Fl. 25 μm CgluDop were incubated with 100 μm Pup-Fl and 100 μm ATP spiked with 200 mCi/mmol of [α-32P]ATP. The time point at 2 min was not taken into account because of the different running behavior. B, after an initial lag phase of ∼12 min, during which ATP gets hydrolyzed, the depupylation reaction reaches maximal speed. At 30 min ∼90% of Pup-Fl is depupylated and ∼40% of ATP is left. The depupylation reaction progress was monitored by fluorescence anisotropy under the same conditions as in A, omitting [α-32P]ATP. C, chemical structure of the model substrate Pup-Fl.
      Figure thumbnail gr6
      FIGURE 6Depupylation lag phase disappears in the presence of ADP and Pi. 1.25 μm CgluDop was incubated with 5 μm Pup-Fl and 100 μm ATP, ADP, or ADP + 10 mm Pi. The depupylation time course in the presence of ADP and Pi does not exhibit the lag phase observed in the presence of ATP. ADP in the absence of Pi does not enable depupylation of Pup-Fl.

      Dop Activity Depends on ADP and Pi Concentrations

      To assess the ADP and phosphate concentration dependence of the Dop activity, we used Pup-Fl to record depupylation time traces at varying concentrations of ADP or Pi, with the other component held constant, respectively (Fig. 7). In the presence of 0.5 mm ADP, the rate of depupylation under steady-state conditions follows a saturation curve with respect to Pi concentration featuring half-maximal activity at 73 ± 11 μm Pi. When the Pi concentration is held constant at 5 mm, the depupylation activity increases with the concentration of ADP, showing a half-maximal rate at 31 ± 6 μm ADP. Measuring the depupylation rate as a function of ATP concentration, we obtained a half-maximal rate at 2.3 ± 0.3 μm ATP.
      Figure thumbnail gr7
      FIGURE 7The depupylation activity of Dop is dependent on the concentrations of ADP and Pi. The steady-state rates of Dop-catalyzed Pup-Fl cleavage were measured as a function of Pi (A), ADP (B), or ATP (C) concentrations. Averages of three experiments are presented with the corresponding standard deviations (error bars). Data are fitted to r([x]) = rmax × [x]/(Kapp + [x]), where [x] indicates the Pi, ADP, or ATP concentration. Kapp is given with its fitting standard error. 0.25 μm Dop and 5 μm Pup-Fl were used.

      Discussion

      The two enzymes involved in the pupylation and depupylation of proteins in actinobacteria, the ligase PafA and the depupylase Dop, are evolutionarily related. Both belong to the large family of carboxylate-amine ligases, enzymes that catalyze the formation of an amide linkage between a carboxylate and an amine via an acylphosphate intermediate (
      • Iyer L.M.
      • Burroughs A.M.
      • Aravind L.
      Unraveling the biochemistry and provenance of pupylation: a prokaryotic analog of ubiquitination.
      ,
      • Abbott J.J.
      • Pei J.
      • Ford J.L.
      • Qi Y.
      • Grishin V.N.
      • Pitcher L.A.
      • Phillips M.A.
      • Grishin N.V.
      Structure prediction and active site analysis of the metal binding determinants in γ-glutamylcysteine synthetase.
      ). Accordingly, PafA and Dop are structural homologs and the residues forming the active site are highly conserved (
      • Özcelik D.
      • Barandun J.
      • Schmitz N.
      • Sutter M.
      • Guth E.
      • Damberger F.F.
      • Allain F.H.
      • Ban N.
      • Weber-Ban E.
      Structures of Pup ligase PafA and depupylase Dop from the prokaryotic ubiquitin-like modification pathway.
      ,
      • Cerda-Maira F.A.
      • Pearce M.J.
      • Fuortes M.
      • Bishai W.R.
      • Hubbard S.R.
      • Darwin K.H.
      Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis.
      ). A defining feature of the active site of carboxylate-amine ligase family members is the nucleotide binding site. The role of ATP in the ligation reaction is to activate the carboxylate for nucleophilic attack by the amine and to drive the otherwise thermodynamically unfavorable ligation reaction. For the Pup ligase PafA, the activation of Pup occurs by formation of a γ-glutamylphosphate mixed anhydride intermediate at the C-terminal glutamate of Pup (
      • Guth E.
      • Thommen M.
      • Weber-Ban E.
      Mycobacterial ubiquitin-like protein ligase PafA follows a two-step reaction pathway with a phosphorylated pup intermediate.
      ). This intermediate is poised in the active site, protected from hydrolysis, for reaction with a lysine ϵ-amino group of an incoming pupylation substrate. As a close homolog of PafA, Dop features a nearly identical ATP-binding site (
      • Özcelik D.
      • Barandun J.
      • Schmitz N.
      • Sutter M.
      • Guth E.
      • Damberger F.F.
      • Allain F.H.
      • Ban N.
      • Weber-Ban E.
      Structures of Pup ligase PafA and depupylase Dop from the prokaryotic ubiquitin-like modification pathway.
      ), however, thermodynamically, ATP turnover is not required, since breakage of an amide linkage is entropically favorable. In accordance with that, amide bond cleavage catalyzed by Dop is not accompanied by stoichiometric turnover of ATP, neither during deamidation nor during depupylation (
      • Striebel F.
      • Imkamp F.
      • Sutter M.
      • Steiner M.
      • Mamedov A.
      • Weber-Ban E.
      Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes.
      ,
      • Imkamp F.
      • Striebel F.
      • Sutter M.
      • Ozcelik D.
      • Zimmermann N.
      • Sander P.
      • Weber-Ban E.
      Dop functions as a depupylase in the prokaryotic ubiquitin-like modification pathway.
      ). Nevertheless, our structural analysis clearly identified ADP and Pi in the active site of Dop, indicating that ATP does not merely play a structural role to maintain active site configuration. Although it was previously shown that the ATP analog ATPγS is able to support a very low level of deamidase activity (
      • Striebel F.
      • Imkamp F.
      • Sutter M.
      • Steiner M.
      • Mamedov A.
      • Weber-Ban E.
      Bacterial ubiquitin-like modifier Pup is deamidated and conjugated to substrates by distinct but homologous enzymes.
      ), this might be due to the tendency of this analog to exhibit some cleavage of the bond to the γ-phosphate. In contrast, the ATP analog AMP-PCP employed in this study is not known to undergo hydrolysis (
      • Bystrom C.E.
      • Pettigrew D.W.
      • Remington S.J.
      • Branchaud B.P.
      ATP analogs with non-transferable groups in the γ position as inhibitors of glycerol kinase.
      ). The fact that Dop does not exhibit any activity with this nucleotide analog (Fig. 1) indicates that cleavage of the bond to the γ-phosphate of ATP and, therefore, the presence of ADP and phosphate in the active site of Dop is crucial for catalysis. This is further supported by our finding that radiolabeled [α-32P]ATP is turned over to [α-32P]ADP independent of substrate turnover (Fig. 4, upper panels). Another mechanistically revealing observation is that ADP and phosphate but not ADP alone can support Dop activity (Figs. 1 and 6). Together, these lines of evidence strongly point to a mechanistic role of inorganic phosphate in the depupylase/deamidase reaction. We propose that the role of the phosphate in the active site of Dop is the formation of a transient phospho-Pup intermediate (Fig. 8), a scenario also supported by the evolutionary relationship with the carboxylate-amine ligase family, where exactly such an intermediate is formed in the forward reaction. An inorganic phosphate oxygen attacks the side chain carbonyl carbon of the Pup C-terminal glutamine or, in the case of depupylation, the carbonyl carbon of the isopeptide bond between Pup and substrate, thereby bringing about the cleavage of the amide bond. The ammonium/amine leaving group dissociates from the enzyme, and in the next step water, likely activated for nucleophilic attack by Asp-94 in the active site, hydrolyzes the phospho-Pup intermediate, thereby releasing Pup from the enzyme. The importance of this aspartate during catalysis has been demonstrated previously, as mutation to either alanine (
      • Özcelik D.
      • Barandun J.
      • Schmitz N.
      • Sutter M.
      • Guth E.
      • Damberger F.F.
      • Allain F.H.
      • Ban N.
      • Weber-Ban E.
      Structures of Pup ligase PafA and depupylase Dop from the prokaryotic ubiquitin-like modification pathway.
      ) or asparagine (
      • Cerda-Maira F.A.
      • Pearce M.J.
      • Fuortes M.
      • Bishai W.R.
      • Hubbard S.R.
      • Darwin K.H.
      Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis.
      ) abolishes Dop activity in vitro for AcelDop and in vivo for Dop from Mycobacterium tuberculosis (MtbDop). In agreement with the lack of a thermodynamic requirement for ATP hydrolysis, inorganic phosphate remains in the active site and is ready for another round of catalysis. This mechanism is similar to the reaction where GS catalyzes the conversion of glutamine to glutamate in the presence of ADP and arsenate (
      • Eisenberg D.
      • Gill H.S.
      • Pfluegl G.M.
      • Rotstein S.H.
      Structure-function relationships of glutamine synthetases.
      ).
      Figure thumbnail gr8
      FIGURE 8Proposed catalytic mechanism of Dop. Active site residues are numbered according to AcelDop and shown in a simplified representation omitting Mg2+ coordinating residues Glu-8, Tyr-92, His-155, and His-241. Mg2+ at position n2 is omitted at the beginning of the reaction for clarity and added with the first step (compare with B).
      Superposition of the Dop active site containing ADP and Pi with structures of other members of the carboxylate-amine/ammonia ligase family in complex with phosphorylated inhibitors results in excellent congruence of Pi with the phosphate group of the inhibitors (Fig. 2C). This lends strong support to the existence of a phosphorylated Pup intermediate during the depupylation reaction catalyzed by Dop, which resembles the well characterized intermediates from the other family members (
      • Biterova E.I.
      • Barycki J.J.
      Structural basis for feedback and pharmacological inhibition of Saccharomyces cerevisiae glutamate cysteine ligase.
      ,
      • Krajewski W.W.
      • Jones T.A.
      • Mowbray S.L.
      Structure of Mycobacterium tuberculosis glutamine synthetase in complex with a transition-state mimic provides functional insights.
      ,
      • Hibi T.
      • Nii H.
      • Nakatsu T.
      • Kimura A.
      • Kato H.
      • Hiratake J.
      • Oda J.
      Crystal structure of γ-glutamylcysteine synthetase: insights into the mechanism of catalysis by a key enzyme for glutathione homeostasis.
      ,
      • Hothorn M.
      • Wachter A.
      • Gromes R.
      • Stuwe T.
      • Rausch T.
      • Scheffzek K.
      Structural basis for the redox control of plant glutamate cysteine ligase.
      ), including the phosphorylated Pup from PafA (
      • Guth E.
      • Thommen M.
      • Weber-Ban E.
      Mycobacterial ubiquitin-like protein ligase PafA follows a two-step reaction pathway with a phosphorylated pup intermediate.
      ).
      Intriguingly, only in the presence of Pup, and more specifically in the presence of the Pup C-terminal residue in the active site of Dop, are ADP and Pi formed efficiently. Dop in absence of Pup or in the presence of a Pup variant shortened by one residue hydrolyzes ATP only very slowly (Fig. 4). Pup thus supports the activation of water in the active site needed for the attack on the γ-phosphate of ATP. This occurs either directly or indirectly by slight rearrangements of active site residues, such as, for example, Asp-94. The requirement of Pup binding for efficient ATP cleavage might serve as a protection mechanism against uncontrolled ATP hydrolysis by free Dop.
      An earlier study suggested that Asp-95 of MtbDop (corresponding to Asp-94 in AcelDop) participates in catalysis by forming a covalent mixed anhydride intermediate with Pup (
      • Burns K.E.
      • McAllister F.E.
      • Schwerdtfeger C.
      • Mintseris J.
      • Cerda-Maira F.
      • Noens E.E.
      • Wilmanns M.
      • Hubbard S.R.
      • Melandri F.
      • Ovaa H.
      • Gygi S.P.
      • Darwin K.H.
      Mycobacterium tuberculosis prokaryotic ubiquitin-like protein-deconjugating enzyme is an unusual aspartate amidase.
      ). This hypothesis was based on the observation that a nucleophilic Pup derivative acting as an irreversible trap, Pup-DON (6-diazo-5-oxo-l-norleucine), forms a covalent bond with Asp-95 in the active site of MtbDop. Pup-DON features a highly reactive aliphatic diazo group that, after addition of a proton to the ϵ-carbon and elimination of N2, forms a carbenium ion that readily reacts with any nucleophiles in the vicinity, such as, for example, carboxylates. This is in perfect agreement with a mechanism in which this aspartate acts as catalytic base to activate water. It should also be noted in this context that the ϵ-carbon in the Pup-DON trap, which is attacked by the nucleophile, is positioned one bond length deeper in the active site than the side chain carbonyl carbon of Pup (Cϵ versus Cδ), a position that is never occupied by the isopeptide bond or the carbamoyl group of glutamine. Pup with a C-terminal asparagine was found to resist deamidation, indicating the significance of the length of the side chain and the precise position of the carbonyl carbon in the active site (
      • Cerda-Maira F.A.
      • Pearce M.J.
      • Fuortes M.
      • Bishai W.R.
      • Hubbard S.R.
      • Darwin K.H.
      Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis.
      ).
      Further support for our proposed mechanism is given by the sequence of events with ATP hydrolysis taking place first and creating a lag phase in the depupylation reaction. Depupylation reaches maximal velocity only after the ADP and phosphate have been generated in the active site (Figs. 5 and 6). In agreement with this interpretation, in the presence of ADP and Pi, the lag phase is absent.
      In an earlier study it was shown that, during the cycle of catalysis, 18O-water (H218O) is incorporated only into Pup and not into Dop (
      • Burns K.E.
      • McAllister F.E.
      • Schwerdtfeger C.
      • Mintseris J.
      • Cerda-Maira F.
      • Noens E.E.
      • Wilmanns M.
      • Hubbard S.R.
      • Melandri F.
      • Ovaa H.
      • Gygi S.P.
      • Darwin K.H.
      Mycobacterium tuberculosis prokaryotic ubiquitin-like protein-deconjugating enzyme is an unusual aspartate amidase.
      ). Furthermore, the authors showed that hydroxylamine can act as a nucleophile to form Pup-hydroxamate, indicating that during the Dop-catalyzed reaction an activated carbonyl must exist. These findings are in agreement with our proposed mechanism, as the resolution of the phosphorylated Pup intermediate by water (H218O) or hydroxylamine directly leads to the experimentally identified Pup species, 18O-labeled Pup or Pup-hydroxamate, respectively.
      Taken together, our findings on the fate of ATP in the Dop active site, previous mutational and biochemical studies of Dop, and the high degree of structural conservation between members of the carboxylate-amine ligase family, in particular the nearly identical active site configuration of Dop and PafA, strongly indicate that Dop catalyzes the cleavage of the isopeptide bond by a mechanism representative of the reverse reaction of PafA (Fig. 8). In a first step, Dop hydrolyzes ATP to produce ADP and Pi, which remain bound in the active site. This at the same time prevents Dop from acting as a ligase. In the next step, the active site-bound Pi attacks the isopeptide bond and forms the transient phosphorylated Pup intermediate. During this step Arg-227 plays a critical role, likely by stabilizing the transition state with its guanidyl group. In the last step, the resolution of the phosphorylated Pup intermediate is mediated by nucleophilic attack of water, activated by the active site catalytic residue Asp-94, on the carbonyl carbon of Pup. The two tetrahedral transition states that flank the transiently formed phospho-Pup intermediate are further stabilized by the Mg2+ ion coordinated by residues Glu-10, Asp-94, and Glu-99 in the active site.
      Our experimental results demonstrate that Dop activity can be supported by binding ATP that is then cleaved in the active site or by direct binding of ADP and Pi. Bacterial cytosolic concentrations in an exponentially growing culture lie at around 0.5 mm for ADP and around 8–10 mm for ATP (
      • Bennett B.D.
      • Kimball E.H.
      • Gao M.
      • Osterhout R.
      • Van Dien S.J.
      • Rabinowitz J.D.
      Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli.
      ). However, whereas ADP changes only moderately, ATP decreases significantly in stationary phase, leading to a variation in the ATP/ADP ratio from 10 to 3 depending on the growth conditions (
      • Tran Q.H.
      • Unden G.
      Changes in the proton potential and the cellular energetics of Escherichia coli during growth by aerobic and anaerobic respiration or by fermentation.
      ). Single-cell measurements in continuously growing bacterial cultures showed a distribution of ATP concentrations with a mean value of 1.5 mm (
      • Yaginuma H.
      • Kawai S.
      • Tabata K.V.
      • Tomiyama K.
      • Kakizuka A.
      • Komatsuzaki T.
      • Noji H.
      • Imamura H.
      Diversity in ATP concentrations in a single bacterial cell population revealed by quantitative single-cell imaging.
      ). The concentration of inorganic phosphate in the bacterial cytosol lies at about 10 mm (
      • Albe K.R.
      • Butler M.H.
      • Wright B.E.
      Cellular concentrations of enzymes and their substrates.
      ). Taking into account this information, both binding of ATP followed by cleavage or binding of ADP and Pi can occur depending on the conditions. As both can support the activity, should the ratio of ATP to ADP change significantly, this would not result in a significant change of Dop activity.
      Our biochemical analysis supported by X-ray crystallographic data provides the framework for understanding the mechanism of the depupylase/deamidase enzyme Dop and thus provides insight that might be exploited for the rational design of antituberculosis drugs aimed at interfering with the pupylation enzymes. Furthermore, it contributes to our understanding of the close evolutionary relationship between the two opposing players in the pupylation system, the Pup ligase PafA and the depupylase Dop.

      Experimental Procedures

      Chemicals and Reagents

      Chemicals were obtained from Sigma unless otherwise noted. 5-FAM Lys was provided by AnaSpec. [α-32P]ATP was obtained from Hartmann Analytic (Braunschweig, Germany) at a specific activity of 15 TBq (400 Ci)/mmol. Polyethyleneimine TLC plates were provided by VWR International. ADP was further purified by ion-exchange chromatography (6-ml Resource Q column, GE Healthcare Life Sciences) and desalted by size exclusion chromatography (100 ml Superose 12 column, GE Healthcare Life Sciences).

      Protein Expression and Purification

      Dop from A. cellulolyticus (AcelDop) was expressed from isopropyl β-d-1-thiogalactopyranoside-inducible pET21 vector in E. coli Rosetta (DE3) cells (Invitrogen) with a C-terminal tobacco etch virus (TEV) protease cleavage site-His6 fusion and purified by affinity chromatography using a 5-ml Hi Trap IMAC HP column (GE Healthcare Life Sciences) charged with Ni2+. After washing the protein-charged column with 50 ml of buffer W (50 mm HEPES-NaOH, pH 8, at 23 °C, 500 mm NaCl, 40 mm imidazole), protein was eluted with buffer W containing 300 mm imidazole. Protein-containing fractions were pooled and dialyzed for 1 h at 4 °C against buffer D (50 mm HEPES-NaOH, pH 8, at 4 °C, 150 mm NaCl). The C-terminal histidine tag was cleaved at the TEV protease cleavage site by addition of His-tagged TEV protease and further dialysis for 15 h. TEV protease was removed via affinity chromatography. AcelDop was further purified by size exclusion chromatography using a Superdex 75 column in 20 mm HEPES-NaOH, pH 8, at 20 °C and 50 mm NaCl. Corynebacterium glutamicum Dop (CgluDop) with an N-terminal His6-TEV protease cleavage site fusion was expressed from pET24 and purified similarly to AcelDop. After size exclusion chromatography, CgluDop was further purified by anion exchange chromatography with a Mono Q HR 10/10 column (GE Healthcare Life Sciences) to reduce the amount of E. coli adenylate kinase impurity. A linear gradient from 0 to 1 m NaCl in 20 mm Tris-HCl, pH 7, at 23 °C was used to elute CgluDop. Buffer was changed to buffer R (50 mm HEPES-NaOH, pH 8, at 23 °C, 150 mm NaCl) either by PD-10 desalting columns (GE Healthcare Life Sciences) or by Amicon Ultra centrifugal filters (Merck Millipore).

      Crystallization of Dop

      Crystallization of AcelDop was carried out in sitting drop vapor diffusion plates at a protein concentration of 8–12 mg/ml at 20 °C by mixing 2 μl of protein solution with 1 μl of reservoir solution. AcelDop formed crystals in reservoir solutions consisting of 18–23% (w/v) PEG 3350, 100 mm Bistris propane, pH 8.25–9.0, at 20 °C, 200 mm KSCN, 20 mm MgCl2, and 5 mm ATP (pH 8). Before flash cooling the crystals with liquid nitrogen, PEG 400 was added in 5% (v/v) steps to a final concentration of 30% (v/v) by using reservoir solution supplemented with PEG 400.

      Data Collection, Processing, Structure Determination, and Refinement

      A data set was collected at beamline X06SA of the Swiss Light Source (Paul Scherrer Institut, Villigen, Switzerland) at 100 K and data were indexed and integrated using XDS (
      • Kabsch W.
      XDS.
      ). Initial analysis of data was performed using POINTLESS (
      • Evans P.
      Scaling and assessment of data quality.
      ) and PHENIX.xtriage (
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • Richardson J.S.
      • Terwilliger T.C.
      • Zwart P.H.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ). Scaling was subsequently done by AIMLESS (
      • Evans P.R.
      • Murshudov G.N.
      How good are my data and what is the resolution?.
      ). The structure was solved by molecular replacement in PHASER-MR (
      • McCoy A.J.
      • Grosse-Kunstleve R.W.
      • Adams P.D.
      • Winn M.D.
      • Storoni L.C.
      • Read R.J.
      Phaser crystallographic software.
      ) using the previously solved Dop structure (PDB code 4b0r (
      • Özcelik D.
      • Barandun J.
      • Schmitz N.
      • Sutter M.
      • Guth E.
      • Damberger F.F.
      • Allain F.H.
      • Ban N.
      • Weber-Ban E.
      Structures of Pup ligase PafA and depupylase Dop from the prokaryotic ubiquitin-like modification pathway.
      )) as a search model. After an initial refinement step in PHENIX (
      • Adams P.D.
      • Afonine P.V.
      • Bunkóczi G.
      • Chen V.B.
      • Davis I.W.
      • Echols N.
      • Headd J.J.
      • Hung L.W.
      • Kapral G.J.
      • Grosse-Kunstleve R.W.
      • McCoy A.J.
      • Moriarty N.W.
      • Oeffner R.
      • Read R.J.
      • Richardson D.C.
      • Richardson J.S.
      • Terwilliger T.C.
      • Zwart P.H.
      PHENIX: a comprehensive Python-based system for macromolecular structure solution.
      ), well defined density was obtained in the active site indicating that ADP and Pi are bound rather than ATP (Fig. 3A). Attempts to refine the structure with ATP resulted in strong difference peaks. Additionally, the anomalous difference Fourier map was calculated and supports the location of the phosphorus atoms with a distance of 4.2 Å between the ADP β-phosphate and the Pi (Fig. 3B). The model was further improved by iterative model building in COOT (
      • Emsley P.
      • Lohkamp B.
      • Scott W.G.
      • Cowtan K.
      Features and development of Coot.
      ) and refinement in PHENIX. Statistics are summarized in Table 1. An additional strong electron density peak near the Pi was interpreted as a sodium ion (n4) based on coordination geometry and hydrogen bond distances. Structural alignments were done by cealign in PyMol (
      • Schrödinger L.L.C.
      ) using only the highly conserved residues around the active site.
      TABLE 1Data collection and refinement statistics
      AcelDop·ADP·Pi
      PDB ID5LRT
      Crystal form
       Space groupP31 2 1
       Unit cell72.398
       Dimensions (Å)72.398
      215.25
       Angels α, β, γ (°)90 90 120
       Molecules/ASU1
      Data collection
       Wavelength (Å)0.99998
       Resolution (Å)35.88–1.85
      (1.88–1.85)
       Total reflections506,641 (23 851)
       Unique reflections56,899 (2,622)
       Multiplicity8.9 (9.1)
       Completeness (%)1.00 (1.00)
       Mean I/σ(I)25.52 (1.50)
       Wilson B-factor (Å2)39.17
      Rmerge0.044 (1.425)
      Rmeas0.047 (1.51)
       CC1/21 (0.607)
      Refinement
       Reflections used56,895 (2,622)
       Reflections used Rfree2,880 (125)
      Rwork0.1665 (0.3185)
      Rfree0.1977 (0.3695)
      Model composition
       Non-hydrogen atoms3,996
       Macromolecules3,689
       Ligands83
       Water224
       Protein residues464
      Root mean square deviations
       Bonds0.027
       Angles1.13
       Dihedrals14.97
      Ramachandran plot
       Favored (%)97
       Allowed (%)2.6
       Outliers (%)0.21
       Rotamer outliers (%)0.52
       Clashscore4.54
       Average B-factor49.53
       Macromolecules49.25
       Ligands52.69
       Water53.07
       Number of TLS groups11

      Gel-based Depupylation Assays

      3 μm MtbPanB-CgluPup (produced as described in Ref.
      • Imkamp F.
      • Striebel F.
      • Sutter M.
      • Ozcelik D.
      • Zimmermann N.
      • Sander P.
      • Weber-Ban E.
      Dop functions as a depupylase in the prokaryotic ubiquitin-like modification pathway.
      and further purified to reduce an adenylate kinase impurity as described for CgluDop) was incubated with 3 μm CgluDop at 30 °C in buffer R supplemented with 20 mm MgCl2, 0.5 mm nucleotides and additional 10 mm Na/K-phosphate, pH 7, at 23 °C in the case of ADP. Still present minute amounts of adenylate kinase activity could be reduced by addition of 0.3 mm Ap5A. The formation of PanB was monitored by SDS-PAGE and Coomassie staining and analyzed densitometrically by GelAnalyzer software version 2010a (
      • Lázár I.
      ). The fraction of PanB is expressed in relationship to total PanB (sum of PanB and PanB-Pup). Pup was not taken into account because of poor staining.

      Fluorescence Anisotropy-based Depupylation Assay of Pup-Fl

      Pupylation of 5-FAM Lys and the depupylation assay were carried out as described (
      • Hecht N.
      • Gur E.
      Development of a fluorescence anisotropy-based assay for Dop, the first enzyme in the pupylation pathway.
      ). For the generation of Pup-Fl, 1 mm 5-FAM Lys and 250 μm CgluPupE were incubated in the presence of 10 μm CgluPafA with 5 mm ATP. Depupylation was carried out in buffer R for data in Fig. 5 or in buffer A (100 mm Bistris propane-HCl, pH 7.5, at 23 °C, 150 mm NaCl, and 0.05% Tween 20) for the data in Figs. 6 and 7. The reactions were supplemented with 10 or 20 mm MgCl2 and varying concentrations of ATP, ADP, and Pi. Single time courses are shown in Fig. 6. The trace in Fig. 5 represents the average of three time courses. All traces for the depupylation rate dependence on Pi, ADP, and ATP were set up in triplicate and the initial velocities were determined by a linear fit for the linear part of the individual time traces. The depupylation rate dependence on the concentration r([x]) was fitted with OriginPro 9 (OriginLab Corporation) to the function,
      r([x])=rmax×[x]/(Kapp+[x])
      (Eq. 1)


      where [x] indicates the Pi, ADP, or ATP concentration.

      Radioactive Assay to Monitor ATP Hydrolysis

      Assays were performed as described (
      • Guth E.
      • Thommen M.
      • Weber-Ban E.
      Mycobacterial ubiquitin-like protein ligase PafA follows a two-step reaction pathway with a phosphorylated pup intermediate.
      ). In brief, 30 μm CgluDop was incubated with 190 μm CgluPupE, 250 μm CgluPupQ, or 250 μm CgluPupGG and 100 μm ATP (100 mCi/mmol of [α-32P]ATP) at 23 °C in reaction buffer R. A molecular dynamics phosphorimaging screen (GE Healthcare) was exposed to the dried TLC plates for 1–2 h and subsequently scanned using a Typhoon Trio phosphorimaging system (GE Healthcare). Images were analyzed using ImageJ software version 1.48 (
      • Rasband W.S.
      ) to determine the ratio, r, of non-hydrolyzed ATP to total nucleotide (ATP and ADP) for each reaction time point, t. To correct for the background hydrolysis of Dop without Pup-Fl (−Pup-Fl), the fraction of non-hydrolyzed ATP, f, at each time point, t, during the depupylation of Pup-Fl was normalized to the corresponding background (f(t) = r(t, +Pup-Fl)/r(t, −Pup-Fl)).

      Author Contributions

      M. B. and E. W. B. conceived the project and wrote the manuscript. M. B., C. V., and C. L. purified Dop and carried out crystallization experiments. M. B., M. L., and N. B. analyzed all crystallographic data. M. B. and C. L. performed the gel-based Dop activity assays, M. B. and C. V. performed the radiochemical and fluorescence anisotropy-based Dop activity assays.

      Acknowledgments

      We thank B. Blattmann and C. Stutz-Ducommun for support with initial screening and acknowledge the staff of X06SA at the Swiss Light Source (SLS, Paul Scherrer Institut, Villigen, Switzerland) for support with data collection.

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