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Structures of Pup Ligase Pafa and Depupylase Dop from the Prokaryotic Ubiquitin-Like Modification Pathway

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Structures of Pup Ligase Pafa and Depupylase Dop from the Prokaryotic Ubiquitin-Like Modification Pathway ARTICLE Received 9 May 2012 | Accepted 19 Jul 2012 | Published 21 Aug 2012 DOI: 10.1038/ncomms2009 Structures of Pup ligase PafA and depupylase Dop from the prokaryotic ubiquitin-like modification pathway Dennis Özcelik1,*, Jonas Barandun1,*, Nikolaus Schmitz1, Markus Sutter1, Ethan Guth1, Fred F. Damberger1, Frédéric H.-T. Allain1, Nenad Ban1 & Eilika Weber-Ban1 Pupylation is a posttranslational protein modification occurring in mycobacteria and other actinobacteria that is functionally analogous to ubiquitination. Here we report the crystal structures of the modification enzymes involved in this pathway, the prokaryotic ubiquitin- like protein (Pup) ligase PafA and the depupylase/deamidase Dop. Both feature a larger amino-terminal domain consisting of a central β-sheet packed against a cluster of helices, a fold characteristic for carboxylate-amine ligases, and a smaller C-terminal domain unique to PafA/Dop members. The active site is located on the concave surface of the β-sheet with the nucleotide bound in a deep pocket. A conserved groove leading into the active site could have a role in Pup-binding. Nuclear magnetic resonance and biochemical experiments determine the region of Pup that interacts with PafA and Dop. Structural data and mutational studies identify crucial residues for the catalysis of both enzymes. 1 ETH Zurich, Institute of Molecular Biology & Biophysics, CH-8093, Switzerland. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to E.W-B. (email: [email protected]). NATURE COMMUNICATIONS | 3:1014 | DOI: 10.1038/ncomms2009 | www.nature.com/naturecommunications © 2012 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2009 he pupylation pathway1–3 has an important role during the C-terminal glutamate and the ε-amino group of a substrate lysine3,14. intracellular persistence of Mycobacterium tuberculosis (Mtb). To carry out the respective reactions, both enzymes, which were TIt supports resistance of this pathogen towards oxidative and bioinformatically classified as belonging to the carboxylate-amine nitrosative stress encountered inside the host macrophages4,5. Mtb ligase family7, use ATP as a cofactor, but only the Pup ligase PafA kills 2 million people every year and, with the emergence of multi- turns over ATP3. In addition to performing the deamidation of drug-resistant strains, new therapeutic approaches are urgently PupQ to PupE3, Dop has an important role as depupylase by remov- needed. The molecular components of the pupylation pathway, ing Pup from modified lysine residues15,16. PafA catalyses a two- therefore, are promising targets for drug development. step reaction proceeding through a phosphorylated intermediate Pupylation is a posttranslational protein-tagging system that formed at the C-terminal glutamate of Pup before transfer of Pup to marks target proteins for degradation by proteasomes in Mtb the substrate acceptor lysine14,17. The detailed reaction mechanism and other actinobacteria1,2. The pupylation gene locus also for Dop remains unknown. occurs sporadically in other bacterial lineages6,7. In analogy to In this work, we present the crystal structures of the two enzymes ubiquitination, a small (60–70 residues), prokaryotic ubiquitin-like involved in the pupylation/depupylation pathway. Biochemical protein (Pup) is covalently attached to lysine residues of target pro- analysis of PafA and Dop variants, designed on the basis of the teins via an isopeptide bond1–3. Pup shares no structural homol- molecular architecture of the active sites, provides mechanistic ogy with ubiquitin and is disordered in its free form8–10. Pup is insight. We demonstrate that the C-terminal half of Pup interacts recognized by the amino-terminal coiled-coil domains of the pro- with Dop and PafA and propose that a conserved groove observed teasomal ATPase Mpa10,11, leading to ATPase-driven unfolding in both enzymes may bind this part of Pup, thereby placing Pup’s of pupylated substrate proteins followed by degradation inside the C-terminal residue near the active site. proteasome12,13. Although functionally analogous to ubiquitination, pupyla- Results tion occurs by a chemically distinct pathway3. In mycobacteria, Crystallization and structure determination. We solved the pupylation involves the sequential action of two homologous but crystal structures of full-length Dop (57 kDa) from Acidothermus catalytically different enzymes. First, the enzyme Dop (deamidase cellulolyticus (DopAcel) and full-length PafA (54 kDa) from of Pup) converts the C-terminal glutamine of Pup (PupQ) to a Corynebacterium glutamicum (PafACglu). The structure of Dop glutamate (PupE), thereby rendering it ligation-competent3. Then, was solved by multiple isomorphous replacement and refined to a the enzyme PafA (proteasome accessory factor A) catalyses the resolution of 2.6 Å without ATP and 2.85 Å with ATP (Table 1). The formation of an isopeptide bond between the side chain of PupE’s final electron density map showed continuous density except for a Table 1 | Data collection and refinement statistics. Dop PtCl4 Dop UOAc Dop DopATP PafAADP Data collection Radiation source SLS X06SA SLS X06SA SLS X06SA SLS X06SA SLS X06SA Radiation wavelength (Å) 1.0722 1.6000 1.00 1.00 1.00 Space group P212121 P212121 P212121 P212121 P212121 Cell dimensions a, b, c (Å)* 66.68 66.43 66.55 65.36 61.85 71.01 71.73 71.47 70.41 118.09 97.24 95.93 96.12 93.52 161.41 Resolution (Å) 57–3.5 49–3.5 48–2.6 47–2.85 49–2.15 Rmerge (%) 5.4 (17.2) 4.6 (13.5) 7.1 (61.5) 6.2 (43.6) 4.0 (51.2) < I > / < σ(I) > 21.9 (8.5) 25.3 (10.0) 15.59 (2.01) 15.69 (2.89) 18.47 (2.70) Completeness (%) 99.9 (100) 100.0 (100) 99.2 (99.7) 98.4 (98.4) 98.4 (99.7) Redundancy 8.97 8.94 3.6 3.4 3.7 Refinement Resolution (Å) 2.6 2.85 2.15 No. of reflections 14513 10376 65412 Rwork/Rfree 0.194/0.238 0.189/0.246 0.177/0.219 No. of atoms Protein 3615 3615 7388 Ligand/ion 1 Mg2 + 1 ATP 2 ADP 2 Mg2 + 2 Mg2 + Water 29 5 433 B-factors Protein 57.27 56.18 53.8 Nucleotide/Mg2 + — 94.38/61.92 43.38/51.34 Water 46.92 33.0 54.8 RMSD Bond lengths (Å) 0.003 0.003 0.008 Bond angles (°) 0.808 0.779 1.202 PDB code 4B0R 4B0S 4B0T Values in parentheses are for highest-resolution shell. *Unit cell angles: α, β, γ = 90.0°, 90.0°, 90.0°. NATURE COMMUNICATIONS | 3:1014 | DOI: 10.1038/ncomms2009 | www.nature.com/naturecommunications © 2012 Macmillan Publishers Limited. All rights reserved. NatUre cOMMUNicatiONS | DOI: 10.1038/ncomms2009 ARTICLE Dop PafA a C C-terminal C-terminal domain N domain N C β12 β11 α4 β10 β7 β2 β6 β3 β1 β4 Dop-loop α1 β3/4-loop β3/4-loop α2 α3′ Conserved Conserved groove groove RNH O O O 2 O O Dop•ATP PafA PafA Pup-Cγ Pup-Cγ Pup-Cγ Pup-Cγ Pup-Cγ + – – 2– 2– NHR O O ATP ADP O-PO PO NHR H2O NH3R 4 4 b β1 α1 Dop-loop β2 β3 β4 α2 Ac_Dop 127 Cg_Dop Mtb_Dop Ac_PafA Cg_PafA 98 Mtb_PafA β5 β6 α3 α3′ β7 Ac_Dop 248 Cg_Dop Mtb_Dop Ac_PafA Cg_PafA 228 Mtb_PafA α4 α5 β8 β9 α6 α7 α8 Ac_Dop 377 Cg_Dop Mtb_Dop Ac_PafA Cg_PafA 362 Mtb_PafA α9 α10 α11 α12 β10 β11 β12 α13 α13′ α14 Ac_Dop 503 Cg_Dop Mtb_Dop Ac_PafA Cg_PafA 482 Mtb_PafA Figure 1 | Dop and PafA are close structural homologues. (a) The crystal structures of deamidase/depupylase Dop (green) from A. cellulolyticus with ATP and Pup ligase PafA (blue) from C. glutamicum with ADP. Front views are shown, looking into the β-sheet cradle (yellow for Dop and purple for PafA). The dotted lines indicate structurally undefined regions. Selected β-strands and α-helices are labelled in the Dop structure. Other structural features are indicated in both Dop and PafA. The catalysed reaction mechanism of Dop and PafA is depicted below the corresponding structures. (b) Schematic depiction of secondary structure elements above the amino-acid sequence alignments of Dop and PafA from A. cellulolyticus, C. glutamicum and M. tuberculosis, respectively. The backbone is indicated by a grey line, linking α-helices (green, α1-14) and β-strands (yellow, β1-12) common to both enzymes, whereas differing structural elements in PafA are indicated as purple β-strands or blue α-helices, respectively. Backbone regions not visible in the structures are shown as dashed grey lines. disordered region between residues 36 to 80. The structure of PafA responding strand and helix in the monomer, based on comparison with ADP bound was solved by molecular replacement using the with the structure of Dop. Therefore, the PafA structure is displayed Dop structure and refined to a resolution of 2.15 Å (Table 1). and discussed throughout this manuscript as the catalytically active PafA crystals contain two molecules per asymmetric unit, form- monomer (chain A: N52-A477 and chain B: S2-S51). ing a stable dimer by swapping of the N-terminal strand-helix motif (β1 and α1, Supplementary Fig. S1). However, the position of the Dop and PafA feature two tightly interacting domains. DopAcel exchanged strand and helix is equivalent to the position of the cor- and PafACglu (38% sequence identity) are globular proteins with NatUre cOMMUNicatiONS | 3:1014 | DOI: 10.1038/ncomms2009 | www.nature.com/naturecommunications © 2012 Macmillan Publishers Limited. All rights reserved. ARTICLE NatUre cOMMUNicatiONS | DOI: 10.1038/ncomms2009 a ADP ATP β3 β3 E16 E8 β4 β4 β1 β1 R418 b R433 β6 β6 H231 H211 W440 W453 β7 R239 R219 ATP β7 ADP N157 β1 β1 β3 R227 R90 N132 R60 β3 H241 S101 Y92 H221 E16 β4 E8 H155 E99 H130 Y62 R201 E216 E10 E70 D94 R205 β4 E18 H95 D64 Glu R185 Glu H68 H97 R199 Figure 2 | Active site of Dop and PafA.
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