Structure of bacterial LigD 3′-phosphoesterase unveils a DNA repair superfamily

Pravin A. Nair, Paul Smith, and Stewart Shuman1

Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065

Edited by Stephen C. Kowalczykowski, University of California, Davis, CA, and approved May 28, 2010 (received for review May 2, 2010)

The DNA D (LigD) 3′-phosphoesterase (PE) module is a con- tides or phosphates from DNA or RNA, it was proposed to served component of the bacterial nonhomologous end-joining exemplify a unique family of 3′ end-healing . Here we (NHEJ) apparatus that performs 3′ end-healing reactions at DNA put this idea to the test and determined the atomic structure double-strand breaks. Here we report the 1.9 Å crystal structure of the Pseudomonas PE domain. of Pseudomonas aeruginosa PE, which reveals that PE exemplifies a unique class of DNA repair . PE has a distinctive fold Results and Discussion in which an eight stranded β barrel with a hydrophobic interior Crystallization and Structure Determination. Whereas our attempts supports a crescent-shaped hydrophilic on its outer sur- to crystallize the full-length PE domain (aa 1–187) were unsuc- face. Six essential side chains coordinate manganese and a sulfate cessful, we noted that inclusion of trace amounts of chymotrypsin mimetic of the scissile phosphate. The PE active site and mechanism in the precipitant solution resulted in growth of tiny crystals. Tak- are unique vis à vis other end-healing enzymes. We find PE homo- ing this cue, we conducted crystallization trials with recombinant logs in archaeal and eukaryal proteomes, signifying that PEs PE (17–187), a catalytically active initiating at comprise a DNA repair superfamily. a chymotryptic cleavage site. Crystals grew in the presence of PEG, ammonium sulfate, and manganese, but diffracted poorly. nonhomologous end-joining ∣ 3′ end-healing Additive screening identified yttrium as uniquely effective in pro- moting growth of diffraction quality crystals. Diffraction data at 1.9 Å and 2.5 Å resolution were collected for native and methyl- NA ligase D (LigD) is the key agent of the bacterial nonho- BIOCHEMISTRY Dmologous end-joining (NHEJ) pathway of DNA double- mercury-treated crystals in space group P1. Phases were obtained strand break (DSB) repair (1). LigD is a single polypeptide by single isomorphous replacement with anomalous Hg/Y/Mn consisting of three autonomous catalytic domain modules: an scattering (see SI Methods in SI Appendix). The structure was ∕ ATP-dependent ligase (LIG), a polymerase (POL), and a 3′-phos- ultimately refined at 1.92 Å resolution with R Rfree values of 0 148∕0 199 phoesterase (PE). The POL domain incorporates dNMP/rNMPs . . and excellent geometry (SI Appendix: Table S1). at DSB ends and gaps prior to strand sealing by the LIG domain The P1 crystals contained two PE protomers in the asymmetric – (2–6) and is responsible, in large part, for the mutagenic out- unit. The model of the A protomer comprised aa 34 185 (with a – comes of bacterial NHEJ in vivo (7). The PE domain provides 4-aa gap at residues 98 101); the B protomer consisted of aa – a3′ end-healing function, whereby it cleans up “dirty” DSBs with 30 187 (with a single aa gap at residue 99). Absence of an exten- 3′-phosphate ends (8). PE also trims short 3′-ribonucleotide sive protomer interface in the crystal was consistent with the tracts (produced by POL) to generate the 3′ monoribonucleotide monomeric state of PE in solution (8). The salutary effects of ends that are the preferred substrates for sealing by bacterial yttrium on PE crystallization were evident in the refined struc- NHEJ (3, 8). The biochemical properties and atomic ture, which revealed four yttrium atoms bridging the A protomer structures of the LIG and POL domains highlighted their mem- to two vicinal PE protomers in the lattice (SI Appendix: Fig. S1). bership in the covalent nucleotidyltransferase and archaeal/ The yttrium cations were chelated by pairs or trios of carboxylate eukaryal primase-polymerase families respectively (5, 9, 10). side chains located on the surfaces of adjacent PE protomers By contrast, the PE domain appears to be sui generis. (SI Appendix: Fig. S1). The A and B protomers in the P1 lattice α The properties of the PE domain elucidated initially for Pseu- had virtually identical tertiary structures (rmsd of 0.33 Å at 145 domonas LigD also apply to the PE modules of Agrobacterium carbon positions). We also collected 2.3 Å diffraction data on a and Mycobacterium LigD (8, 11, 12). Specifically, PE displays a crystal in space group C2 and solved that structure by molecular distinctive manganese-dependent 3′-/3′- replacement (SI Appendix: Table S1). There were no significant ′ differences in the PE structure in space group C2 vis à vis the activity, entailing two component steps: (i) the 3 -terminal nu- α cleoside is removed to yield a primer strand with a ribonucleoside P1 structure (rmsd of 0.27 Å at 145 C positions). The descrip- tions of the PE structure and active site that follow are those of 3′-PO4 terminus; (ii) the 3′-PO4 is hydrolyzed to a 3′-OH (Fig. 1A). PE activity is acutely dependent on the presence the A protomer in the P1 crystal. and length of a 5′ single-strand tail on a duplex primer-template substrate, thus implicating PE in 3′ end repair at gaps or recessed Overview of the PE Tertiary Structure. The PE domain comprises a central 8-stranded antiparallel β barrel surrounded by two α DSBs. Structure probing of Pseudomonas PE in solution revealed 3 an apparently disordered N-terminal 29-aa segment, punctuated helices and a 10 helix (Fig. 2A). The secondary structure ele- by a cluster of trypsin- and chymotrypsin-sensitive sites (Fig. 1B), ments are displayed above the PE amino acid sequence in Fig. 1B flanking a seemingly well folded (i.e., protease insensitive) C-terminal domain (13). Deletion of the protease-sensitive Author contributions: P.A.N., P.S., and S.S. designed research; P.A.N. and P.S. performed N-terminal peptide had no effect on the phosphodiesterase activ- research; P.A.N., P.S., and S.S. analyzed data; and P.A.N., P.S., and S.S. wrote the paper. ity of PE, though monoesterase activity was reduced. Mutational The authors declare no conflict of interest. analyses identified an ensemble of conserved side chain func- This article is a PNAS Direct Submission. tional groups within the protease-resistant module that were Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, essential for phosphoesterase activity (Fig. 1B, highlighted in www.pdb.org (PDB ID codes 3N9B and 3N9D). yellow) and thus candidates to comprise the active site (8, 11, 1To whom correspondence should be addressed. E-mail: [email protected]. 13). Because LigD PE has no apparent primary structural or This article contains supporting information online at www.pnas.org/lookup/suppl/ mechanistic similarity to any other proteins that remove nucleo- doi:10.1073/pnas.1005830107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1005830107 PNAS Early Edition ∣ 1of6 Downloaded by guest on September 27, 2021 Fig. 1. The bacterial LigD PE family of 3′ end repair enzymes. (A) Two-step 3′ end-healing activity of the LigD PE domain. The reaction mechanism entails an initial phosphodiesterase step that incises the 3′-diribonucleotide linkage of the primer template to generate a monoribonucleotide-3′-phosphate-terminated primer strand and release a monoribonucleoside. The phosphomonoesterase activity then releases inorganic phosphate and leaves a 3′-monoribonucleotide primer-template end-product. Both steps depend on manganese. The time course of the reaction of the Pseudomonas PE domain with a diribonucleotide- terminated primer-template (ribonucleotides colored red) is shown at left. The D10R2 primer strand (composed of 10 deoxynucleotides and two ribonucleo- 0 32 tides) is labeled at the 5 -PO4 end with P(indicated by the asterisk). The radiolabeled reaction products were analyzed by denaturing gel electrophoresis. The 11 1 experiment (8) establishes the precursor-product relationship between the fast migrating D11R1p species and the D R OH end-product. (B) Conservation of primary and secondary structure among bacterial LigD PE domains. The amino acid sequence of the Pseudomonas aeruginosa (Pae) LigD PE domain is aligned to the PE domains of two Agrobacterium tumefaciens (Atu) LigD paralogs (D1 and D2) and the PE domain of Mycobacterium tuberculosis (Mtu) LigD. Positions of amino acid side chain identity/similarity in all four proteins are denoted by solid black dots above the sequence. Six conserved residues shown previously to be essential for PaePE activity are highlighted in yellow. Protease-sensitive sites in the N-terminal peptide of PaePE are denoted by blue arrows. The secondary structure elements of the PE fold are shown above the sequence, with β strands depicted as arrows and helices as cylinders. The eight strands that comprise the PE β barrel (Fig. 2A) are numbered. Conserved residues located in the hydrophobic interior of the β-barrel are denoted by black arrowheads below the sequence.

and the folding topology is diagrammed in SI Appendix: Fig. S2. composed of an 8-stranded antiparallel β barrel of quite different The PE β barrel has a narrow aperture filled with hydrophobic folding topology (15, 16). The TTM barrel has a wider aperture residues (Fig. 2B) that are conserved in other LigD PE domains and an extremely hydrophilic interior that binds the divalent (these are denoted by black arrowheads in Fig. 1B). Among these cation and polyphosphorylated substrate on which the TTM are a constellation of tryptophans that, in addition to their van enzymes act (17). Whereas the TTM active site is inside the β der Waals contacts, donate hydrogen bonds from their indole ni- barrel, the PE active site is clearly located on the solvent-exposed trogens to PE main chain carbonyls (e.g., Trp108, Trp113, and surface of the β barrel, as demarcated by the positions of closely Trp141) and form π-cation pairs with overlying lysine/arginine spaced manganese and sulfate ligands (Fig. 2A). The PE active side chains (e.g., Trp108-Lys136, Trp155-Arg145, and Trp141- site architecture is discussed in detail below. Lys159 pairs) (Fig. 2B), said pairs being conserved among bacterial PE domains (Fig. 1B). Thus, there appears to be a char- Active Site and Catalytic Mechanism. The PE β barrel supports a acteristic pattern of amino acid side chain and main chain inter- crescent-shaped hydrophilic active site on its outer surface actions that stabilize the PE β barrel. (Fig. 2A) that binds the essential manganese and a sul- Indeed, a three-dimensional homology search of the protein fate anion that we regard as a steric and electrostatic mimetic of data bank using DALI (14) highlights the PE domain as a unique the scissile phosphate. The active site architecture and pertinent protein fold. The five top DALI “hits,” with relatively feeble Z atomic interactions are depicted in Fig. 3. The electron density scores of 3.6 to 4.2, were members of the triphosphate tunnel me- map highlights an octahedral coordination complex about the talloenzyme (TTM) superfamily of phosphohydrolases, which are manganese ion, which is filled by His42-Nδ, His48-Nϵ, Asp50-

2of6 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1005830107 Nair et al. Downloaded by guest on September 27, 2021 Fig. 2. Tertiary structure of the PE domain. A stereo view of the Pseudomo-

nas LigD PE structure is show in A. The fold comprises an 8-stranded antipar- BIOCHEMISTRY allel β barrel flanked by a 310 helix and two α-helices. The N and C termini (residues 34 and 185, respectively) are indicated. The β strands are numbered Fig. 3. PE active site. (A) Stereo view of the active site. The main chain ribbon as in Fig. 1B. The active site, demarcated by protein-bound manganese is colored green. Selected amino acid side chains and main chain atoms, and (magenta sphere) and sulfate (stick model) ligands, is located in a cres- the sulfate anion (a putative mimic of the scissile phosphate), are rendered as cent-shaped groove on the outer surface of the β-barrel. B shows a detailed stick models with beige carbons. Manganese and waters are depicted as view of the hydrophobic interior of the PE β-barrel, viewed from the “back” magenta and red spheres, respectively. Interatomic contacts are indicated opening relative to the view in A (note the manganese is now on the left). by dashed lines.(B) Stereo view of a finely sampled 2.0 Å composite omit Selected amino acid side chains and main chain atoms are rendered as stick density map of the PE active site contoured at 1.1 σ (black mesh; grid spacing models with beige carbons. Hydrogen bonds are indicated by dashed lines. 0.25 Å). The red mesh is the anomalous difference density for the manganese ion contoured at 15 σ. Oϵ, a sulfate oxygen, and two waters (Fig. 3B). The structure teaches us that a likely catalytic role of the metal ion is to aid secondary structure elements comprising the active site (Fig. 3A). in substrate binding and then stabilize the developing negative Interruption of this triad by mutating Gln40 to alanine reduced charge on the scissile phosphate in the presumptive associative phosphodiesterase activity by 2.5-fold, and activity was virtually transition state. The structure also accounts for the characteristic abolished (<1% of wild type) by replacing Gln40 with glutamate specificity of PE for manganese (and cobalt, cadmium, or copper) (11); the latter mutation would antagonize the native hydrogen- as the metal cofactor, and its inability to utilize magnesium (8), bonding contacts of Gln40-Nϵ to the Tyr88 and Phe91 main-chain insofar as the reliance on two histidine nitrogens as metal ligands carbonyls) (Fig. 3A). “ ” “ ” will favor soft metal interactions in contrast to the hard The Tyr88 OH coordinates a sulfate-bound water in the PE oxygen-based contacts preferred by magnesium. The functional active site (Fig. 3B). Changing Tyr88 to alanine suppressed PE relevance of the three manganese-binding side chains is already diesterase and monoesterase activities, to 7% and 1% of wild established by mutational studies, to wit: H42A, H48A, and type, respectively (13). It is noteworthy that the conservative D50A mutants of Pseudomonas PE are catalytically inert Y88F mutant retained activity as a 3′-phosphodiesterase, but (8, 13). Moreover, each metal-binding residue is strictly essential, was inactive as a 3′-phosphomonoesterase (11). This separation insofar as conservative mutants H42N, H42Q, H48N, H48Q, of function suggests that the water-bridged contact between Tyr88 D50E, and D50N are also unreactive (11). OH and the scissile phosphate is specifically essential for monoe- The sulfate anion is bound in the active site via a network of direct and water-mediated contacts to the metal and to amino ster hydrolysis. Accordingly, we speculate that the water inter- ϵ posed between Tyr88-OH and two of the sulfate oxygens is the acid side chains His84, Arg52, and Tyr88 (Fig. 3A). His84-N ′ makes a direct contact to the sulfate (Fig. 3A). His84 is strictly immediate nucleophile in the 3 phosphomonoesterase reaction essential for PE function, i.e., the H84A, H84N, and H84Q and it relies on Tyr88 for proper orientation during an in-line mutants are inert (8, 13). We surmise that His84 promotes attack. Also, we surmise that the drastic loss of both phosphoes- via transition state stabilization. Arg52 is poised at terase activities when the Tyr88 side chain is subtracted (in Y88A the center of a hydrogen bonding network involving all three or Y88S) reflects the structural contributions of the many van der guanidinium nitrogens, which variously coordinate three of the Waals contacts (3.5–3.8 Å) between the Tyr88 phenyl ring carbon sulfate oxygens (via two waters) and directly engage Ser61-Oγ atoms and the atoms of the surrounding Arg52, His84, Gln40, and Gln40-Oϵ (Fig. 3A). Arg52 is strictly essential for PE and Phe91 side chains. function, i.e., the R52A, R52K, and R52Q mutants are inert In sum, the PE crystal structure localizes the previously (8, 11). Although we surmise that Arg52 participates directly defined essential side chains at the active site and suggests in phosphoester hydrolysis, it is also likely to play an additional catalytic and/or structural roles for each of the active site residues structural role via the Ser61-Arg52-Gln40 triad that tethers the consistent with available structure-function data. The structure

Nair et al. PNAS Early Edition ∣ 3of6 Downloaded by guest on September 27, 2021 also hints at a plausible model for how PE might bind to a DNA configuration in LigD PE (Table 1). Thus, we see no evidence primer-template substrate. The image in SI Appendix: Fig. S3A to indicate convergence of PE with any known phosphoesterase showing the molecular surface and electrostatics of the PE do- catalytic site. main highlights two points: (i) the active site occupies a concave depression on the protein surface that might accommodate the A PE Superfamily in Bacteria, Archaea, and Eukarya. Having installed duplex segment of the primer-template; and (ii) the positive elec- LigD PE as the founder of an enzyme family, we queried whether trostatic potential on the protein surface in front of the sulfate the PE module might have achieved broader use in nature—in might complement that of the electronegative DNA phosphodie- functional and phylogenetic contexts outside the polyfunctional ster backbone. Using an idealized B-form DNA duplex with a LigD polypeptide that anchors the NHEJ systems found in scores 3′-phosphomonoester terminus on the primer strand and a 5′ of bacterial taxa. Reasoning that PE might have existed as a single-stranded extension on the template strand, we manually free standing catalytic module prior to its fusion with LIG and docked the DNA ligand in the active site depression, by super- POL to form LigD, we surveyed bacterial proteomes for PE imposing the 3′-phosphate of the primer strand on the sulfate homologs that were similar in size to the Pseudomonas PE anion. As seen in SI Appendix: Fig. S3B, the resulting docked module. We thereby identified 21 stand-alone PE enzymes that model entailed no gross steric clashes. Also, this orientation of contained the full set of active site residues. The bacterial taxa the primer-template positioned the Tyr88-coordinated water encoding stand-alone PE enzymes span eight different phyla nearly apical to the primer strand terminal O3′, the leaving atom (SI Appendix: Table S3). in the 3′ phosphomonoesterase reaction. PE homologs are not confined to bacteria. For example, we detected stand-alone PE domains in the proteomes of seven Uniqueness of LigD PE Versus Other End-Healing Enzymes. Although archaeal species, representing five genera of the phylum Eur- LigD PE displays no overt amino acid sequence similarity to any yarchaeota (i.e., Methanoculleus, Methanocella, Methanosarcina, known polynucleotide end-healing enzymes, it was unclear if the and Archaeoglobus, and uncultured methanogen RC-1) (Fig. 4A). PE domain truly defined a new repair enzyme family. This situa- The archaeal PE proteins are similar in size (121–199 aa) to the tion now appears to be the case, given its unique fold, distinctive autonomous bacterial PE module, they have strictly conserved active site, and biochemical specificities vis à vis other phosphoes- counterparts of the six PE active site residues (highlighted in terases that repair DNA/RNA ends. Table 1 compares and con- yellow in Fig. 4A), and they have conserved counterparts of trasts PE to the several families of 3′ end-healing enzymes that the signature residues in the interior of the PE β barrel (denoted have been characterized biochemically and structurally, and are by arrowheads in Fig. 4A). Six of the archaeal PE proteins also variously dedicated to DNA or RNA repair pathways. For exam- have conserved counterparts of the N-terminal PE peptide, sug- ple, bacteriophage and mammalian polynucleotide 5′-kinase/ gesting that they are likely to possess 3′ phosphodiesterase and 3′-phosphatase (Pnkp) are members of the DxD acylphosphatase monoesterase activities àlathe bacterial LigD PE domains. superfamily that repair the 2′,3′ cyclic phosphate and 3′-phosphate The Archaeoglobus profundus PE lacks the N-terminal peptide, ends of broken RNA and DNA strands, respectively, via a covalent and is thereby likely to be proficient as a 3′ phosphodiesterase, aspartyl-phosphoenzyme intermediate (18–20). Whereas phage with weaker monoesterase activity, similar to the N-terminal and mammalian Pnkps are bifunctional 50∕30 end-healing en- deletants of Pseudomonas PE (13). zymes, the yeast DNA 3′-phosphatase exemplifies a stand-alone We inspected each archaeal genomic PE locus for potential acylphosphatase module (21). III and its homologs clustering of genes involved in DNA repair or modification. are members of the DNase I superfamily; they remove 3′-mono- The Methanoculleus, Methanosarcina, and methanogen RC-I phosphates from DNA ends and also have 3′ exonuclease and aba- PEs had no such neighbors. The Archaeoglobus PE gene is imme- sic activities (22, 23). Tyrosine-DNA-phosphodiesterase, diately downstream of a cooriented ORF encoding a putative a member of the D superfamily, hydrolyzes trapped DnaG-type DNA primase, which could signify a functional rela- covalent topoisomerase IB-DNA adducts and DNA 3′-phopho- tionship between these two enzymes. Notably, the Methanocella glycolate lesions via a covalent enzyme-(histidinyl-Nϵ)-3′-phos- paludicola PE is encoded by the proximal ORF of a cooriented phoryl-DNA intermediate (24). three ORF cluster comprising PE, an ATP-dependent DNA The bacterial clade of Pnkps can be viewed as the RNA repair ligase, and a LigD-like polymerase. We surmise that the archaeon analogs of the polyfunctional bacterial LigD DNA repair enzyme, M. paludicola has a three-component equivalent of the trifunc- insofar as bacterial Pnkps comprise three catalytic modules in a tional bacterial LigD enzyme. single polypeptide: a 3′ healing enzyme (belonging to the dinuclear Equally remarkable was the appreciation that PE homologs metallophosphoesterase superfamily), a 5′ end-healing enzyme, are extant in eukarya, specifically in fungi. The PE-encoding fungi and an RNA ligase (25, 26). Yeast and plant tRNA ligases are represent seven genera from phylum Ascomycota (Aspergillus, also multidomain RNA repair proteins with ligase and dual Neosartorya, Talaromyces, Penicillium, Coccidioides, Phaeosphaer- end-healing modules, including a 3′ end-healing enzyme (2′,3′ ia, and Verticillium) and a single taxon from phylum Basidiomy- cyclic phosphodiesterase) that belongs to the 2H superfamily (27). cota (Cryptococcus neoformans). An alignment of Pseudomonas Most remarkably, even though many of the known 3′ repair PE to its fungal homologs highlights strict conservation of five enzymes are metal-dependent (or specifically manganese-depen- of the active site constituents and most of the signature residues dent, in the case of bacterial Pnkp), none of their active sites and in the interior of the PE β barrel. The Tyr88 active site moiety of metal coordination complexes resemble the apparently unique bacterial PE is conserved in Cryptococcus PE, but substituted

Table 1. Distinct families of polynucleotide 3′ end-healing enzymes Enzyme Superfamily Metal cofactor / ligands DNA/RNA repair LigD PE PE Mn / 2His, Asp DNA Bacteriophage Pnkp DxD acylphosphatase Mg / 3Asp RNA Mammalian Pnkp yeast DNA 3′-phosphatase DxD acylphosphatase Mg / 3Asp DNA Bacterial Pnkp Dinuclear metallophosphoesterase 2Mn / 3His, 3Asp, Asn RNA Exonuclease III DNase I 2Mg / Glu, Asp, 2Asn DNA tRNA ligase (CPD) 2H none DNA TDP1 none DNA

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Fig. 4. PE family members in archaea and eukarya. (A) Archaeal PE homologs. PaePE is aligned to the homologous polypeptides encoded by Methanoculleus marisnigri (Mma), Methanocella paludicola (Mpa), uncultured methanogenic archaeon RC-I (RC-I), Methanosarcina barkeri (Mba), Methanosarcina mazei (Mma), Methanosarcina acetivorans (Mac), and Archaeoglobus profundus (Apr). (B) Eukaryal PE homologs. PaePE is aligned to the homologous polypeptides encoded by Aspergillus nidulans (Ani), Aspergillus clavatus (Acl), Neosartorya fischeri (Nfi), Talaromyces stipitatus (Tst), Penicillium marneffei (Pma), Penicillium chrysogenum (Pch), Coccidioides immitis (Cim), Phaeosphaeria nodorum (Pno), Verticillium albo-atrum (Val), and Cryptococcus neoformans (Cne). Positions of amino acid side chain identity/similarity are denoted by dots above the aligned sequences. Conserved PE active site residues are highlighted in yellow. The secondary structure elements of the PE fold are shown above the sequences. Conserved residues located in the hydrophobic interior of the PaePE β-barrel are denoted by black arrowheads below the aligned sequences.

by asparagine in the other fungal PE homologs (Fig. 4B). The MnCl2. Diffraction data at 1.9 to 2.5 Å resolution were collected at National activity and physiology of the fungal PE homologs merits further Synchrotron Light Source (NSLS), Brookhaven NY. Phases for a Hg-soaked attention, especially as several of the PE-encoding fungi are triclinic crystal were determined by using a modified Single Isomorphous human pathogens. Replacement with Anomalous Scattering (SIRAS) method that included In conclusion, our elucidation of the atomic structure of LigD contributions from anomalous scatterers in a native P1 crystal dataset. Elec- PE has revealed a DNA repair enzyme family that is distributed tron density maps revealed two PE molecules in the unit cell. The final refined ∕ ¼ 0 148∕0 199 broadly among taxa in all three phylogenetic domains. model at 1.92 Å resolution (R Rfree . . ) included 305 amino acids from the two PE protomers, with excellent geometry (SI Appendix: Table S1). Materials and Methods We also solved via molecular replacement the 2.3 Å structure of PE crystal- ∕ ¼ 0 212∕0 263 Detailed methods are provided in SI Appendix and are summarized briefly lized in space group C2 (R Rfree . . ). The crystallographic data and here. The Pseudomonas PE domain was produced in as a refinement statistics are compiled in SI Appendix: Table S1. The coordinates His10Smt3 fusion and then isolated from a soluble bacterial lysate by Ni-affi- for the P1 and C2 structures have been deposited in PDB under ID codes 3N9B nity chromatography. After tag removal by treatment with Smt3 protease and 3N9D. Ulp1, the tag-free PE domain was purified by phosphocellulose and gel filtration chromatography. PE crystals were grown by vapor diffusion at room ACKNOWLEDGMENTS. This work was supported by National Institutes of temperature against buffer containing 30% PEG-5000-MME, 100 mM MES Health (NIH) Grant GM63611. S.S. is an American Cancer Society Research (pH 7.0), 200 mM ammonium sulfate, 10 mM yttrium chloride, 2 mM Professor.

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