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Structure of Bacterial Ligd 3′-Phosphoesterase Unveils a DNA Repair Superfamily

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Structure of Bacterial Ligd 3′-Phosphoesterase Unveils a DNA Repair Superfamily 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 ligase 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 enzymes. 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 enzyme. 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 active site 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 phosphodiesterase 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 ligases (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′-ribonuclease/3′-phosphatase 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).
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