A Conserved Lysine Residue in the Crenarchaea-Specific Loop is Important for the Crenarchaeal Splicing Endonuclease Activity

doi:10.1016/j.jmb.2010.10.050

J. Mol. Biol. (2011) 405, 92–104 Contents lists available at www.sciencedirect.com

Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b

A Conserved Lysine Residue in the Crenarchaea-Specific Loop is Important for the Crenarchaeal Splicing Endonuclease Activity Maho Okuda 1,2 , Tomoo Shiba 1 , Daniel-Ken Inaoka 1 , Kiyoshi Kita 1 , Genji Kurisu 3 , Shigeru Mineki 2 , Shigeharu Harada 4 , Yoh-ichi Watanabe 1 ⁎ and Shigeo Yoshinari 1 ⁎ 1

Department of Biomedical Chemistry, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan 2 Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba 278-8510, Japan 3 Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Meguro-ku, Tokyo 153-8902, Japan 4 Department of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Sakyo-ku, Kyoto 606-8585, Japan Received 2 July 2010; received in revised form 26 October 2010; accepted 27 October 2010 Available online 2 November 2010 Edited by R. Huber Keywords: Crenarchaea; splicing endonuclease; subunit structure; intron; tRNA splicing

In Archaea, splicing endonuclease (EndA) recognizes and cleaves precursor RNAs to remove introns. Currently, EndAs are classified into three families according to their subunit structures: homotetramer, homodimer, and heterotetramer. The crenarchaeal heterotetrameric EndAs can be further classified into two subfamilies based on the size of the structural subunit. Subfamily A possesses a structural subunit similar in size to the catalytic subunit, whereas subfamily B possesses a structural subunit significantly smaller than the catalytic subunit. Previously, we solved the crystal structure of an EndA from Pyrobaculum aerophilum. The endonuclease was classified into subfamily B, and the structure revealed that the enzyme lacks an N-terminal subdomain in the structural subunit. However, no structural information is available for crenarchaeal heterotetrameric EndAs that are predicted to belong to subfamily A. Here, we report the crystal structure of the EndA from Aeropyrum pernix, which is predicted to belong to subfamily A. The enzyme possesses the N-terminal subdomain in the structural subunit, revealing that the two subfamilies of heterotetrameric EndAs are structurally distinct. EndA from A. pernix also possesses an extra loop region that is characteristic of crenarchaeal EndAs. Our mutational study revealed that the conserved lysine residue in the loop is important for endonuclease activity. Furthermore, the sequence characteristics of the loops and the positions towards the substrate RNA according to a docking

*Corresponding authors. E-mail addresses: [email protected]; [email protected]. Present addresses: G. Kurisu, Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan; S. Yoshinari, Nucleic Acid Synthetic Biology Research Team, Systems and Structural Biology Center (SSBC), RIKEN, Yokohama, Kanagawa 230-0045, Japan. Abbreviations used: EndA, splicing endonuclease; APE-EndA, EndA from Aeropyrum pernix; BHB motif, bulge–helix– bulge motif; PAE-EndA, EndA from Pyrobaculum aerophilum; A280, absorbance at 280 nm; SIRAS, single isomorphous replacement with anomalous scattering; AFU-EndA, EndA from Archaeoglobus fulgidus; NEQ-EndA, EndA from Nanoarchaeum equitans; WT, wild type. 0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.

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model prompted us to propose that crenarchaea-specific loops and an extra amino acid sequence at the catalytic loop of nanoarchaeal EndA are derived by independent convergent evolution and function for recognizing noncanonical bulge–helix–bulge motif RNAs as substrates. © 2010 Elsevier Ltd. All rights reserved.

Introduction Most RNA primary transcripts are subjected to multiple steps of processing to be mature and functional: primary transcripts of tRNA genes are trimmed to have correct 5′ and 3′ termini; many nucleotides at specific positions are modified according to the tRNA species; and -CCA-3′ terminal sequences are added if they are lacking.1 In addition, all domains of life have tRNA genes with introns that must be removed. In bacteria, the introns are removed autocatalytically by Group I self-splicing mechanisms.2,3 In the cases of eukaryotic nuclearencoded tRNA and archaeal tRNA, tRNA introns are removed by a specific set of enzymes.4 Among eukaryotic species, yeast is the beststudied organism for tRNA splicing. In yeast, the first step of tRNA intron removal, the cleavage of phosphodiester bonds between an exon and an intron, is performed by tRNA intron endonuclease.4 The enzyme consists of four different polypeptides: Sen2p, Sen34p, Sen15p, and Sen54p.5 Sen2p and Sen34p possess RNase activity, and Sen54p is considered to specify the position of the introns by recognizing structural features present in the tRNA bodies.5,6 A similar enzyme is also present in human cells.7 The genes for human tRNA intron endonuclease subunits have been reported to be the causative genes for a neural disorder.8 In Archaea, the cleavage of phosphodiester bonds between an exon and an intron is processed by a splicing endonuclease (EndA),4,9 which is also involved in intron removal in rRNA.10 Furthermore, the enzyme is considered to be involved in the removal of mRNA introns 11–13 and in rRNA maturation14–16 through recognition of a bulge– helix–bulge (BHB) motif composed of a couple of 3-nucleotide bulges split by a 4-bp helix.4,9 The subunits in eukaryotic tRNA intron endonuclease and archaeal EndA are considered to share common ancestors.17,18 EndAs known to date can be classified into three types according to differences in their subunit structures. 9 Homotetrameric (α4) enzymes consist of four identical polypeptides with a molecular weight of 18,000–20,000. Homodimeric (α 2 ) enzymes are composed of two identical polypeptides with a molecular mass of 30,000–45,000. X-ray structures for these two types have already been solved.19–21 By comparing the structures, it has been suggested that the gene for the homodimeric

enzyme was generated by duplication of the endA gene of the homotetramer type, and that the duplicated genes fused to make one large subunit.20 The third type, the heterotetramers (α2β2), is a recent discovery by several groups.22–24 Enzymes of this type were considered to consist of two sets of polypeptides, catalytic (α) and structural (β) subunits, and this prediction was confirmed by solving the X-ray structures.25,26 Euryarchaeal species possess either homodimeric or homotetrameric EndAs. On the other hand, crenarchaeal and nanoarchaeal species possess heterotetrameric EndAs. One exception to this rule is the euryarchaeon Methanopyrus kandleri, which is predicted to possess a heterotetrameric form of EndA; however, this has not been experimentally verified. Recently, there has been growing evidence that EndAs with different subunit structures have different substrate specificities. Homotetrameric and homodimeric EndAs cleave only RNAs with canonical BHB motifs. On the other hand, heterotetrameric EndAs can recognize and cleave RNA substrates with noncanonical BHB motifs.12,24,25,27–29 The crenarchaeal heterotetrameric EndAs can be further classified into two subfamilies based on the size of the structural subunit.25 The first subfamily, subfamily A, possesses a structural subunit similar in size to the catalytic subunit, whereas the second subfamily (subfamily B) possesses a structural subunit significantly smaller than the catalytic subunit. Previously, we solved the crystal structure of an EndA from Pyrobaculum aerophilum (PAEEndA),25 which is classified into subfamily B. On the other hand, an EndA from Aeropyrum pernix (APEEndA), which has introns in pre-tRNAs,30 prerRNAs,31 and pre-mRNAs11,12 with BHB motifs, has been predicted to belong to subfamily A. No structural information is available for this type. Furthermore, among subfamily A structural subunits, the similarity of amino acid sequence in the N-terminal region is relatively low (Fig. S1). In this study, we solved the crystal structure of APE-EndA and showed that the endonuclease retains the N-subdomain of the structural subunit. As a result of the crystal structure study, we found that the enzyme also possesses an inserted loop structure that is characteristic of crenarchaeal heterotetrameric EndAs. Therefore, we also assessed the functional importance of the loop by mutating each amino acid residue to alanine.

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Results Expression and purification of APE-EndA For the structural study, we prepared constructs for His-tagged proteins of the wild-type (WT) sequence of the catalytic subunit of APE-EndA and a substitution mutant at His133 of the catalytic (α) subunit to Ala. His133 is a conserved residue corresponding to one of the catalytic residues identified in previous studies.21,25,32 We included the H133A mutant because it is potentially useful for preparing an enzyme–RNA substrate complex in future work. The two subunits of APE-EndA were simultaneously expressed in Escherichia coli and purified by heat treatment and two successive column chromatographies. For protein complexes with a 6×His-tag at the N terminus of the structural subunit, we successfully purified 6×His-APE-EndA (WT) and 6×His-APE-EndA(H133A). After gel filtration, the peak absorbance at 280 nm (A280) overlapped the peak of endonuclease activity (for the WT; data not shown), and the fractions contained two polypeptides, indicating their formation of a complex. The molecular mass of the protein in these fractions was calculated to be 83.9 ± 3.7 kDa (data not shown), which is consistent with the expected value of the enzyme in a heterotetrameric form consisting of two catalytic and two structural subunits. We found that the recombinant APE-EndA digested an A. pernix cbf5 pre-mRNA fragment with a conventional BHB motif11 (Fig. 1a) and a short model substrate containing a BHB motif29 (Fig. 1b, B3) and its loop-number mutants, while the H133A mutant of the catalytic subunit showed a significantly reduced endonuclease activity under our standard assay conditions (Fig. 1a). A euryarchaeal dimeric EndA from Archaeoglobus fulgidus (AFU-EndA) disfavored the digestion of the RNA at the bulge with the altered loop number (Fig. 1b, lanes B0–B2 and lanes B4–M2) rather than the conventional 3 nt (Fig. 1b, lane B3), while APE-EndA was more tolerant of such substrates (Fig. 1b, see lanes B4 and M1). This substrate specificity agrees with earlier reports.24,27 AFU-EndA cleaved the pre-mRNA into the products with the expected sizes (Fig. 1a). This was not surprising because the enzyme is believed to cleave RNAs other than pre-tRNA, such as prerRNAs at their terminus.16 In the cleavage of the pre-mRNA with AFU-EndA, an extra band (shown by an asterisk) from a likely cleavage at an unexpected position was observed, suggesting that APE-EndA may have more accurate cleavage specificity towards non-tRNA substrates than AFU-EndA.

Crystal structure of 6×His-APE-EndA(H133A) To obtain further structural insight on APE-EndA, we screened conditions for crystallization of 6×HisAPE-EndA(WT) and 6×His-APE-EndA(H133A) using the fractions after gel filtration. For 6×His-APE-EndA(H133A), the initial screening revealed that one reservoir solution described in Materials and Methods resulted in hexagonalshaped crystals with good diffraction signals. Unfortunately, we could not obtain any crystal for 6×His-APE-EndA(WT). From the crystals, we collected a diffraction data set up to 1.7-Å resolution with 98.3% completeness. The crystal belongs to a rhombohedral crystal system, space group R3, with unit-cell parameters a = b = 95.3, c = 253.8 Å. The crystal structure of 6×His-APE-EndA(H133A) was solved by the single isomorphous replacement with anomalous scattering (SIRAS) method with Ptsoaked crystals. The final refined model has an Rwork of 21.8% and an Rfree of 22.9% for the resolution range of 30 to 1.7 Å, and it has good stereochemical quality. A summary of the data collection and refinement is shown in Table 1. The refined structure contains all amino acid residues except for several N-terminal residues of each subunit (2 for chain A, 6 for chain B, 9 for chain C, and 8 for chain D). Moreover, we could not fit the 20 N-terminal amino acid residues derived from the 6×His-tag sequences in chains A and C into the electron density map. The asymmetric unit contains one heterotetrameric enzyme unit, α2β2, composed of two catalytic subunits (α, chains B and D) and two structural subunits (β, chains A and C) that are related by a noncrystallographic 2-fold axis (Fig. 2a). Overall structure of APE-EndA The heterotetrameric APE-EndA has a rectangular parallelepiped-like structure with approximate dimensions of 80 Å × 55 Å × 35 Å (Fig. 2a). A functional unit can be defined as a heterodimeric form composed of one structural subunit and one catalytic subunit. This functional unit is equivalent to one subunit of a homodimeric EndA. Two functional units of APE-EndA (chains A + B and chains C + D) do not show any significant structural differences (RMSD = 0.47 Å for 339 Cα atoms). The structural subunit consists of seven β-strands (β1–β7) and five α-helices (α1–α5; Fig. 2b). A close look at the structure reveals that the structural subunit can be divided in two subdomains. The N-terminal subdomain consists of three β-strands that form a mixed antiparallel/parallel β-sheet (order: β2-β1-β3; underlined strands are parallel strands) and three α-helices (α1, α2, and α3) that

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Fig. 1. Cleavage of synthetic RNA precursors with EndAs. (a) Cleavage of an A. pernix cbf5 pre-mRNA fragment. The products were analyzed by 8.3 M urea–6% PAGE. The products were assigned according to the expected sizes. Asterisk shows a product with an unexpected size. (b) Sequence and cleavage of Mini-BHB29 and its bulge number mutants. In conventional BHB B3 sequence, the cleavage sites are shown as arrowheads. In M1 and M2 sequences, substituted residues from B3 are shown in red. The products were analyzed by 8.3 M urea–20% PAGE.

surround the β-sheet. There are two disulfide bonds in the domain (Cys11–Cys62 and Cys27–Cys32). In the C-terminal subdomain of the structural subunit,

four β-strands form a mixed antiparallel/parallel β-sheet (order: β4-β5-β6-β7), with two α-helices (α4 and α5) placed around the β-sheet.

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Table 1. Data collection and refinement statistics

Data collection statistics Beamline Wave length (Å) Space group Unit cell a (Å) b (Å) c (Å) α (°) β (°) γ (°) Resolution (Å) Observed reflections Unique reflections Completeness (%) Rmerge I/σI Phasing statistics No. of Pt sites Overall mean FOM

Refinement statistics Resolution (Å) Reflections No. of non-hydrogen atoms Protein Solvent Rwork Rfree RMSD from ideal values Bond (Å) Angle (°) Average B-factor (Å2) Ramachandran plot (%) Most favored Additional allowed Generously allowed Disallowed

APE-EndA (H133A)

APE-EndA (H133A)-Pt-soaked

PF BL17A 1.0000 R3

PF BL17A 1.0717 R3

95.3 95.3 253.8 90 90 120 50 to 1.70 (1.73–1.70) 452,698 94,288 98.5 (85.1) 7.2 (34.6) 14.0 (3.1)

96.0 96.0 256.3 90 90 120 50 to 2.30 (2.4–2.30) 407,249 39,146 95.6 (100) 9.1 (28.9) 14.5 (9.4)

15 0.536 (after solvent flattering) 30 to 1.70 88,180 5542 549 (water), 60 (glycerol), 2 (Cl−) 21.8 22.9 0.016 1.6 14.1 94.3 5.7 0 0

The catalytic subunit consists of five α-helices (α6–α10) and nine β-strands (β8–β16; Fig. 2b). The catalytic subunit can also be divided in two subdomains. In the N-terminal subdomain of the catalytic subunit, four β-strands form a mixed antiparallel/parallel β-sheet (order: β10-β9-β8β11) that is surrounded by three α-helices (α6–α8). In the C-terminal subdomain of the catalytic subunit, five β-strands form a mixed antiparallel/ parallel β-sheet (order: β12-β13-β14-β15-β16) that is surrounded by two α-helices (α9 and α10). Between the structural and catalytic subunits, an antiparallel intermolecular β-sheet that is important for dimerization (β5–β14 interaction) is formed by β5 (structural subunit) and β14 (catalytic subunit). The interface between the two functional units is rich in both hydrophobic and electrostatic interac-

tions. The interaction between the L10 loop in the structural subunit and the receptor pocket in the catalytic subunit is also conserved.33 The N-terminal subdomain, which is missing in the PAE-EndA, interacts with the opposing catalytic subunit through hydrophobic interactions from tryptophan 69, valine 73, and isoleucine 76 in α-helix 3. Comparison with other EndAs—overall structure The archaeal EndAs can be distinctly classified into three families: homodimers (α2), homotetramers (α4), and heterotetramers or dimers of heterodimers (α2β2). Crystal structures of the homodimeric EndA from A. fulgidus (AFU-EndA; PDB accession number 1RLV)20 and EndA from Thermoplasma acidophilum (PDB accession numbers 2OHC and 2HOE)21 have been reported. In addition, a crystal structure of the AFU-EndA in complex with an RNA substrate has been revealed (PDB accession number 2GJW).34 Furthermore, one crystal structure for a homotetrameric EndA from Methanocaldococcus jannaschii (PDB accession number 1A79)19 and two crystal structures for the active heterotetramer [one from P. aerophilum (PAE-EndA; PDB accession number 2ZYZ)25 and another from Nanoarchaeum equitans (NEQ-EndA; PDB accession number 3IEY)26] have also been solved. The EndA from A. pernix (APE-EndA; this work) is a dimer of the heterodimer family (α2β2). Structural alignment revealed that the structure of APE-EndA is similar to that of PAE-EndA (RMSD = 2.07 Å for 506 Cα atoms; Fig. S2a). A large difference between the two structures is that PAE-EndA lacks the entire N-terminal subdomains of the structural subunits, whereas APE-EndA retains the subdomains (Fig. S2a, dotted circles in red). The NEQEndA structure (as a heterotetramer) aligns with the APE-EndA structure less well (RMSD = 2.57 Å for 488 Cα atoms) than the PAE-EndA structure (not shown). Structural alignment of AFU-EndA with the APE-EndA structure showed modest similarity (RMSD = 2.44 Å for 540 Cα atoms; Fig. S2b). Less structural similarity with APE-EndA was seen for EndA from T. acidophilum (RMSD = 2.93 Å for 456 Cα atoms; not shown) and EndA from M. jannaschii (RMSD = 2.91 Å for 586 Cα atoms; Fig. S2c). The N-subdomains of the structural subunits among EndAs share the same structural characteristics of folding.19–21 They are basically composed of one large antiparallel/parallel β-sheet surrounded by three α-helices. The second and third α-helices are located next to each other. The N-subdomain of APEEndA is no exception, suggesting that, at least, the Nsubdomain of APE-EndA shares a common origin with those of homodimeric and homotetrameric EndAs, and the catalytic subunits of crenarchaeal EndAs.

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Fig. 2. Crystal structure of APE-EndA. (a) Crystal structure of 6×His-APE-EndA(H133A). One heterotetramer unit in a unit cell is shown. One heterotetramer is composed of two structural subunits (β subunit; green and magenta) and two catalytic subunits (α subunit; cyan and yellow). (b) A functional heterodimeric unit of 6×His-APE-EndA(H133A), chains A (green) and B (cyan). Dotted-circled N and C indicate the N and C termini of the chains. Red color in chain B indicates the position of the crenarchaea-specific loop in the structure. The secondary structures of the polypeptides are also labeled.

Comparison with other EndAs—catalytic subunit (domain) In our previous report on the PAE-EndA crystal structure, we discovered that crenarchaeal EndAs possess specific inserted loops in their catalytic subunits, and showed that the loop is involved in the enzymatic activity.25 To examine if this is also the case for APE-EndA, which is of crenarchaeal origin, we structurally aligned the catalytic subunits and catalytic domains of the solved structures. When the X-ray structures were superposed, there was a clear difference between the EndAs of crenarchaeal origin and those of other origins (euryarchaeal or nanoarchaeal). The EndAs of

crenarchaeal origin possess loops of 10 to 13 amino acids inserted at a particular position (Figs. 2b and 3b–d). An amino acid sequence alignment of the EndAs also supports the idea that the loops are inserted in a crenarchaea-specific manner (Fig. 3a, shaded in violet). Interestingly, when the amino acid residues of the extra loops (Fig. 3d, shown as sticks) at the border of the gap (Fig. 3a, highlighted in colors) were mapped on the superimposed crystal structures, the positions of the edges of the inserted loops seemed almost identical. The amino acid sequences of the inserted loops also share some common characteristics. The loops are rich in charged residues, and a lysine residue at the third position of the loop (numbered

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Fig. 3. Crenarchaea-specific loop. (a) A part of amino acid sequence alignment of the catalytic subunits or catalytic domains of the EndAs with defined X-ray structures. Shaded part shows the inserted amino acid sequences that are specific for the crenarchaeal EndAs. Conserved and semiconserved lysines are marked with asterisks. Underlined residues are structurally presented in (b)–(d). (b–d) Structural superposition of the underlined residues in (a). Color assignments are as follows: green, APE1646; cyan, PAE2269; magenta, AF0900; yellow, NEQ205; orange, MJ1424; gray, Ta1191; red, ST0358 (PDB accession number 2CV8); and blue, SSO0439. For (b), all structures are superposed. The same superposed structures are divided into (c) EndAs of non-crenarchaeal origins and (d) EndAs of crenarchaeal origins. In (d), residues at the border of the loops [highlighted in colors in (a)] are shown as sticks.

from the N-terminus of the loop, marked as asterisk in Fig. 3a) is conserved. In the complex of AFU-EndA with substrate RNA, the substrate was situated along the border between two subunits of the enzyme.34 The interactions between the substrate RNA and the enzyme occurred in bulge regions in the RNA. The catalytic residues interact with residues surrounding the cleavage sites.34 In APE-EndA, the conserved Lys in the crenarchaea-specific loop is within 23 Å of the catalytic triad (Y125, Ala in place of H133 in this study, and K164), suggesting that, at least, some of the residues in the loop may interact with the bulge region of the substrate RNA. In fact, when APE-EndA was superposed on AFU-EndA in the RNA–enzyme complex, the crenarchaea-specific loop was located near the bulge region in the bound RNA (see Fig. 4a and Fig. S3).

The conserved lysine residue is responsible for the enzymatic activity Previously, as the first step to investigate the region characteristics of the crenarchaeal enzyme, we prepared mutants with partial or entire deletions of the crenarchaea-specific loop of PAE-EndA, and showed that they lost nuclease activity towards an RNA substrate with a conventional BHB motif.25 Although these mutants maintained a heterotetrameric subunit structure, the deletion might cause an effect on the enzyme other than a missing region. Thus, to examine the functional importance of the crenarchaea-specific loop with minimal effect on other regions, we mutated each amino acid residue in the loop of APE-EndA to alanine if it was a non-alanine or non-glycine residue. The mutants we made were as follows:

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Fig. 4. Splicing endonuclease activity of mutants at the crenarchaea-specific loop. (a) Superposition of APE-EndA with AFU-EndA (in complex with substrate RNA, 2GJW). AFU-EndA is not shown. The catalytic triad (Y125, Ala in place of H133 in this study, and K164; shown in red) and two residues (K44 and R46; shown in green) in the crenarchaea-specific loop are shown as capped sticks. The crenarchaea-specific loop is shown in green. (b) APE-EndAs introduced with the indicated mutations were subjected to an EndA assay. H133A was also assayed to confirm that the mutant lost EndA activity. (c) Position of the crenarchaea-specific loop towards substrate RNA. Green indicates the crenarchaea-specific loop of APE-EndA. The thick orange ribbon represents a backbone of substrate RNA, and bars with a scarlet to violet color gradient indicate bases of the RNA. K44 and R46 of APE-EndA are shown as a stick model. B-1 to B-3 indicate the corresponding bases, which consist of a 3-base bulge in the BHB motif. The EndAs cleave a phosphodiester bond between B-2 and B-3.

E43A, K44A, P45A, R46A, D49A, F50A, and E51A. The mutants were expressed, purified by heat treatment and metal-affinity chromatography, and subjected to an EndA assay using Sulfolobus tokodaii tRNATrp precursor as a substrate. The results are shown in Fig. 4b. In this assay, the WT APE-EndA cleaved the substrate RNA at two sites and

produced fragments (intron, 5′-exon, and 3′-exon) as expected. Changing E43, D49, and E51 to alanine did not affect the enzymatic activity. However, mutations of K44, P45, R46, and F50 to Ala decreased the endonuclease activity. In the case of K44A, practically no EndA activity was observed. This result clearly showed that the amino acid

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residues in the loop have important roles in the enzymatic activity.

Discussion Crenarchaeal heterotetrameric EndAs can be divided into two subfamilies according to the size of structural subunit.25 EndAs of subfamily B have smaller structural subunits harboring 90–120 amino acid residues. An example is PAE-EndA. The crystal structure has revealed that the enzyme lacks the N-subdomain of the structural subunit,25 in agreement with the fact that the structural subunit is approximately half the size of the catalytic subunit. EndAs of subfamily A possess structural subunits similar to catalytic subunits in size (approximately 180 amino acid residues). APE-EndA is categorized in this subfamily. In the present study, we determined the crystal structure of the APE-EndA with H133A mutation. The APE-EndA possesses an N-terminal subdomain in the structural subunit that is missing in PAE-EndA (Fig. S2a). Therefore, the difference in residue number in the structural subunit is due directly to the structure of crenarchaeal EndA. The small (96 amino acid residues for PAE-EndA) structural subunit lacks the N-terminal subdomain, whereas the large (170 amino acid residues for APE-EndA) structural subunit consists of both N- and C-terminal subdomains. At the moment, we have no idea why there exist two subfamilies of heterotetrameric EndAs with different subdomain structures. We tried to make an APEEndA mutant lacking the N-subdomain of the structural subunit. It was expressed in significantly low amounts in E. coli cells and became insoluble during preparation (data not shown). Therefore, the N-subdomain might be important for the stability and/or solubility of the protein in solution for crenarchaeal subfamily A EndAs. In the structural analysis in this study, some of the residues in the third α-helix of the domain (W69, V73, I76) were shown to interact with the amino acids of the opposing catalytic subunit. These interactions might be important for the enzyme to be active and soluble in solution. The N-subdomain also contains two disulfide bonds, which might be important for the stability of the protein. We previously solved the crystal structure of PAEEndA (subfamily B), which is of crenarchaeal origin. At that point, we could obtain only one structural coordinate of the EndA of crenarchaeal origin (2CV8, inactive dimer form of the catalytic subunit from S. tokodaii; see Ref. 23 for the enzymatic activity) from the public domain. Now, we have added the structure of active APE-EndA from subfamily A of crenarchaeal enzymes. We also personally obtained a coordinate file for a crystal structure of an inactive dimer of the catalytic subunit from S. solfataricus.24

A comparison of the four protein structures reveals that they harbor a crenarchaeal EndA-specific extra loop that exists in neither euryarchaeal nor nanoarchaeal EndAs (Fig. 3b–d). This loop has been shown to be involved in the enzymatic activity of PAE-EndA.25 The sequence of the loops has some characteristic features. They are rich in positively charged residues. A lysine residue at the third position is conserved, and a lysine at the fifth position is semiconserved among the crenarchaeal EndAs for which crystal structures are known (Fig. 3a). By our mutational study, we revealed that the conserved lysine at the third position of the loop has a crucial function for the enzymatic activity. The semiconserved fifth position (lysine or arginine) is also involved in the activity. Since these amino acid residues are not the ones predicted to be involved in hydrolysis of the phosphodiester bond of the substrate, we are considering that these residues could be involved in substrate binding to the enzyme. In fact, when we superposed the structures of APE-EndA and AFU-EndA (in complex with substrate RNA, 2GJW)34 and assessed the positions of the positively charged residues in the crenarchaea-specific loop, we found them closely positioned to the substrate RNA (K44 and R46 in Fig. 4c). Crenarchaeal EndAs digest substrates with variant BHB motifs (Fig. 1b). The charged residues at various positions in the crenarchaea-specific loop may serve to influence the interaction between the enzyme and its various substrates, leading to broader substrate specificity of the crenarchaeal enzyme. Recently, a substitution of K35 (equivalent to K44 in the crenarchaea-specific loop of APEEndA) in PAE-EndA to Ala or Glu was found to nullify enzymatic activity, while a K35R mutant maintained nuclease activity (S.Y. unpublished data), suggesting that the positive charge of the conserved Lys residue could interact with any functional group in the substrate RNA, such as a phosphate residue. Crenarchaeal and nanoarchaeal EndAs have the common characteristics to accept and cleave RNAs with noncanonical BHB-motifs as substrates.12,24,25,27–29 Coincidentally, we found the same characteristic features of the crenarchaeaspecific loop in an extra sequence in nanoarchaeal EndA at a loop in which resides a histidine residue that is responsible for hydrolysis of the phosphodiester bond. The extra amino acid sequence is also rich in charged residues and harbors 3 lysines in the sequence (Fig. 5a). When we superposed the structure of NEQ-EndA onto AFU-EndA (in complex with substrate RNA, 2GJW)34 and added the position of the nanoarchaea-specific extra sequence in Fig. 3c, the basic residues in the extra sequence were also closely situated to the substrate RNA (K93, K94, K96, and R97 Fig. 5b). From this observation, we propose here that the positively

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Fig. 5. Nanoarchaea-specific insertion in relation to crenarchaea-specific loop. (a) Partial amino acid sequence alignment of the catalytic subunits or catalytic domains of EndAs with defined X-ray structures. Histidine residues in red indicate that they are conserved and responsible for the catalytic activity. When H133 in APE1646 was mutated to alanine, the mutant lost EndA activity, as shown in Fig. 4. (b) Positions of the crenarchaea-specific loop and nanoarchaeal extra sequence towards substrate RNA. The residues for the NEQ-EndA extra sequence indicated in cyan were added to the model shown in Fig. 4c. K93, K94, K96, and R97 of the NEQ-EndA catalytic subunits are shown as a stick model.

charged residues in the crenarchaea-specific loop and the nanoarchaeal extra sequence work as novel substrate RNA biding sites that do not exist in euryarchaeal EndAs. In addition, as an example of convergent evolution, these novel RNA binding sites that were acquired independently from different lineages might provide broader substrate specificity for the enzymes. On the other hand, Mitchell et al. suggested that the broad substrate specificity of nanoarchaeal EndA might be due to an inherent flexibility in the active site to accommodate noncanonical RNA bulge structures.26 Further study is needed to test these two possibilities.

Materials and Methods

Nomura and Prof. Y. Sako of Kyoto University). The PCR product was digested with NdeI and BamHI and inserted into the NdeI and BglII sites in the multi-cloning site 2 of pETDuet-1 (Novagen). The resultant plasmid was named pETDuet-APE1646. APE0685 coding the open reading frame amplified by PCR was inserted between the NdeI and BamHI sites of pET15b. Then the APE0685 coding region with a 6×His tag sequence in pET15b was amplified by PCR with pETupstream and pETterminator primers. The product was digested with NcoI and BamHI and inserted between the NcoI and BamHI sites in the multi-cloning site 1 of pETDuet-APE1646. The resultant plasmid was called pETDuet-6×His-APE-EndA(WT). To make plasmids for expression of APE-EndA mutants, pETDuet-6x His-APE-EndA was subjected to a QuikChange (Stratagene) reaction using the primer sets listed in Table S1.

Construction of expression plasmids Coexpression of subunits of APE-EndA or its mutants An open reading frame coding APE1646 was amplified by PCR using A. pernix K1 genomic DNA as a template (the genomic DNA was generously provided by Dr. M.

E. coli strain Rosetta-gami (DE3) (Novagen) was transformed with each plasmid constructed above. A

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colony was picked and the bacteria were grown in 1 L of LB broth [1% (w/v) Bacto-tryptone, 0.5% (w/v) Bactoyeast extract, 0.5% (w/v) NaCl] at 37 °C supplemented with 100 μg/ml carbenicillin and 34 μg/ml chloramphenicol. When the optical density at 600 nm (OD600) reached 0.5, IPTG was added to give a final concentration of 0.5 mM, and then the culture was continued at 37 °C for 3 h. A typical culture after 3 h gave an OD600 reading of approximately 1.0. The cells were harvested by centrifugation at 8000g for 10 min, washed, and suspended in 1/25 (40 ml) of the culture volume of buffer A [20 mM Tris–HCl (pH 8.0), 50 mM NaCl]. Purification of the recombinant APE-EndAs The cells in the suspension were lysed by 100 μg/ml of hen egg lysozyme, and the viscosity was reduced by sonication. Afterwards, the cell lysate was incubated at 80 °C for 20 min, followed by centrifugation at 20,000g for 10 min. The soluble protein in the supernatant was subjected to further purification of the enzyme. The collected supernatant (37 ml) was gently agitated with 0.75 ml of bed volume of TALON metal-affinity resin (Clontech, Co2+-chelated agarose) at 4 °C for 40 min. After incubation, the resin was recovered by centrifugation (700g for 10 min) and washed twice with buffer B [50 mM NaPO4 (pH 8.0), 300 mM NaCl] + 10 mM imidazole. Then the resin was packed into the column, and the His-tagged protein was eluted with a linear gradient of 18–300 mM imidazole in buffer B at a flow rate of 1 ml/min. After fractionating by 1.5 ml, the fractions were subjected to SDS-PAGE. The fractions (115–210 mM imidazole) containing 6×His-APE-EndA were pooled and dialyzed against buffer C [20 mM Tris–HCl, 750 mM KCl, and 10% (w/v) glycerol]. For structural analysis, the dialyzed fraction was concentrated to an A280 reading of approximately 7 by use of Amicon Ultra-4 [Millipore; molecular weight cutoff: 10,000]. After concentration, 0.5 ml of protein solution was loaded onto Superdex 200 10/300 (GE Healthcare), which was equilibrated with buffer C at a flow rate of 0.5 ml/ min. Peak fractions of A280 containing 6×His-APE-EndA were used for crystallization experiments. For estimation of the molecular weight of APE-EndA, gel filtration standards (Bio-Rad Laboratories) were used. Preparation of AFU-EndA The AFU-EndA open reading frame (AF0900) was amplified by PCR from A. fulgidus genomic DNA (America Type Culture Collection 49558D-5) as template, digested with NdeI and BamHI at the terminus, and cloned into the site between NdeI and BamHI in pET-15b (Novagen). Expression and purification were carried out essentially as described above but without purification with gel filtration. Crystallization of 6×His-APE-EndA, X-ray data collection, and processing Peak fractions of the gel filtration were pooled and concentrated to an A280 reading of approximately 10 by

the use of Amicon Ultra-4 (molecular weight cutoff: 10,000). Screening for crystallization conditions by sitting-drop vapor-diffusion technique was examined using CrystalClear P Strips (Douglas Instruments). Concentrated 6×His-APE-EndA protein solutions of 1 μl were mixed with an equal volume of reservoir solution, and the drop of the resultant protein solution was allowed to reach equilibration with 100 μl of the reservoir solution. Initial crystallization conditions for the reservoir solution were screened at 22 °C using Crystal Screen,35 Crystal Screen II (Hampton Research), and Wizard Screens I, II, and III (Emerald BioStructures). Out of 242 conditions, hexagonal crystals were observed under several conditions for the H133A variant (no crystal could be obtained with the WT protein). Among them, the best crystal in size and thickness was obtained under the following conditions: Wizard Screen II No. 36, 0.2 M NaCl, 0.1 M phosphate-citrate (pH 4.2), and 10% (w/v) polyethylene glycol 3000. For phasing, the crystals were introduced to platinum by soaking in reservoir solution supplemented with 2 mM K2PtCl4 for 24 h before being picked in a nylon loop. For collecting the diffraction data, a crystal was picked in a nylon loop and soaked briefly in reservoir solution supplemented with 20% (v/v) glycerol and then frozen by submerging it rapidly in liquid nitrogen. Diffraction data sets for native crystals (λ = 1.000 Å) and single-wavelength anomalous diffraction data sets for Pt-soaked crystals (λ = 1.0717 Å) were collected under liquid nitrogen-cooled conditions of 100 K at beamline BL17A (Photon Factory, Tsukuba, Japan) using a Quantum-270 detector (Advanced Detector System Corporation). All data sets were indexed, integrated, and scaled using HKL2000.36 Initial attempts to solve the crystal structure using the molecular replacement method with known EndAs were unsuccessful. Therefore, the crystal structure of APEEndA was determined by the SIRAS method. SHELXC/ D/E37 programs implemented in the HKL2MAP38 graphical interface were used to find and refine the 15 platinum sites in the SIRAS data sets, producing a phase set with a figure of merit of 0.54 at 1.7 Å (Table 1). The electron density map was readily interpretable and the phases were input for refinement. The initial model automatically built in ARP/wARP39 allowed us to trace about 619 residues (90%) of APE-EndA (687 residues in the final model). Manual building of the remaining model and further crystallographic refinement were performed with the COOT program40 and Refmac5.41 In the final refinement, using an amplitude-based twin refinement41 with a twinning fraction of 0.054 (twinning operator k, h, − l) and TLS parameters42 (four TLS groups) dramatically improved the Rfree (26.0% to 22.9%). The final model was checked by PROCHECK,43 revealing good stereochemical parameters with no residues outside of the allowed regions of the Ramachandran plot. The statistics for data collection and refinements are summarized in Table 1. Structural analyses and graphic presentations Structural alignments were done by the “align” function of PyMol44 (version 1.2r2). Graphic presentations of the structures were developed with the same software.

Crenarchaeal Splicing Endonuclease Structure and Its Activity

Splicing endonuclease assay A typical endonuclease reaction in 10 μl of total volume containing 10 pmol of Mini-BHB29 or S. tokodaii tRNATrp precursor transcript23 including 50,000 cpm of the 32Plabeled transcript and 2.5 pmol of EndA was performed as described previously25 with modifications. After incubation at 70 °C for 20 min, the reaction was stopped by adding an equal volume of FDE [90% (v/v) formamide, 10 mM ethylenediaminetetraacetic acid (pH 8.0), 0.02% (w/v) bromophenol blue, 0.02% (w/v) xylene cyanol], and the product RNAs were resolved by 8.3 M urea–10% PAGE. Radioactivity was detected by exposing the gel to ImagePlate (Fujifilm) and then scanning the exposed plate with BAS-2500 (Fujifilm). For the pre-mRNA fragment,11 the assay was conducted essentially as described previously.23. No radioactive nucleotide was used for the substrate preparation, and the cleavage products were analyzed with ethidium bromide staining after gel electrophoresis. Accession code The coordinates and the structural factors of the EndA with the H133A mutation from A. pernix [APE-EndA (H133A)] have been deposited in the PDB with accession code 3AJV.

Acknowledgements We thank Dr. M. Nomura and Prof. Y. Sako (Kyoto University) for the gift of Aeropyrum pernix K1 genomic DNA. We are also grateful to Prof. H. Li (Florida State University) for the structural coordinates of archaeal EndAs before their deposition to the PDB. We also appreciate the staff members of the beamline BL17A at Photon Factory for their help with X-ray diffraction data collection. This study is partly supported by a Grant-in-Aid for Creative Scientific Research of Japan Society for the Promotion of Science (to K.K. and Y.W.) and the Targeted Proteins Research Program (to K.K.) of the Japanese Ministry of Education, Science, Culture, Sports and Technology (MEXT).

Supplementary Data Supplementary data to this article can be found online at doi:10.1016/j.jmb.2010.10.050

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