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1 Department of Biological Chemistry, Institute of Molecular Biology, and
2 Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Copenhagen, Denmark
Reprint requests to: Anders Kadziola, Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark; e-mail: anders{at}ccs.ki.ku.dk; fax: +45-35-32-02-99.
(RECEIVED October 8, 2003; FINAL REVISION November 28, 2003; ACCEPTED December 1, 2003)
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Abstract |
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5' exoribonucleases that also includes polynucleotide phosphorylase (PNPase). RNase PH is involved in the maturation of tRNA precursors and especially important for removal of nucleotide residues near the CCA acceptor end of the mature tRNAs. Wild-type and triple mutant R68Q-R73Q-R76Q RNase PH from Bacillus subtilis have been crystallized and the structures determined by X-ray diffraction to medium resolution. Wild-type and triple mutant RNase PH crystallize as a hexamer and dimer, respectively. The structures contain a rare left-handed 
-motif in the N-terminal portion of the protein. This motif has also been identified in other enzymes involved in RNA metabolism. The RNase PH structure and active site can, despite low sequence similarity, be overlayed with the N-terminal core of the structure and active site of Streptomyces antibioticus PNPase. The surface of the RNase PH dimer fit the shape of a tRNA molecule. Keywords: crystal structure; maturation of tRNA; ribonuclease; RNase PH; tRNA precursor
Abbreviations: RNase, ribonuclease • PNPase, polynucleotide phosphorylase • Tris, 2-amino-2-hydroxymethyl-1,3-propanediol • MES, 2-[N-morpholino]ethanesulfonic acid • PEG, polyethylene glucol
3 These authors have contributed equally to this work. 
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.03477004.
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Introduction |
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5' exoribonucleases that also includes PNPase, EC 2.7.7.8
[EC]
(Mian 1997; Zuo and Deutscher 2001). These two enzymes are the only known examples of phosphorolytic exoribonucleases, which catalyze the cleavage of RNA substrates using inorganic orthophosphate. The reaction releases nucleoside diphosphates rather than the nucleoside monophosphates produced by hydrolytic RNases. The best characterized biological function of RNase PH is the trimming of tRNA precursors at their 3' ends. This process involves the action of several exo- and endonucleases (Deutscher 1995). The exonucleases have overlapping specificities. Deleting a single gene encoding one of these enzymes in bacteria does not affect growth (Kelly and Deutscher 1992a). Despite the overlapping specificities of these enzymes, Li and Deutscher (1996) have shown that RNase PH together with RNase T are the prime enzymes responsible for removing the 2 nt closest to the CCA acceptor end of the tRNAs. They can substitute each other’s function, but the lack of both enzymes in E. coli leads to accumulation of 3'-extended tRNAs in the cells and severe retardation of cell growth (Kelly et al. 1992). Other, but less understood functions of RNase PH are indicated by the observation that the lack of both RNase PH and PNPase in E. coli leads to very slow growth of the cells and a defect in ribosome assembly (Zhou and Deutscher 1997) and by the observation that high expression of Bacillus subtilis RNase PH in E. coli suppresses several cold-sensitive phenotypes (Craven et al. 1992) by a mechanism that is not at all understood.
Most of the biochemical characterization of RNase PH has been done with the enzyme isolated from E. coli (Poulsen et al. 1984; Jensen et al. 1992). In our hands, this enzyme did only generate poorly diffracting crystals. Instead we produced and characterized the enzyme from B. subtilis (Craven et al. 1992). The sequence of this enzyme is 50% identical to the sequence of the E. coli RNase PH. Recently, the structure of RNase PH from Aquifex aeolicus has been published (Ishii et al. 2003). In the present work, we report the crystal structures of wild-type and triple mutant R68Q-R73Q-R76Q RNase PH from B. subtilis.
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). Within experimental uncertainty, the monomeric structures of crystal forms I, II, and III are identical where they overlap. The 245-residue RNase PH monomer is a single domain subunit with a mixture of -helix and -strand secondary structure elements ordered in layers as a -sandwich. We have assigned nine -strands and four -helices numbered in sequential order (Figs. 1, 4). Strands 1 to 5 and 6 to 9 form a mixed and an antiparallel sheet, respectively. The two -sheets are structurally separated by helices 1, 2, and 3 and helix 4 packs across the other face of sheet 6–9. The hexameric form displays P32 symmetry; that is, it has three twofold axes perpendicular to a threefold axis. This symmetry creates two types of interfaces between the monomers. For one of the interfaces, the interactions between subunits A and B are clearly closer than for the other between subunits A and F. It is therefore natural to consider the hexamer as a trimer of dimers. The dimerization interface (AB) contains as a key feature an edge-to-edge antiparallel hydrogen bond interaction between the two 9 strands from both subunits A and B, resulting in the formation of a 6–9:9–6 eight-stranded antiparallel -sheet across the dimer (Fig. 2A). In addition, four salt bridges contribute to the dimerization: Glu 89A/B-Arg 92B/A and Glu 89A/B-Arg 96B/A. The trimerization of dimers involves face-to-face interactions between sheets 1–5 from subunits A and F (Fig. 2B). In crystal form II, 10 salt bridges are observed (see after next section) and are partly responsible for this interaction. One of the two SO42- ions per monomer, in crystal forms I and II, is found at the N-terminal end of helix 2, where it binds to the main-chain atoms Thr 125-N and Arg 126-N and to the side-chain atoms T125-O
1 and Arg 126-N
2. The second SO42- ion binds to side-chain atoms Trp 58-N
1, Thr 60-O1 and Arg 99-N1. The structures have been deposited with the Protein Data Bank with accession codes 1OYP, 1OYR, and 1OYS.
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). The high identity between the two enzymes was also reflected in an excellent root-mean-square deviation of 0.77 Å using 228 pairs of C atoms with a maximum distance of 2.0 Å, when overlayed. Although the connectivity of the secondary structure elements is very similar, the A. aeolicus enzyme contains two additional short -helices (1, 2) and three extra -strands (10, 11, 12). The extra -strands are located at the C terminus of the enzyme, which is 10 amino acid residues longer than the B. subtilis enzyme. The -helix 2 is located in the central region of the hexameric structure, which, in crystal forms I and III of B. subtilis, are disordered. We show that this region contains conserved arginine residues that are important for maintaining the hexameric structure and propose that upon binding of the substrate RNA the hexamer is destablized.
Conserved arginine residues are important in assembly of the hexameric structure
The structure of crystal form II shows a long loop, Leu 66-Ser 84, which is unresolved in crystal forms I and III. During purification of the protein, this region was recognized to contain a cleavage site (probably a trypsinlike protease site) resulting in a heterogeneous sample (less than 5% were cleaved). The amino acid sequence in this region of the protein contains three arginyl residues, Arg 68, Arg 73, and Arg 76, of which the latter two are conserved among all RNase PH sequences. The three arginyl residues appear to be structurally important, stabilizing the hexamer by forming electrostatic interactions with acidic amino acid residues from the neighboring dimers. In crystal form II, the following 10 salt bridges between dimers are observed: Arg 68A/F-Glu 25F/A, Arg 73A/F-Glu 62F/A, Arg 73A/F-Asp 115F/A, Arg 76A/F-Glu 62F/A, and Arg 76A/F-Asp 117 F/A. We have substituted the arginyl residues in the loop region with glutaminyl residues by site-directed mutagenesis, resulting in the triple mutant enzyme R68Q-R73Q-R76Q. The spontaneous cleavage during purification of the wild-type enzyme is not observed in the R68Q-R73Q-R76Q enzyme. In contrast to wild-type RNase PH, the mutant enzyme crystallized as a dimer with nothing reminiscent of the hexamer form, yielding crystal form III.
The change in quaternary structure was consistent with results from size exclusion chromatography experiments, which also revealed a native molecular weight of the triple mutant enzyme corresponding to that of a dimer (data not shown). Thus, the substitution of the arginyl residues with glutaminyl residues in the triple mutant R68Q-R73Q-R76Q enzyme results in an enzyme with an apparently altered quaternary structure and clearly demonstrates that the three arginyl residues, Arg 68, Arg 73, and Arg 76, are important for maintaining the hexameric structure.
Comparison of RNase PH with the more distantly related polynucleotide phosphorylase
The crystal structure of another member of the RNase PH superfamily, Streptomyces antibioticus PNPase, has been solved by Symmons et al. (2000). In addition to the 3' 5' phosphorylase activity shared with RNase PH, the S. antibioticus PNPase has a guanosine 3'-diphosphate-5'-triphosphate pppGpp synthetase activity. The monomer of S. antibiotocus PNPase consists of 757 amino acids and is thus considerably larger than the RNase PH monomer. The structural core of PNPase has two homologous domains interrupted by an all helical domain. Two RNA-binding domains are located at the C-terminal end of the protein, the KH (Burd and Dreyfuss 1994) and the S1 (Bycroft et al. 1997) domains. These domains have been shown to be dispensable for catalytic activity. Comparison of the two PNPase core domain sequences shows that they have diverged extensively since presumed gene duplication gave origin to the PNPase family (Symmons et al. 2002). The binding of the inhibitory orthophosphate analog tungstate to the second core domain suggests that the phosphorolytic activity resides in this domain. It had earlier been shown that the second core domain of PNPase aligns to RNase PH (Mian 1997). Despite relatively low sequence similarity of 17%, the connectivity of the secondary structure elements is similar in the two enzymes (Fig. 4C) and both enzymes form an overall structure of a bulky disk with a central channel (Fig. 2B). The quaternary structure is maintained by forming a trimer of monomers in PNPase and by forming a trimer of dimers in RNase PH. This places the three PNPase active sites on the same face of the disk, whereas in RNase PH the twofold symmetry of the dimer places three active sites on either side of the disk, as also has been noted by Symmons et al. (2002). We have superimposed the entire RNase PH structure with the second core domain of PNPase. The structures could be overlayed with a root-mean-square deviation of 1.1 Å using 184 pairs of C atoms with a maximum distance of 2.0 Å. It should be mentioned that as the two core domains of S. antibioticus PNPase have arisen from gene duplication, the first core domain of S. antibioticus PNPase also shows structural similarity to RNase PH.
The N-terminal part of RNase PH contains a fold common to other enzymes in RNA metabolism
The RNase PH structure was submitted to the Dali-server (Holm and Sander 1993) that searches for structural homology in the Protein Data Bank. This search revealed that the N-terminal portion: Gly 26 to Ala 147 of RNase PH has structural similarity to other enzymes involved in RNA metabolism. Two structures were found with very low Z scores: the protein part of the ribozyme ribonuclease P (Z = 3.2; Stams et al. 1998) and domain IV of elongation factor G Ef-G. (Z = 2.2; Al-Karadaghi et al. 1996). These structures are shown in Figure 3. The common fold consists of a four-stranded -sheet covered on one side with two -helices and displays a rare left-handed crossover motif. This topology has also been observed in the second domain of DNA gyrase B, a type II topoisomerase, and the C-terminal domain of ribosomal protein S5 (Murzin 1995), and argues for the evolution of a module adapted for polynucleotide binding. Photo-cross-linking studies of precursor tRNA molecules to RNase P holoenzyme have shown that the -sheet forms a platform for binding of the tRNA molecules (Niranjanakumari et al. 1998). However, in RNase PH, the -sheet is used for trimerization contacts between dimers to form the hexamer and can thus only be accessible to tRNA binding when the hexamer dissociates to dimers.
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) and Arg 126 at the N terminus of helix 2. This corresponds to the binding of the phosphate ion in the A. aeolicus structure (binds to Thr 125 at the N terminus of 4) and thus strongly confirms the identification of the active site. This region also contains the highly conserved Gly 123 of RNase PH (Gly 460 of PNPase). Close by is helix 3 with the acidic motif DX4EDX5D (Zuo and Deutscher 2001), which is completely conserved in RNase PH (D175, E180, D181, D187) and PNPase (D508, E513, D514, D520) families. In the more distantly related members of the RNase PH family only the first aspartic acid is completely conserved (Mian 1997). In the RNase PH crystal form II, the carboxylate groups of Asp 181 and Asp 187 are pointing towards each other with a poorly resolved residual density in between (Fig. 5). This may represent a partially occupied Cd2+ ion site (not included in the model) as the crystals were grown in its presence. The role of these residues could therefore be binding of the divalent metal ion Mg2+, which is required for enzymatic activity (Kelly and Deutscher 1992b). However, Asp 181 and Asp 187 could also be the cleaving site of the phosphodiester bond by carrying one common catalytic proton. The active site residues, Thr 125, Arg 126, Asp 181, Asp 187, and the sulfate ion are shown in Figure 5. Connected to the DX4EDX5D motif is the RX3RX5R motif (Zuo and Deutscher 2001), which is also completely conserved in RNase PH (R2, R6, R12) and PNPase (R339, R343, R349). It has been proposed by Symmons et al. (2002) that this motif in PNPase could be involved in RNA binding. In RNase PH, however, this is only possible in an unfolded mode of the protein, as salt-bridge interactions are observed between Arg 2-Glu 180, Arg 6-Asp 4, and Arg 12-Asp 175, indicating a role as structural stabilization of helix 3.
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we have monitored conserved residues in a space-filling model of the RNase PH dimer. A groove extends from the active site in molecule A (marked by the active site sulfate ion), then passes by the second sulfate ion of molecule B and continues over the RNA binding -motif also located in molecule B. The groove conforms to the shape of a tRNA molecule with the 3' end (CCA sequence) pointing down into the active site. Not only does this strongly argue that the dimer is the active form, but we also envisage that the hexamer is displaced upon substrate binding, due to interactions between the -motif and the substrate tRNA. This is consistent with the studies on the R68Q-R73Q-R76Q mutant. This model is different from the model of tRNA binding by Ishii et al. (2003), in which only the tRNA acceptor stem interacts with the dimer.
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Substrate RNA binding
We have no biochemical data addressing the number of subsites present on RNase PH for the binding of tRNA. However, the density of a sulfate ion in proximity to the conserved residues Trp 58, Thr 60, and Arg 99 could be a potential binding site for phosphate groups in the RNA phosphodiester backbone. In addition, the highly conserved region of helix 1, RX5RX3RX2R (Arg 86, Arg 92, Arg 96, Arg 99), could also be thought as stabilizing the phosphodiester backbone of the tRNA substrate. Actually helix 1 contributes to the walls of the tRNA binding groove shown in Figure 6. The RX5RX3RX2R region is comparable to the RNR signature motif, RNX3RX2R, in ribozyme RNase P, which also has been argued to be implicated in RNA binding by recognizing the tertiary structure of the catalytic RNA (Spitzfaden et al. 2000). As mentioned above, it is likely that RNase PH recognizes the tertiary structure of the precursor tRNA molecule, and, thus, comparable to RNase P, the conserved arginines of helix 1 could be involved in recognizing the tertiary structure of the precursor tRNA molecules. Interestingly, the arginines of helix 1 in RNase PH and the RNR motif of RNase P are located in the "RNA-binding domain" found in both proteins, where they are located in the left-handed -crossover. The recognition of tRNA tertiary structure by RNase PH through the interactions of amino acids in the left-handed crossover could argue for the existence of this domain in the RNase PH superfamily.
The active site
The sulfate ion at positions Thr 125 and Arg 126 superimposes well with the partially occupied phosphate analog, tungstate in S. antibioticus PNPase and the phosphate ion in A. aeolicus RNase PH and thus establishes the location of the active site. In support, we have found a poorly resolved residual density between Asp 181 and Asp 187 in helix 3 close to the active site that may represent the binding of a divalent metal ion Mg2+ that is required for catalysis (Fig. 5). Arg 86, which Ishii et al. (2003) showed by structural and biochemical analysis played an important role in the phosphorolysis reaction, is placed in the same C position in the B. subtilis enzyme; the side-chain, however, does not bind in a well ordered way to the sulfate ion and thus did not provide us with any information.
Quaternary structure
Both B. subtilis and A. aeolicus RNase PH crystallize as a hexamer. Docking of a substrate tRNA molecule to the B. subtilis enzyme suggests that the substrate interacts with the dimer (Fig. 6). In the model of Ishii et al. (2003), tRNA binding, although different from our model, also only involve the dimer. So, what is the functional role of a hexameric form? We suggest that the hexamer has a protective role, protecting the highly flexible loop between strand 4 and helix 1, which is used for substrate recognition, from cleavage by a trypsinlike protease. This is supported by the observed electrostatic interactions between neighboring dimers and the arginyl residues of the flexible loop maintaining the hexameric structure and from the crystallization of the triple mutant R68Q-R73Q-R76Q enzyme, which crystallizes as a dimer. However, studies on the molecular weight of E. coli and B. subtilis RNase PH have shown that the enzyme has a tendency to aggregate at higher protein concentrations, and thus the presence of the hexamer could be caused by the high concentration of protein under the crystallization conditions.
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Crystallization and data collection
Two crystal forms, I and II, of native B. subtilis RNase PH have been obtained at 20°C by sitting drop vapor diffusion. The protein concentrations were 12.5 mg/mL and 37.5 mg/mL, respectively, and 2 M (NH4)2SO4 was used as precipitant. For crystal form I, pH was adjusted to 6.0 by adding small amounts of 1.0 M NH4HCO3 to a 3.0 M (NH4)2SO4 stock solution. For crystal form II, pH was adjusted to 8.5 using a 0.1 M Tris-HCl buffer, and 20 mM CdCl2 was present in the precipitant solution. In both cases, the sitting drops composed of 5-µL protein solution and 5-µL precipitant solution were equilibrated against a 1.0-mL reservoir of precipitant solution. Crystals appeared within a week. Both crystal forms belong to the orthorhombic space-group P212121 with unit cell dimensions: a = 51.3 Å, b = 163.6 Å, c = 166.8 Å, and a = 99.8 Å, b = 146.5 Å, c = 152.3 Å for crystal forms I and II, respectively. A third crystal form, III, has been obtained for the triple mutant protein R68Q-R73Q-R76Q. Mutant protein at 15.3 mg/mL was crystallized using a precipitant composed of 1 M Na-citrate (pH 6.2) and 10% PEG400. Sitting drops were composed of 3 µL protein solution, 1 µl 10% -octyl glycoside, and 3 µL of precipitant and equilibrated against 1 mL of precipitant solution in the reservoir. Crystal form III belongs to the tetragonal space-group P41212 with cell dimensions: a = b = 62.2 Å and c = 203.0 Å. Data collection statistics are summarized in Table 1.
Structure determination
The structure was determined by isomorphous replacement using crystal form II. Heavy-atom derivatives were prepared by soaking with HgCl2. Five different derivative crystals, containing from 2 to 6 Hg sites, were made by soaking in various heavy-atom concentrations. Heavy atoms were refined and initial phases were calculated using the program MLPHARE (Collaborative Computational Project 1994). These phases were subjected to solvent flattening and histogram matching using the program DM (Collaborative Computational Project 1994). In the first electron density map, a Trigonal Planar Pseudo Residue (TPPR) model was constructed using the graphics program TURBO-FRODO (Roussel and Cambillau 1992). This model showed sixfold P32 symmetry that was incorporated as noncrystallographic symmetry averaging in DM. The electron density was much improved and major parts of the amino acid sequence could be assigned to the electron density. The structures of crystal forms I and III were determined by molecular replacement with the program AMoRe (Navaza 1994) using the monomer structure of crystal form II. Similar to crystal form II, crystal form I contains a hexamer in the asymmetric unit. The hexamer is organized as a trimer of dimers. Crystal form III of mutant protein R68Q-R73Q-R76Q contains one molecule per asymmetric unit, but one of the crystallographic twofold axes generates the dimer found as part of the hexamers in crystal forms I and II.
Refinement
All structures were refined with the program CNS (Brünger et al. 1998) using torsion angle dynamics. Rebuilding between rounds of refinement was performed with the graphics program TURBO-FRODO (Roussel and Cambillau 1992) using 3mFobs - 2DFcalc and mFobs - DFcalc electron density maps (Read 1986; Collaborative Computational Project 1994). For crystal forms I and II, sixfold noncrystallographic symmetry restraints were applied during refinement, and electron density maps were accordingly averaged for rebuilding (Kleywegt and Read 1997). In crystal form II, three additional Cd2+ ions were detected in the packing of the nonaveraged map and included in the model. Refinement statistics are given in Table 1.
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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level and the mFobs - DFcalc difference density is shown with strong lines and contoured at 4