Monomeric solution structure of the helicase-binding domain of Escherichia coli DnaG primase Xun-Cheng Su, Patrick M. Schaeffer, Karin V. Loscha, Pamela H. P. Gan, Nicholas E. Dixon and Gottfried Otting Research School of Chemistry, Australian National University, Canberra, Australia All organisms replicate DNA by copying one strand (the leading strand) in a continuous manner, whereas the other DNA strand (the lagging strand) is replicated in a discontinuous manner by the synthesis of short Okazaki fragments that are later joined into a continu- ous strand [1]. During DNA replication, a helicase sep- arates the double-stranded DNA into single strands, and replication of the leading strand and synthesis of the Okazaki fragments is initiated by RNA primers made by the specialized RNA polymerase, primase. The first primase to be identified and characterized was that from Escherichia coli. In E. coli, the replicative helicase and primase are encoded by the dnaB and dnaG genes, respectively. The DnaB helicase forms a hexameric ring structure with up to three molecules of the DnaG primase attached [2–4]. DnaG is composed of three main domains comprising an N-terminal zinc-binding domain for interaction with single-stranded DNA, a central domain responsible for primer synthesis, and a C-terminal domain (residues 434–581; DnaG-C) that binds to the DnaB helicase. The binding interaction with DnaB locates DnaG in the correct position to lay down primers on newly formed single-stranded DNA as the DnaB helicase progresses along the DNA. Pri- mases are essential for DNA synthesis and are there- fore targets for the development of new antibiotics [5]. No 3D structure has been determined for full-length DnaG, but crystal structures have been obtained for the N-terminal domain from Bacillus stearothermophilus Keywords DnaB; DnaG; domain swap; NMR structure; primase Correspondence G. Otting, Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia Fax: +61 2 61250750 Tel: +61 2 61256507 E-mail: Gottfried.Otting@anu.edu.au Database The NMR chemical shifts and coordinates of the structure have been submiited to the BioMagResBank (accession code 6284) and Protein Data Bank (accession code 2HAJ) (Received 28 July 2006, revised 7 September 2006, accepted 11 September 2006) doi:10.1111/j.1742-4658.2006.05495.x DnaG is the primase that lays down RNA primers on single-stranded DNA during bacterial DNA replication. The solution structure of the DnaB-helicase-binding C-terminal domain of Escherichia coli DnaG was determined by NMR spectroscopy at near-neutral pH. The structure is a rare fold that, besides occurring in DnaG C-terminal domains, has been described only for the N-terminal domain of DnaB. The C-terminal helix hairpin present in the DnaG C-terminal domain, however, is either less sta- ble or absent in DnaB, as evidenced by high mobility of the C-terminal 35 residues in a construct comprising residues 1–171. The present structure identifies the previous crystal structure of the E. coli DnaG C-terminal domain as a domain-swapped dimer. It is also significantly different from the NMR structure reported for the corresponding domain of DnaG from the thermophile Bacillus stearothermophilus. NMR experiments showed that the DnaG C-terminal domain does not bind to residues 1–171 of the E. coli DnaB helicase with significant affinity. Abbreviations DnaB(1–171), residues 1–171 of E. coli DnaB helicase; DnaB-N, the N-terminal domain (residues 24–136) of E. coli DnaB helicase; DnaG-C, the C-terminal domain of DnaG primase (residues 434–581 of the E. coli protein); DTPA-BMA, diethylenetriamine pentaacetic acid- bismethylamide; P16, the C-terminal domain of Bacillus stearothermophilus DnaG (residues 452–597). FEBS Journal 273 (2006) 4997–5009 ª 2006 The Authors Journal compilation ª 2006 FEBS 4997 [6], the central RNA polymerase domain from E. coli [7,8], the two-domain fragment comprising both the N-terminal and RNA polymerase domains from Aquifex aeolicus [9], and the C-terminal helicase-binding domain from E. coli [4]. In addition, the structure of the C-terminal domain from B. stearothermophilus (P16) has been determined by NMR spectroscopy [10]. Despite their conserved function, the crystal struc- ture of E. coli DnaG-C [4] and the subsequent solution structure of B. stearothermophilus P16 [10] show sub- stantial differences, including a different number of helices with different helix boundaries and a different spatial arrangement of the C-terminal helices. These differences are important, because the C-terminal helix hairpin is critical for the binding of DnaG to DnaB [10,11]. In P16, the C-terminal helices are only loosely held in place by the rest of the structure [10]. In both structures, the N-terminal helices are packed in a fold similar to that of the N-terminal domain of DnaB (residues 24–136; DnaB-N) [12,13], and the DnaG-C crystal structure shows the C-terminal helices from different monomers entwined via intermolecular con- tacts in a way reminiscent of the fold of DnaB-N. The dimer structure was distorted by crystal contacts, resulting in noticeably different backbone conforma- tions and different orientations of the C-terminal heli- ces in each of the two monomers [4]. However, both gel filtration and analytic ultracen- trifugation experiments at neutral pH showed that DnaG-C was monomeric [4], and it was difficult to ascribe any functional significance to the dimer. In addition, NMR spectroscopic analysis showed little evidence for dimer formation in solution. Some NOEs were observed that were consistent with the dimer interface observed in the crystal structure, and these were interpreted as evidence for a monomer–dimer equilibrium [4], but they could also arise from intramo- lecular contacts in solution that are not present in the monomers in the crystal structure. In order to resolve these difficulties and the discrepancies between the structure of P16 (which is monomeric in solution) and the different conformers in the crystal structure of DnaG-C, we here report the solution structure of E. coli DnaG-C determined under conditions where the protein is strictly monomeric. This new structure differs from the conformers observed in the single crystal, reveals a fold even closer to that of DnaB-N than the crystal conform- ers, and shows no evidence for the presence of two independent subdomains as in P16. The conforma- tional rigidity of the monomeric DnaG-C structure was confirmed by 15 N-relaxation, coupling constant and solvent accessibility measurements. The structure identifies the crystal structure of DnaG-C as a domain-swapped dimer that probably has no func- tional significance. The close fold conservation between DnaG-C and DnaB-N prompted us also to investigate a longer N-terminal construct of DnaB, DnaB(1–171), for the presence of a C-terminal helix hairpin as present in DnaG-C. DnaB(1–171) comprises the complete N-ter- minal domain and hinge regions of DnaB identified by proteolysis [14], and includes peptide segments that have previously been shown by mutation analyses to modulate the interaction between DnaG and DnaB [3,11,15,16]. Consequently, we also probed the interac- tion between DnaG-C and DnaB(1–171). Results Aggregation state of DnaG-C DnaG-C is prone to self-aggregation at high protein concentration and in the absence of salt [17]. Ultra- centrifugation experiments at 0.06 and 0.29 mm pro- tein concentration in the presence of 100 mm NaCl yielded M r values of 16 500 and 14 100, respectively, indicating that the single species present was the monomer (calculated M r ¼ 16 701; supplementary Fig. S1). To verify the monomeric state of the protein under the conditions used for NMR structure deter- mination (0.4 mm DnaG-C, pH 6.1, 100 mm NaCl, 25 °C), the rotational correlation time of DnaG-C was determined from the ratio of transverse and lon- gitudinal 15 N relaxation rates. The rotational correla- tion time s m was found to be 11 ± 1 ns, based on average values of R 1 ¼ 0.99 ± 0.13 s )1 and R 2 ¼ 20.41 ± 1.68 s )1 for the structurally well-defined part of the protein (Fig. 1). Increased R 1 and decreased R 2 relaxation rates indicated increased mobility and structural disorder for about 12 and three residues at the N-terminus and C-terminus of the construct, respectively, in agreement with the narrow 1 H-NMR line widths reported earlier for these residues [17]. Negative [ 1 H] 15 N NOEs were observed for residues 437–441 at the N-terminus, demonstrating mobility on the subnanosecond timescale, whereas the NOE was greater than 0.7 for residues 453–578, indicating structural rigidity for this part of the protein (data not shown). The rotational correlation time of rigid protein structures can be predicted from the atomic coordi- nates using hydronmr [18]. The rotational correlation times predicted for the individual monomers and the dimer in the crystal structure of DnaG-C [4] were about 17 and 36 ns, respectively, and thus much longer Solution structure of E. coli DnaG-C X C. Su et al. 4998 FEBS Journal 273 (2006) 4997–5009 ª 2006 The Authors Journal compilation ª 2006 FEBS than the value of 11 ns derived from the 15 N-relaxa- tion times in solution. However, rotational correlation times of, respectively, 11 and 12 ns were predicted for the corresponding domain from B. stearothermophilus [10] and for the monomeric DnaG-C structure repor- ted here. These data and the uniformity of the relaxa- tion rates along the amino acid sequence (Fig. 1) supported the notion of DnaG-C being a monomeric, structurally compact domain with no evidence for seg- mentation into subdomains as observed in the crystal structure [4] and reported for P16 [10]. Structure determination The solution structure of E. coli DnaG-C was deter- mined using NOEs and backbone dihedral angle restraints derived from chemical shifts. All NOEs were interpreted as intramolecular NOEs. The resulting monomeric structure fulfilled all assigned NOEs with- out significant residual violations (Table 1). The fold exposes all charged amino acid side chains to the sol- vent and buries all hydrophobic side chains that are highly conserved among different bacterial species (Fig. 2). The side chain solvent accessibility averaged over the different NMR conformers is 16% or less for any of the conserved hydrophobic side chains, except for the side chain of Leu484, which is almost 30% sol- vent exposed. The conservation of Leu484 may be explained by its contacts with Leu519, which is a strictly conserved residue (Fig. 2). Insertions and dele- tions in the sequence alignment of Fig. 2 are all con- fined to loop regions, indicating that the secondary structure of DnaG-C is conserved among DnaG mole- cules from many different bacterial species. h1 h2 h3 h4 h5 h6 h7 1.5 1.0 0.5 0.0 R 1 s -1 440 460 480 500 520 540 560 580 Residue number 0 30 20 10 R 2 s -1 Fig. 1. 15 N-relaxation rates measured for 15 N ⁄ 13 C-labeled DnaG-C. The data were measured at a 1 H-NMR frequency of 800 MHz, using a 0.4 m M solution of DnaG-C in NMR buffer at 25 °C. Upper panel, R 1 relaxation rates. Lower panel, R 2 relaxation rates. Error bars indicate the error reported by the fitting routine in SPARKY [40]. Table 1. Structural statistics for the NMR conformers of E. coli DnaG primase C-terminal domain (DnaG-C). Parameter Value Number of assigned NOE cross-peaks a 2400 Number of nonredundant NOE upper-distance limits 2151 Number of dihedral-angle restraints 154 Intraprotein AMBER energy (kcalÆmol )1 ) ) 4575 ± 1176 Maximum NOE-restraint violations (A ˚ ) 0.17 ± 0.06 Maximum dihedral-angle restraint violations (°) 3.1 ± 3.1 rmsd for N, C a and C¢ (A ˚ ) b,c 0.8 ± 0.2 rmsd for all heavy atoms (A ˚ ) b,d 1.2 ± 0.2 Ramachandran plot appearance e Most favored regions (%) 85.7 Additionally allowed regions (%) 11.8 Generously allowed regions (%) 1.4 Disallowed regions (%) f 1.1 a Stereospecific resonance assignments were obtained for 26 pairs of C b H 2 groups, two pairs of C c H 2 and C d H 2 groups, and six pairs of C c H 3 and C d H 3 groups. b For residues 449–576. c 0.5 ± 0.1 A ˚ for residues 449–525. d 0.9 ± 0.1 A ˚ for residues 449–525. e From PRO- CHECK NMR [37]. f All residues in disallowed regions were located in loop regions or at the C-terminus of the structure. No residue was consistently found in disallowed regions. X C. Su et al. Solution structure of E. coli DnaG-C FEBS Journal 273 (2006) 4997–5009 ª 2006 The Authors Journal compilation ª 2006 FEBS 4999 The fold of DnaG-C comprises six helices arranged as in the N-terminal domain of DnaB (Fig. 3A,B). Pairwise comparison using the CE server [19] gave an rmsd between the two proteins of 3.3 A ˚ for 101 aligned residues. No other protein in the Protein Data Bank has a similar fold (other than P16 from B. stearothermophilus; see below). Comparison with the crystal structure of DnaG-C The crystal structure of dimeric DnaG-C [4] contains two DnaG-C molecules with different orientations and boundaries of helix 6 (Fig. 3D,E), showing that this helix can be separated from the core of the structure. The solution structure of DnaG-C identifies the crystal Fig. 2. Sequence alignment of DnaG-C with homologs from different bacterial species. The sequence numbering of E. coli DnaG-C is shown at the top, together with the helix boundaries of DnaG-C determined in this work. Conserved hydrophobic residues are shaded dark gray. The amino acid sequence of DnaG-C from B stearothermophilus is shown at the bottom together with the helix boundaries reported by Syson et al. [10] The following sequences from DnaG-C proteins are shown (abbreviation, species, GenBank number): E. coli, Escherichia coli, 130908; S. enterica, Salmonella enterica subsp. enterica serovar Paratyphi A, str. ATCC 9150, 56129407; Y. pestis, Yersinia pestis CO92, 15978733; P. luminescens, Photorhabdus luminescens subsp. laumondii TTO1, 36787269; E. carotovora, Erwinia carotovora subsp. atroseptica SCRI1043, 49610155; B. aphidicola, Buchnera aphidicola str. Sg (Schizaphis graminum), 21622949; C. blochmannia, Candidatus blochmannia pennsylvanicus str. BPEN, 71795953; V. parahe, Vibrio parahaemolyticus RIMD 2210633, 28805388; H. somnus, Haemophilus somnus 2336, 46156266; P. multocida, Pasteurella multocida subsp. multocida str. Pm70, 12721596; I. loihiensis, Idiomarina loihiensis L2TR, 56180311; P. profundum, Photobacterium profundum SS9, 46912067; X. fastidiosa, Xylella fastidiosa Dixon, 71164362; L. pneumophila, Legionella pneumophila, 1575484; P. syringae, Pseudomonas syringae pv. tomato str. DC3000, 28851001; B. stearo, Bacillus stearothermo- philus, 78101045. The sequences were identified and aligned in a BLAST search [41], except for the sequence of B. stearothermophilus, which was aligned on the basis of its secondary structure elements. Solution structure of E. coli DnaG-C X C. Su et al. 5000 FEBS Journal 273 (2006) 4997–5009 ª 2006 The Authors Journal compilation ª 2006 FEBS structure of DnaG-C as a domain-swapped dimer, where helix 6 from one protein molecule binds to the core of the other in a manner similar to that in which helix 6 binds to the core of the structure in the mono- meric solution structure (Figs 3A and 4). The two conformers in the crystal structure vary not only with regard to helix 6 (Fig. 3D,E) but also in the part comprising helices 1–5, with a backbone rmsd of 2.0 A ˚ for residues 449–525. The differences are mostly due to a displacement of helix 5 and variability in the loop region between helices 2 and 3. The backbone rmsd for the same residues with respect to the solution structure is 1.8 ± 0.1 and 2.4 ± 0.1 A ˚ for crystal con- formers I and II, respectively. The largest differences are in the loop region between helices 2 and 3, suggest- ing that this region is flexible. Whereas 15 N-HSQC spectra of DnaG-C at pH 4.6, 6.1 and 8.1 displayed virtually the same chemical shifts, some of the cross-peaks in the spectrum recor- ded at pH 4.6 (the pH used for crystallization) were exceedingly weak, especially in the loop regions between helices 2 and 7 (supplementary Figs S2 and S3). This indicates the presence of chemical exchange phenomena at low pH in the millisecond time regime. Increased mobility of the loop regions at pH 4.6 and 8.1 was also suggested by the observation of enhanced 15 N-relaxation rates (supplementary Fig. S4). There- fore, the domain swap observed in the crystal structure may have been due to the use of a pH value below the isoelectric point of the protein (5.0). As comparable NMR line widths and 15 N-relaxation rates were observed for the regular secondary structure elements at all three pH values, the domain-swapped dimer is not the major conformational species even at low pH. Comparison with P16 from B. stearothermophilus Except for the C-terminal helices, the solution struc- ture of P16, the DnaG-C domain from B. stearo- thermophilus [10], shows the same overall fold as the present solution structure of E. coli DnaG-C (Fig. 3A,C). However, the similarity is less striking h1 h2 h3 h4 h5 h6 h7 h1 h2 h3 h4 h5 h6 h6 h7 DE crystal conformer I crystal conformer II DnaG-C DnaG-C h1 h2 h3 h4 h5 h6 h7 h1 h2 h3 h4 h5 h6 h1 h2 h3 h4 h5 h6 h7 h8 A B C DnaG-C DnaB-N P16 solution structure Fig. 3. Ribbon representations of DnaG-C and related proteins. (A) E. coli DnaG-C. The short 3 10 helix between helices 2 and 3 was found in fewer than half of the NMR conformers and was therefore not labeled. It was also found in conformer II but not conformer I of the crystal structure [4]. (B) N-terminal domain of E. coli DnaB (residues 24–136) [12]. (C) B. stearothermophilus DnaG-C (fragment P16) [10]. (D) Con- former I of the crystal structure dimer of E. coli DnaG-C [4]. (E) Conformer II of the crystal structure dimer of E. coli DnaG-C [4]. X C. Su et al. Solution structure of E. coli DnaG-C FEBS Journal 273 (2006) 4997–5009 ª 2006 The Authors Journal compilation ª 2006 FEBS 5001 than anticipated based on the functional similarity of DnaG-C domains, with a backbone rmsd of 3.2 A ˚ for 88 aligned residues from the globular part of P16 (cor- responding to residues 449–543 of E. coli DnaG-C), which excludes helices 7 and 8 of P16 (Fig. 1). Helices 6 and 7 of P16 do not form a single continuous helix as in E. coli DnaG-C, but are connected by a flexible linker, entailing a very different orientation of the C-terminal helix hairpin with respect to the core of the structure [10]. A H541 H541 R448 R448 K447 K447 NN I530 I530 C C B C F535 L464 L454 E532 E532 L454 L464 F535 Fig. 4. Stereo views of the solution and crystal structures of DnaG-C. (A) Superposi- tion of the backbone atoms of residues 447–581 of the 20 NMR conformers of DnaG-C representing the NMR structure (Table 1). Numbers identify sequence posi- tions as in Fig. 2. The 15 flexible N-terminal residues were not plotted. (B) Stereo view of the DnaG-C conformer closest to the mean structure of the 20 conformers shown in (A), using a heavy atom representation. The polypeptide backbone is drawn as a rib- bon and the flexible N-terminal 15 residues are omitted for clarity. The following colors were used for the side chains: blue, Arg, Lys, His; red, Glu, Asp; yellow, Ala, Cys, Ile, Leu, Met, Phe, Pro, Trp, Val; gray, Asn, Gln, Ser, Thr, Tyr. Darker-shaded bold lines indi- cate the side chains of Lys447, Lys448, Ile530 and His541. (C) Domain-swapped dimer in the crystal structure of DnaG-C [4]. Only residues 447–528 of conformer I and residues 527–580 of conformer II of the crystal structure are shown, with white and magenta ribbons tracing the backbones of the respective conformers. Darker-shaded bold lines indicate the side chains of Lys447, Lys448, Ile530 and His541. The side chain of Ile530 is buried in the dimer interface by packing against Ile530 from the other monomer (not shown). The side chains of Glu532, Phe535, Leu454 and Leu464 are labeled. NOEs between these residues are explained by the monomeric solution structure, but are also predicted by intermolecular interactions in the dimer of the crystal structure [4]. Solution structure of E. coli DnaG-C X C. Su et al. 5002 FEBS Journal 273 (2006) 4997–5009 ª 2006 The Authors Journal compilation ª 2006 FEBS The structural differences between P16 and E. coli DnaG-C may be explained by the low sequence homology between the two proteins. Although P16 fea- tures 14 of the 16 hydrophobic side chains found with high conservation among DnaG-Cs from different bac- terial species, the structure-based sequence alignment of Fig. 2 resulted in only 13% sequence identity between P16 and E. coli DnaG-C. The low sequence homology also explains why our structure-based sequence alignment is very different from the sequence alignment reported earlier [10]. The flexibility of the linker peptide connecting heli- ces 6 and 7 in P16 (Fig. 3C) and the different breaking points in helix 6 of E. coli DnaG-C observed in the crystal structure (Fig. 3D,E) raise questions about the flexibility of helix 6 of E. coli DnaG-C in solution. Structure verification of helix 6 of DnaG-C An extensive set of H a (i)-H N (i+3) NOEs, 3 J HNHa coupling constants smaller than 6 Hz, and chemical shifts ( 15 N, 13 C a , 13 C b , 1 H a and 13 C¢) indicative of heli- cal secondary structure along the length of helix 6, all suggest that a straight helix as depicted in Fig. 3A is a faithful representation of this helix in DnaG-C under the conditions of the NMR experiments. Measure- ments of the 3 J HNHa coupling constants at 20 lm rather than 0.4 mm protein concentration (data not shown) did not yield significantly increased coupling constants, showing that the structure of helix 6 is not stabilized by concentration-dependent self-association. Although the NMR structure of DnaG-C should be a reliable representation of the average structure in solution, this does not exclude the possibility of small populations of conformers with spontaneously formed transient breaks in helix 6 as a possible prelude to the formation of a domain-swapped dimer. We carefully analyzed the NOESY spectra of DnaG-C with regard to this question. As NOEs strongly emphasize the presence of short internuclear distances, NOE spectra can convey the signature of minor conformational spe- cies if short internuclear distances occur in a minor, but not in the major, conformation. However, the 3D 15 N-NOESY-HSQC spectrum of DnaG-C recorded at 0.4 mm protein concentration on a 800 MHz NMR spectrometer showed no significantly different NOE patterns compared to the corresponding spectrum recorded previously on a 600 MHz NMR spectrometer with a 0.6 mm sample in the same NMR buffer [4]. In particular, strong sequential H N –H N NOEs and weak sequential H a –H N NOEs characteristic of helical sec- ondary structure were found all along helix 6. Further- more, no evidence for a minor population of the domain-swapped dimer could be found, as all NOEs previously thought to be indicative of the domain- swapped dimer [4] were in agreement with the present monomeric structure and independent of protein con- centration between 0.2 and 0.4 mm. The flexibility of helix 6 was further investigated by measurements of the solvent accessibility of amide pro- tons as evidenced by enhanced 1 H-NMR line widths observed in the presence of a soluble paramagnetic relaxation agent. Breaks in this helix would be expec- ted to interrupt the hydrogen bonding pattern and expose some of the amide protons to the solvent. We used Gd[diethylenetriamine pentaacetic acid-bismethyl- amide (DTPA-BMA)] as an uncharged relaxation enhancement agent that does not change the chemical shifts of the protein signals [20]. In addition, we used a low protein concentration (40 lm) to minimize the chance of any self-association. Comparison of the peak heights measured in 15 N-HSQC spectra recorded with and without Gd(DTPA-BMA) revealed pronounced solvent exposure only for loop regions between helices and for the flexible N-terminal residues (Fig. 5). In contrast, the amide protons of helix 6 were among the protected protons. In view of the uncertainty ranges associated with the data points, the slightly enhanced relaxation rates observed for the amide protons of resi- dues 541, 543, and 548 barely indicates significant temporary solvent exposure in a conformational equilibrium. Structure investigation of DnaB(1–171) The striking structural homology between DnaG-C and DnaB-N (Fig. 3A,B) invites the question of whe- ther a longer construct of DnaB-N could display a C-terminal helix hairpin like DnaG-C, considering that it is a feature of all DnaG-C conformers reported to date. Secondary structure prediction of DnaB suggests a helix for residues 153–169 and an extension of helix 6 by 11 amino acids to residue 145. As our original DnaB-N construct was truncated at Glu161, this could have caused the random coil behavior reported from residue 137 onwards [21]. A TOCSY spectrum recorded of DnaB(1–171), how- ever, displayed the same cross-peaks as the TOCSY spectrum reported previously of DnaB(1–161) [21] with additional cross-peaks for the 10 additional C-terminal residues (data not shown). Owing to the increased M r effected by dimerization of the DnaB-N domain [12], the TOCSY spectrum recorded with a long mixing time (80 ms) strongly emphasizes the signals from the mobile residues with narrow line widths. In the TOC- SY spectrum of DnaB(1–171), narrow line widths and X C. Su et al. Solution structure of E. coli DnaG-C FEBS Journal 273 (2006) 4997–5009 ª 2006 The Authors Journal compilation ª 2006 FEBS 5003 random coil chemical shifts were observed for the entire polypeptide segment from residues 137 to 171. Therefore, the C-terminal helix hairpin observed in DnaG-C is not a structural feature of DnaB(1–171). Interaction of DnaG-C with DnaB(1–171) Binding of DnaG-C to DnaB(1–171) was probed by comparing the 15 N-HSQC spectra of 0.13 mm 15 N ⁄ 13 C-labeled DnaG-C in the absence and presence of an equal amount of unlabeled DnaB(1–171). No chemical shift changes or changes in peak intensities were detec- ted. This indicates that any binding between these two domains would be characterized by a dissociation constant of at least 0.5 mm. A dissociation constant of 4.9 lm has been reported for the complex between DnaG-C and full-length DnaB from BIAcore studies [4]. In agreement with the NMR results, no inhibitory interaction between DnaB(1–171) and full-length DnaG could be observed in a BIAcore assay, where a 5 lm solution of DnaB(1–171) was mixed with 285 nm DnaG prior to its injection over a surface displaying single-stranded DNA-bound DnaB hexamer, under conditions used in our earlier studies [4] (data not shown). Furthermore, there was no sign of toxicity of DnaB(1–171) when overexpressed in E. coli, as might have been expected if tight binding of DnaB(1–171) to DnaG were to compete with its interaction with the DnaB hexamer. Discussion The present structure determination of DnaG-C revealed a fold very similar to that of the N-terminal domain of the E. coli DnaB helicase (DnaB-N) [12,13]. The similarity includes helix 6, which is differently ori- ented in the conformers of the domain-swapped dimer (Fig. 3). The structural similarity between DnaG-C and DnaB-N is intriguing, as no other protein is known with this particular fold, and DnaG binds to DnaB. In view of the critical importance of the C-ter- minal helix hairpin of DnaG-C for the interaction with DnaB [4,10], it is tempting to speculate that the domain-swapped dimer observed in the crystal struc- ture of E. coli DnaG-C might serve as a model for the interaction with DnaB-N. Many attempts have been made to pinpoint the interaction between DnaG and DnaB to protein sub- domains. Whereas the interaction seems to be entirely confined to the C-terminal domain of DnaG [4,10], the situation is much less clear for DnaB. For example, mutations in the N-terminal domain of E. coli DnaB have been shown to interfere with the DnaB–DnaG interaction [22], but corresponding mutations in B. ste- arothermophilus had much smaller if any effects [3,16]. 440 460 480 500 520 540 560 580 0.0 0.5 1.0 h1 h2 h3 h4 h5 h6 h7 Residue number Relative intensity Fig. 5. Intensity ratio of backbone amide cross-peaks in 15 N-HSQC spectra of 0.04 mM 15 N ⁄ 13 C-labeled E. coli DnaG primase (DnaG-C) in the presence and absence of 6.0 m M Gd(DTPA-BMA). Solution structure of E. coli DnaG-C X C. Su et al. 5004 FEBS Journal 273 (2006) 4997–5009 ª 2006 The Authors Journal compilation ª 2006 FEBS Apparently inconsistent results could arise from the fact that E. coli DnaB-N is only marginally stable against unfolding [23,24] and easily destabilized by mutations. In B. stearothermophilus, DnaG was found to protect the linker residues between the N-terminal and C-terminal domains of DnaB from digestion with trypsin and pepsin [25]. Mutations of linker residues (I135N, I141T, L156P) also affected the interaction of Salmonella typhimurium DnaB and DnaG [15]. In E. coli, the interaction depends in addition on residues of the C-terminal domain between Tyr210 and Val255 of DnaB [26]. Mutation analysis of linker residues and of residues in the C-terminal domain of B. stearothermo- philus DnaB confirmed the importance of residues in these parts of the protein [3,16]. Unlike in the wild- type protein, the individual N-terminal and C-terminal domains of B. stearothermophilus DnaB do not form a complex with DnaG that is sufficiently stable for isola- tion by gel filtration [25]. The emerging picture is one of an extensive interaction interface between DnaB and DnaG-C involving the N-terminal and C-terminal domains of DnaB as well as the connecting linker [3,16]. Interactions characterized by exceedingly weak bind- ing affinities can be probed sensitively by NMR spectro- scopy. However, attempts to observe an interaction between E. coli DnaG-C and a shorter DnaB-N frag- ment containing the N-terminal 161 residues by NMR spectroscopy were unsuccessful [4]. Our new fragment DnaB(1–171), which includes many of the linker resi- dues, equally showed no binding with DnaG-C or DnaG, illustrating the critical importance of the C-ter- minal domain of DnaB for this interaction. Possibly, the linker between the N-terminal and C-terminal domains of DnaB also assumes a different secondary structure in the full-length protein, considering that we found the C-terminal 35 residues of DnaB(1–171) to be disordered, although secondary structure predictions show high helix propensity for more than half of them. The present structure of monomeric E. coli DnaG-C identifies the earlier crystal structure of the same pro- tein as a domain-swapped dimer, in which helix 5 of one monomer binds to the core of helices formed by helices 1–4 of the other, in a very similar manner as in the monomeric solution structure. The present data suggest that the domain-swapped dimer occurs only at a pH value below the isoelectric point of the protein and plays no role under physiologic conditions. As the present solution structure of DnaG-C accommodates all the NOEs discussed previously [4] in a monomeric structure, there remains no evidence for intermolecular interactions across a dimer interface, and no conform- ational exchange phenomena need to be invoked to explain differences between the NMR data and the crystal structure [4]. The sensitivity of the DnaG-C structure with respect to pH is reflected in much decreased peak intensities for loop residues observed in 15 N-HSQC spectra at pH 4.6 versus those recorded at pH 6.1 or 8.1, and in increased 15 N-relaxation rates for amides in loop regions. These exchange phenomena indicate the pres- ence of alternative conformations, especially at low pH. Considering that carboxylate side chains remain mostly deprotonated at pH 4.6, the low-pH form of the DnaG-C structure may be triggered by protonation of histidine side chains. Of the two histidine residues in DnaG-C, His541 is located in helix 6. In the solu- tion structure, His541 is close to Lys447 and Lys448, which are located near the N-terminus of the domain, whereas these residues are much farther from His541 in the domain-swapped dimer (Fig. 4A,B). Electro- static repulsion could thus drive the separation of helix 6 from the core of the structure. Weak and missing 15 N-HSQC cross-peaks observed for His541 and nearby residues, including residues 445–450, suggest that histidine protonation contributes to the exchange phenomena at pH 4.6 (supplementary Fig. S3). Comparison of the solvent-accessible surface of hydrophobic amino acid side chains in the monomer and the dimer shows only few significant differences, with the most notable difference involving the side chain of Ile530, which is highly solvent exposed in the monomer (Fig. 4B) but buried in the dimer interface. Neither His541 nor Ile530 are conserved in the amino acid sequence (Fig. 2), suggesting that the phenomenon of domain-swapping at low pH may be limited to DnaG-C from E. coli. Considering, in addition, the apparent absence of any interaction between DnaG-C and DnaB(1–171), the domain-swapped dimer of DnaG-C is unlikely to be a model of the DnaG–DnaB interaction. The equivalent DnaG-C domain from B. stearother- mophilus (P16) [10] is a monomer in solution, but helix 6 in this structure is broken into two (Fig. 3C). A flex- ible helix linkage is supported by the presence of Pro556 in P16, which may act as a helix breaker. The corresponding residue in E. coli DnaG-C is Met542, i.e. a residue with high helix propensity. None of the other DnaG-C domains shown in the sequence align- ment of Fig. 2 features a proline residue at this posi- tion, suggesting that a break in helix 6 is not a general feature of DnaGs from different organisms. Therefore, although the present solution structure of E. coli DnaG-C is representative of DnaG-C domains from a large number of bacteria, significant structural variabil- ity seems to have evolved in less closely related species, X C. Su et al. Solution structure of E. coli DnaG-C FEBS Journal 273 (2006) 4997–5009 ª 2006 The Authors Journal compilation ª 2006 FEBS 5005 where the sequence divergence is sufficiently large to render amino acid sequence alignments unreliable [10]. This observation highlights the fact that structures determined for thermophilic or Gram-positive bacteria are not necessarily faithful representations of their homologs in E. coli, the bacterium for which most bio- chemical knowledge has been accumulated. Experimental procedures Sample preparation Unlabeled and uniformly 13 C ⁄ 15 N-labeled DnaG-C contain- ing residues 434–581 was overproduced and purified as pre- viously described [17]. All samples for NMR measurements were prepared in a buffer containing 90% H 2 O ⁄ 10% D 2 O, 10 mm phosphate (pH 6.1), 100 mm NaCl and 1.0 mm dithiothreitol. The protein concentration was 0.4 mm except where indicated otherwise. The DnaB(1–171) deletion mutant was amplified by PCR from plasmid pPS562 containing the dnaB gene [27]. An NdeI site was present at the ATG start codon, and a TAA stop codon followed by an EcoRI site was inserted immedi- ately after codon 171. The amplified fragment was digested and inserted between corresponding restriction sites in the phage T7 promoter-based vector pETMCSI [28] and trans- formed into E. coli strain BL21(DE3)recA [23] for protein expression. Nucleotide sequences were confirmed using an ABI 3730 sequencer (Biomolecular Resource Facility, Aus- tralian National University, Canberra, Australia), following the recommendations of the manufacturer (Applied Biosys- tems, Foster City, CA, USA). DnaB(1–171) was produced and the cells were lysed using a procedure established for other DnaB-N domains [21]. After cell lysis, the protein was purified as described [12], except that the Sephadex G50 column (Amersham Biosciences, Uppsala, Sweden) was equilibrated with 50 mm Tris ⁄ HCl (pH 7.6), 5 mm MgCl 2 and 100 mm NaCl. Peak fractions containing DnaB(1–171) were pooled (20 mL), diluted with an equal volume of MonoQ buffer (50 mm Tris ⁄ HCl at pH 7.6 and 5mm MgCl 2 ), and loaded directly onto a MonoQ (HR 5 ⁄ 5) column (Amersham Biosciences) equilibrated in MonoQ buffer. A linear gradient of NaCl in MonoQ buffer was applied (3.75 mmÆmin )1 , at a flow rate of 0.5 mLÆmin )1 ). DnaB(1–171) eluted as a sharp peak between 52 and 58 min. The protein fractions were pooled and dialyzed in NMR buffer. ESI MS confirmed the identity of the protein and the absence of an N-terminal methionine (observed molecular mass, 18 919; calculated molecular mass 18 920). Analytic ultracentrifugation The molecular weights of DnaG-C samples were deter- mined by equilibrium sedimentation using a Beckman analytical ultracentrifuge XLI with An-60 rotor (Beckman Coulter, Fullerton, CA, USA). The samples were prepared by dialysis against a buffer similar to that used for NMR studies, containing 10 mm sodium phosphate (pH 6.1), 100 mm NaCl, 1 mm dithiothreitol, and 0.1% (w ⁄ v) sodium azide at two different concentrations (1.02 and 4.86 mgÆmL )1 ). The sedimentation equilibrium profile was recorded in triplicate at two different wavelengths (280 and 300 nm) after 18 h at 20 000 r.p.m. and 25 °C. Plots of ln A versus r 2 were linear (supplementary Fig. S1), indica- ting the absence of an equilibrium mixture of species at both concentrations. The average M r was calculated by linear regression using ultrascan data analysis software Version 5 (Beckman Coulter), and an (assumed) partial specific volume of 0.72 mLÆg )1 . NMR measurements NMR measurements of unlabeled DnaB(1–171) were car- ried out in a buffer containing 10 mm Tris ⁄ HCl (pH 6.5), 50 mm NaCl, 5 mm MgCl 2 and 1 mm dithiothreitol. Free DnaB(1–171) was measured at a concentration of 0.22 mm. The interaction with DnaG-C was probed using the same buffer with each protein at 0.13 mm. All NMR spectra were recorded at 25 °C using a Bruker (Karlsruhe, Germany) AV 800 NMR spectrometer equipped with a TCI cryoprobe. The previously reported backbone resonance assignments of DnaG-C [4] were veri- fied and supplemented with side chain resonance assign- ments using 3D CC(CO)NH, HNHA (H)CCH-TOCSY, NOESY- 15 N-HSQC (60 ms mixing time), 13 C-HSQC- NOESY (40 ms mixing time), and 2D NOESY (40 ms mixing time), DQF-COSY, and TOCSY spectra. 3 J HNHa coupling constants were measured at protein con- centrations of 20 and 400 lm, in a CT-HMQC-HN experi- ment [29]. The solvent exposure of protein backbone amides was probed by the decrease in peak intensities observed in 15 N-HSQC spectra caused by 6 mm Gd(DTPA- BMA) [20]. The experiment was carried out at protein con- centrations of 20 and 40 lm. 15 N-relaxation parameters (R 2 , R 1 , and [ 1 H] 15 N-NOE) were measured [30], using relaxation delays of 3, 30, 80, 150, 250, 400, 600, 850 and 1200 ms in the R 1 experiment, and relaxation delays of 8.8, 17.6, 26.4, 35.2, 44.0, 52.8, 61.6, 70.4, 79.2 and 88.0 ms in the R 2 experiment. The rota- tional correlation time s m was estimated from the R 2 ⁄ R 1 ratio [31]. A TOCSY spectrum of DnaB(1–171) was recorded under the same conditions, using a mixing time of 80 ms. Restraints used for the structure calculation In total, 2400 NOE cross-peaks were assigned and integra- ted, resulting in 2151 meaningful distance restraints. Most Solution structure of E. coli DnaG-C X C. Su et al. 5006 FEBS Journal 273 (2006) 4997–5009 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... Soultanas P (2004) DnaG interacts with a linker region that joins the N- and Cdomains of DnaB and induces the formation of 3-fold symmetric rings Nucleic Acids Res 32, 2977–2986 Solution structure of E coli DnaG- C 4 Oakley AJ, Loscha KV, Schaeffer PM, Liepinsh E, Pintacuda G, Wilce MCJ, Otting G & Dixon NE (2005) Crystal and solution structures of the helicase-binding domain of Escherichia coli primase J Biol... DNA primases Annu Rev Biochem 70, 39–80 6 Pan H & Wigley DB (2000) Structure of the zinc-binding domain of Bacillus stearothermophilus DNA primase Structure 8, 231–239 7 Keck JL, Roche DD, Lynch AS & Berger JM (2000) Structure of the RNA polymerase domain of E coli primase Science 287, 2482–2486 8 Podobnik M, McInerney P, O’Donnell M & Kuriyan J (2000) A TOPRIM domain in the crystal structure of the. .. and NMR studies of the helicase interaction domain of Escherichia coli DnaG primase Protein Expr Purif 33, 304–310 FEBS Journal 273 (2006) 4997–5009 ª 2006 The Authors Journal compilation ª 2006 FEBS 5007 Solution structure of E coli DnaG- C X.-C Su et al 18 Garcia de la Torre J, Huertas ML & Carrasco B (2000) HYDRONMR: prediction of NMR relaxation of globular proteins from atomic-level structures and... display and analysis of macromolecular structures J Mol Graph 14, 51–55 Sevilla-Sierra P, Otting G & Wuthrich K (1994) Deter¨ mination of the nuclear magnetic resonance structure of the DNA-binding domain of the P22 c2 repressor (1–76) in solution and comparison with the DNA-binding domain of the 434 repressor J Mol Biol 235, 1003–1020 Goddard TD & Kneller DG (2004) SPARKY 3 University of California, San... catalytic core of Escherichia coli primase confirms a structural link to DNA topoisomerases J Mol Biol 300, 353–362 9 Corn JE, Pease PJ, Hura GL & Berger JM (2005) Crosstalk between primase subunits can act to regulate primer synthesis in trans Mol Cell 20, 391–401 10 Syson K, Thirlway J, Hounslow A, Soultanas P & Waltho JP (2005) Solution structure of the helicase interaction domain of the primase DnaG: a... 4.6 and 6.1 Fig S4 Comparison of 15N-relaxation data for DnaG- C at pH 8.1, 6.1 and 4.6 Solution structure of E coli DnaG- C Fig S5 15N-HSQC spectra of DnaG- C recorded with and without residues 1–171 of E coli DnaB helicase [DnaB(1–171)] This material is available as part of the online article from http://www.blackwell-synergy.com FEBS Journal 273 (2006) 4997–5009 ª 2006 The Authors Journal compilation... activation Structure 13, 609–616 11 Tougu K & Marians KJ (1996) The interaction between helicase and primase sets the replication fork clock J Biol Chem 271, 21398–21405 12 Weigelt J, Brown SE, Miles CS, Dixon NE & Otting G (1999) NMR structure of the N-terminal domain of E coli DnaB helicase: implications for structure rearrangements in the helicase hexamer and its biological function Structure 7,... 13 Fass D, Bogden CE & Berger JM (1999) Crystal structure of the N-terminal domain of the DnaB hexameric helicase Structure 7, 691–698 14 Nakayama N, Arai N, Kaziro Y & Arai K (1984) Structural and functional studies of the dnaB protein using limited proteolysis Characterization of domains for DNA-dependent ATP hydrolysis and for protein association in the primosome J Biol Chem 259, 88–96 15 Stordal... NED, a Federation Fellowship to GO, and a grant for purchase of the 800 MHz NMR spectrometer References 1 Kornberg A & Baker TA (1991) DNA Replication, 2nd edn Freeman, New York, NY 2 Mitkova AV, Khopde SM & Biswas SB (2003) Mechanism and stoichiometry of interaction of DnaG primase with DnaB helicase of Escherichia coli in RNA primer synthesis J Biol Chem 278, 52253–52261 3 Thirlway J, Turner IJ,... Ratnakar PVAL, Mohanty BK & Bastia D (1996) Direct physical interaction between DnaG primase and DnaB helicase of Escherichia coli is necessary for optimal synthesis of primer RNA Proc Natl Acad Sci USA 93, 12902–12907 27 San Martin MC, Stamford NPJ, Dammerova N, Dixon NE & Carazo JM (1995) A structural model for the Escherichia coli DnaB helicase based on electron microscopy data J Struct Biol 114, 167–176 . views of the solution and crystal structures of DnaG- C. (A) Superposi- tion of the backbone atoms of residues 447–581 of the 20 NMR conformers of DnaG- C. studies of the helicase interaction domain of Escherichia coli DnaG primase. Protein Expr Purif 33, 304–310. X C. Su et al. Solution structure of E. coli DnaG- C FEBS