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Research Article 691
Crystal structure of the N-terminal domain of the DnaB hexameric helicase Deborah Fass1†, Cynthia E Bogden1 and James M Berger2*
Background: The hexameric helicase DnaB unwinds the DNA duplex at the Addresses: 1Whitehead Institute, Cambridge, Escherichia coli chromosome replication fork. Although the mechanism by Massachusetts 02142, USA and 2Department of which DnaB both couples ATP hydrolysis to translocation along DNA and Molecular and Cell Biology, Stanley Hall, University of California, Berkeley, California 94720, USA. denatures the duplex is unknown, a change in the quaternary structure of the protein involving dimerization of the N-terminal domain has been observed and †Present Address: Department of Structural may occur during the enzymatic cycle. This N-terminal domain is required both Biology, Weizmann Institute of Science, Rehovot for interaction with other proteins in the primosome and for DnaB helicase 76100 Israel. activity. Knowledge of the structure of this domain may contribute to an *Corresponding author. understanding of its role in DnaB function. E-mail: [email protected]
Results: We have determined the structure of the N-terminal domain of DnaB Key words: DnaB, DNA helicase, DNA replication, domain crystallographically. The structure is globular, highly helical and lacks a close structural relative in the database of known protein folds. Conserved residues Received: 1 February 1999 and sites of dominant-negative mutations have structurally significant roles. Revisions requested: 17 February 1999 Each asymmetric unit in the crystal contains two independent and identical Revisions received: 4 March 1999 Accepted: 11 March 1999 copies of a dimer of the DnaB N-terminal domain. Published: 1 June 1999 Conclusions: The large-scale domain or subunit reorientation that is seen in DnaB by electron microscopy might result from the formation of a true twofold Structure June 1999, 7:691–698 http://biomednet.com/elecref/0969212600700691 symmetric dimer of N-terminal domains, while maintaining a head-to-tail arrangement of C-terminal domains. The N-terminal domain of DnaB is the first © Elsevier Science Ltd ISSN 0969-2126 region of a hexameric DNA replicative helicase to be visualized at high resolution. Comparison of this structure to the analogous region of the Rho RNA/DNA helicase indicates that the N-terminal domains of these hexameric helicases are structurally dissimilar.
Introduction 12 kDa called Fragment III [8]. Fragment II contains sig- The DnaB helicase is an essential protein that is involved nature sequences of nucleotide-binding proteins [9] and in both the initiation and elongation stages of Escherichia retains hexamerization, single-stranded DNA-binding and coli chromosome replication [1]. It is a hexameric ATPase ATPase activities [8,10]. The predicted secondary struc- that uses the hydrolysis of ATP to unwind double-stranded ture around the nucleotide-binding signature sequences DNA [2]. DnaB also contains binding sites for other pro- matches the consensus topography predicted for other teins making it a central component in assembly of the pri- hexameric helicase ATP-binding domains [11], and is mosome, movement of the replication apparatus and likely to comprise a Walker-type ATP-binding fold. Frag- termination of replication [1]. For example, DnaB interacts ment III has no known activity alone but this domain is with its DNA-loading factor DnaC [3,4], with the primase essential for DnaB helicase activity, general priming and DnaG [5] and with the replication termination protein, interaction with DnaC [8,10]. RTP [6]. Furthermore, translocation of DnaB is coordi- nated with the movement of the replication fork through On the basis of negative-stain electron microscopy direct contact to the τ-subunit of the pol III holoenzyme; studies, low-resolution structural models for the DnaB this complex couples the helicase and polymerase activities hexamer reveal six bi-lobed protomers arranged in a ring and appears to stimulate DnaB action as well [7]. around a channel [12,13]. Fragment II has been proposed to correspond to the larger, central lobe, because it is the The domain structure of DnaB has been examined to larger of the two protease-resistant regions and contains locate various functional regions (Figure 1a). Proteolysis of the primary multimerization activity [10,12,13]. The intact DnaB removes the N-terminal 14 residues to yield smaller domain farther from the sixfold axis was assumed the 50 kDa Fragment I [8]. Fragment I can be further to be Fragment III [12,13]. DnaB can display either cleaved into two species: a 33 kDa C-terminal region threefold or sixfold symmetry [13] and dimerization of termed Fragment II and a smaller, N-terminal domain of the Fragment III domain is associated with the switch 692 Structure 1999, Vol 7 No 6
Figure 1 the Fragment III domains in DnaB alone do not appear to protrude radially but instead may lie closer to the oligomer (a) Fragment III Fragment II axis stacked above the Fragment II domains [16]. This Regulatory ssDNA binding, ATPase arrangement is consistent with fluorescence energy transfer T 12 kDa T T 33 kDa experiments that suggest that the Fragment III domains N C 2º oligomerization 'Hinge' 1º oligomerization face the 5′ end of the replication fork arm [17]. Positioning of the N-terminal domains atop the ATPase regions is also (b) α1 α2 PPHSIEAEQSVLGGLMLDNERWDDVA consistent with the known low-resolution structures of ¥¥¥ 30 40 50 phage T7 hexameric helicase/primase protein [18] and the 3 1 10 α3 RuvB branch migration protein [19]; these enzymes also ERVVADDFYTRPHRHIFTEMARLQES ¥¥ 60 70 have a pronounced polarity, with both domains of the bi- α4 α5 lobed protomers circling the hexamer axis. GSPIDLITLAESLERQGQLDSVGGFA ¥¥¥ 80 90 100 α6 α7 The similarities between DnaB and other hexameric heli- YLAELSKNTPSAANISAYADIVRER case assemblies suggest that these enzymes may share ¥¥ 110 120 Structure certain mechanistic features [20]. Hexameric ATPases with Walker-type nucleotide-binding regions, however, Organization of DnaB. (a) The primary domain structure of DnaB. The have been found to play diverse roles in numerous cellular polypeptide chain has been divided into distinct regions by proteolytic events, such as ATP synthesis via a proton gradient [21], mapping [8]. Nomenclature, sizes and positions of these domain boundaries are indicated. (b) Secondary structure of DnaB-III. The termination of transcription [22] and facilitation of vesicle sequence of the DnaB N-terminal domain is shown with helices boxed fusion [23,24]. The molecular motors responsible for each and secondary structure labels assigned. These secondary structural of these functions must be adapted to their particular tasks assignments agree well with previous assignments from NMR [41]. through structural differences between regions outside the nucleotide-binding motifs. A high-resolution view of DnaB, in particular of regions that distinguish it from [12,13]. A similar distribution of symmetry states has other hexameric ATPases, is ultimately necessary to recently been observed for the papillomavirus E1 hexam- understand the details of its reaction mechanism. The eric helicase [14]. N-terminal domain (Fragment III) is critical to the func- tion of DnaB, regulating the conformational consequences Fragments II and III of DnaB are connected by a of ATP binding and hydrolysis for both priming of replica- 45 amino acid linker, which may facilitate the quaternary tion and unwinding of DNA. To begin to address how the structural changes of the enzyme. The rate of protease hexameric ATPase DnaB is customized for its cellular role cleavage at this linker decreases dramatically in the pres- in DNA replication, we have determined the structure of ence of a non-hydrolyzable ATP analog, as compared with Fragment III, which we will refer to as DnaB-III, using ATP or ADP [8], indicating that ATP hydrolysis results in X-ray crystallography. conformational changes affecting accessibility of the hinge. Interestingly, protease treatment to uncouple Frag- Results ments II and III leads to a fivefold increase in the ATPase Structure of the DnaB N-terminal domain activity of Fragment II without affecting ATP binding The DnaB N-terminal domain structure was solved by ([8], but also see [10]), suggesting that the presence of the multiple isomorphous replacement and has been refined to N-terminal domain of DnaB can regulate ATP hydrolysis. 2.3 Å resolution (Figure 2; Table 1). The working R factor Despite having enhanced ATPase activity, the hexameric is 26.3% and the free R factor is 29.3% (Table 2). The Fragment II shows no helicase activity [10], which further overall dimensions of the DnaB-III domain are 25 Å × 25 Å indicates that the Fragment III domain is critical for cou- × 35 Å, which is roughly consistent with the size of the pling ATPase activity to enzyme function. Other globules at the vertices in the electron microscope (EM) members of the replication machinery that require the reconstruction of intact DnaB after correction for typical DnaB N-terminal domain for binding may either bind this losses in measured volume because of artifacts of negative domain directly or may recognize a quaternary structure of staining [12]. The DnaB-III structure is largely helical the enzyme that is assumed only when the Fragment III (Figures 2b,c) as previously reported from circular dichro- domains are present. Temperature-sensitive mutations in ism studies [25]. The domain consists of six α helices, five the hinge region of DnaB have been proposed to alter the of which (α2–α6) are wrapped around a central helix (α1). position of the Fragment III domains or the ability of Although the crystallized fragment contains amino acids these domains to change orientation [15]. 15–128 of the DnaB sequence, the first residue for which electron density could be seen was Pro26; continuous In a recent investigation of the interaction between DnaB density was evident, however, from this residue to the end and DnaC by three-dimensional cryoelectron microscopy, of the expressed fragment. Similar domain limits have Research Article The N-terminal domain of the DnaB hexameric helicase Fass, Bogden and Berger 693
Figure 2
DnaB-III monomer structure. (a) Stereogram showing experimental electron density (green) for DnaB-III (Fobs, experimental phases). Contouring is at 1σ. The refined model is shown in ball-and-stick representation (yellow). (b) The structure of the DnaB-III protomer is shown as a stereogram stick representation [42]. Every fifth Cα position is marked by a sphere and every tenth position is labeled. (c) Same view as in (b), but the molecule is shown as a ribbon representation [43] with the secondary structure elements labeled.
been identified previously by proteolysis (15–128) [10] and and Thr85. Glu33 is both buried and invariant among all by NMR (24–136) [25]. A search through the protein fold Fragment III homologs; this residue forms hydrogen database using the Dali server [26] revealed no appreciable bonds to the backbone amide of Tyr60, to the Nε of His28 similarity to any other proteins. and to the Nδ of the conserved His64. The aromatic ring of Tyr60 stacks against His28 but the opposite face of the The amino acid sequence conservation in the DnaB Frag- Tyr60 ring is exposed to solvent. The invariant Asp82 is ment III domain is less extensive than in the Fragment II partially buried and uses its sidechain carboxylate to initi- domain. The residues in DnaB-III that are conserved ate helix α4 by hydrogen bonding to Oγ of Thr85, to its among bacterial and bacteriophage helicases are generally own backbone amide and to the backbone amides of Ile84 hydrophobic core residues in the structure. Interesting and Thr85. Two additional residues, Pro27 and Glu128, exceptions, however, include Glu33, Tyr60, His64, Asp82, are also conserved but lie at the extreme N terminus and 694 Structure 1999, Vol 7 No 6
Table 1 Table 2
Data collection. Refinement.
Native MeHgCl TmAc –SeMet Native 2 Resolution (Å) 20.0–2.3 Number of reflections Resolution (Å) 3.0 3.5 3.5 3.5 2.3 working 17,850
Rsym* (%) 6.1 12.1 11.9 7.9 6.9 free 1277 Completeness (%) 96.0 92.7 94.6 95.2 98.7 Rwork/Rfree* (%) 0.263/0.293 † Riso (%) – 19.1 21.4 15.5 – Number of atoms Number of sites – 6 4 8 – protein 3198 Phasing power‡ – 2.08 0.95 1.67 – water 43 Cullis R§ – 0.67 0.90 0.73 – rmsd bond lengths (Å) 0.007 Figure of merit (to 3.1 Å) 0.442 rmsd bond angles (°) 1.433
Σ Σ Σ Σ *Rsym = j|Ij–|/ Ij, where Ij is the intensity measurement for reflection *Rwork, free = ||Fobs|–|Fcalc||/ |Fobs|, where the working and free R j and is the mean intensity for multiply recorded reflections. factors are calculated using the working and free reflection sets, † Σ Σ Riso = ||Fph|–|Fp||/ |Fp|, where Fph and Fp are the derivative and native respectively. The free reflections were held aside throughout refinement. ‡ structure factors, respectively. Phasing power =
Crystals of DnaB-III grew in the space group P21 with specific role in chromosomal replication; this region is four molecules in the asymmetric unit arranged as a pair of structurally unrelated to the analogous portion of the Rho dimers (Figure 3). The two dimer interfaces are identical, RNA/DNA hexameric helicase and transcription termina- with each composed of two antiparallel α helices (α4 and tion factor, which contains a five-stranded β barrel housing α6) abutting their symmetry-mates such that the twofold an RNA-binding cleft [28–30]. rotation axis relating the protomers is parallel to the helical axes. This arrangement forms a four-helix bundle at the The requirement for the integrity of the DnaB-III domain dimer interface comprising the following hydrophobic in vivo derives from studies on the highly homologous DnaB residues and their symmetry-mates: Leu83, Ile84, Ala87, enzyme of Salmonella typhimurium. This protein was Phe102 and Ala106. A small α helix, α5, bridges α4 and α6 screened for dominant-negative mutations [31], which are and places Leu96 into the hydrophobic region of this changes that are likely to generate proteins sufficiently interface. In addition, an ion-bridge is observed across the structurally sound to form mixed oligomers with wild-type dimer between the sidechains of Glu88 and Lys′110 protein, yet lack key functions and thereby poison the hexa- (where the prime denotes the dimer-related residue). The meric assembly. Lethal mutations that fall in the DnaB-III sidechain of Glu92 is also hydrogen bonded to the back- region correspond in the E. coli sequence to Asp82→Asn, bone amides of Phe′102 and Ala′103. Ala106→Val and Asn117→Ser. Although these mutations are fairly conservative, they eliminate DnaB function in the The surface area buried per dimer is 465 Å2, or 10.5% of the cell. Asp82 provides a cap for helix α4 and Asn117 helps ini- protomer surface area. Though somewhat small, the size of tiate helix α6; changes in these two residues appear to affect this interface is significantly larger than other packing inter- formation of stable domain secondary structure. Ala106, on faces in the crystal, with the next largest being only 241 Å2, the other hand, is partially surface-exposed and does not or 5.5% of the available surface area. Other dimeric proteins appear to have a key structural role in the DnaB-III are also known to contain small interfaces, such as the 434 monomer, yet this residue is part of the observed dimer cro protein [27], which buries 260 Å2 or 7.4% of its protomer interface, packing against both Phe102 of its own protomer surface area. It can be argued that proteins with such small and Ile84 of the dimer-related molecule. Rather than inhibit interfaces dimerize only in response to an inducer such as the folding of the protomer structure, mutations at this posi- DNA binding. It is possible, however, that the DnaB-III tion may affect the interaction between DnaB-III pro- domain does likewise, dimerizing only at high effective tomers. Modeling the Ala106→Val mutation into the concentrations such as on the surface of the DnaB hexamer. DnaB-III dimer structure does not result in gross steric clashes but in fact provides additional hydrophobic interac- Discussion tions across the interface. Such a mutation may exert its Although the ATPase, oligomerization, and single- effects by altering the equilibrium of DnaB-III domain stranded DNA-binding regions of DnaB are located in dimerization, but not necessarily by inhibiting dimerization. Research Article The N-terminal domain of the DnaB hexameric helicase Fass, Bogden and Berger 695
Figure 3
DnaB-III dimer structure. (a) Structure of the dimer viewed perpendicular to the twofold axis. One protomer is colored blue and the other gold. (b) Structure of the dimer viewed along the twofold axis. Coloring is as in (a). (c) Stereo representation of the dimer interface from the view shown in (b). Segments from one protomer are colored gold and the other blue. Individual residues are labeled. This figure was generated by Ribbons [43].
Analytical ultracentrifugation has shown the isolated [12,13]. Biochemical studies have further suggested that DnaB N-terminal domain to be monomeric at a concentra- the presence of the DnaB-III domain affects the overall tion of 0.2 mM [25]. In the intact DnaB hexamer, stability or conformation of the hexamer [10]. Thus, in the however, these domains have been observed to dimerize context of the hexamer, the effective concentration of 696 Structure 1999, Vol 7 No 6
Figure 4
Illustration of one possible rotation to transform the DnaB-III domain from a
C6-symmetrical to a C3-symmetrical state containing the observed DnaB-III dimer. (a) Approximate positions for a
C6-symmetrical configuration of DnaB-III domains. Each domain is rotated 60° with respect to its neighbor. (b) A combined rotation of 120° per N-terminal domain pair
would generate a C3-symmetrical state in which the N-terminal domains oligomerize about a local twofold axis while the C-terminal domains retain their head-to-tail packing arrangement. The rotation of the DnaB-III domains need not occur around an axis parallel to the hexamer axis, and other rotations leading to alternate orientations for the local twofold axis are possible. Purple and orange ovals represent relative locations of the ATP-binding sites, based on other hexameric ATPases [21,23,24]. Neighboring sites in DnaB are non-equivalent for ATP binding and hydrolysis, as indicated by the two colors. Biochemical studies on DnaB [44] and other hexameric helicases [45,46] have demonstrated that only three high-affinity ATP-binding sites exist per hexamer.
DnaB-III domains may be sufficiently high to promote C-terminal domains (Figure 4). It is important to note that dimerization. Indeed, it is possible that the affinity of this no evidence for D3 symmetry can be seen in electron interface is in the range where its formation both affects micrographs of DnaB [12,13]. The resolution of the elec- and is affected by the quaternary state of the enzyme. tron microscopy is insufficient to resolve the precise orien-
Interestingly, the homologous DnaB-III domain from a tation of individual N-terminal domains. Thus, the C3 thermophilic bacterium forms a dimer at room tempera- symmetry evident in the projections probably arises from ture in solution (D Wigley, personal communication), sug- the maintenance of head-to-tail packing by the ATPase gesting that the monomer–dimer equilibrium is shifted in regions whereas the N-terminal domains may form a true this organism to compensate for the conditions under dimer about a local twofold axis. The linker between the which replication of its DNA occurs. According to this DnaB-III domain and the ATPase domain of DnaB would model, mutations that alter the affinity of dimerization be expected to accommodate these motions [12,13]. such as perhaps the Ala106→Val alteration described above, would be expected to have significant effects on Large-scale domain rearrangements have a precedent in enzyme activity. helicase structures: for the E. coli Rep helicase, two copies of the protomer in the asymmetric unit of the crystals If the quaternary arrangement of DnaB oscillates between differ by a swiveling of one of the Rep domains by 130°
C6-symmetric and C3-symmetric states via dimerization of about a hinge region [32]. It remains to be seen whether the N-terminal domains according to the true twofold the conformational variability observed in dimeric heli- symmetry that we observe in the crystals, dimerization cases such as Rep and hexameric helicases such as DnaB must occur through large-scale conformational changes. reflect any common features in their mechanism of translo- Such structural variations have been observed by electron cation along the DNA or their duplex-unwinding activity. microscopy [13]. In a C6-symmetrical state each N-termi- nal domain would be rotated 60° from its neighbor in the Biological implications hexamer. Given that DnaB-III dimers are related by a The DnaB protein is the hexameric helicase responsible rotation of 180°, the N-terminal domains must undergo for unwinding the DNA duplex during replication of the rotations that total at least 120° per pair relative to the Escherichia coli chromosome. The enzyme is a molecular Research Article The N-terminal domain of the DnaB hexameric helicase Fass, Bogden and Berger 697
motor that can undergo large-scale quaternary structural cacodylate, pH 6.9; 20 mM MgCl2, 8% ethanol. Thin plates × × µ changes as visualized by electron microscopy. Although (~150 150 20 m) of space group P21 and unit cell a = 35.3 Å, b = 66.4 Å, c = 107.0 Å, α = γ = 90°, β = 93.8° grew in 2–3 weeks. several critical aspects of DnaB function have been addressed biochemically and via electron microscopy, a Data collection and phasing thorough understanding of the mechanism by which Crystals were equilibrated in reservoir solution plus increasing amounts DnaB acts on DNA will also rely on atomic-resolution of glycerol (5%, 10 min; 10%, 10 min; 15%, 5 min) before flash-freez- ing in a gaseous N stream at –155°C. Initial data were collected on structural information. 2 Brookhaven beamline X4A at wavelengths of 0.9793, 0.9789 and 0.9667 Å. An attempt was made during this time to use these data for We report an X-ray crystallographic study of the N-ter- multiwavelength anomalous dispersion (MAD) experiments, but the minal domain of the DnaB helicase. This domain is data showed no strong dispersive signal as evidenced from Patterson essential for helicase activity, priming of replication, and analysis. We have attributed this to severe anisotropy readily apparent in the diffraction patterns and to the fact that the diffraction decayed interaction with other factors in the replisome. The struc- visibly during the extended course of data collection to obtain multiple ture of this domain is different from that of the N-termi- P21 data sets. Derivatives were therefore produced by soaking heavy nal domain of the Rho hexameric DNA/RNA helicase, metals into the crystals (methyl mercuric chloride, 1 mM, 3 h; thulium illustrating that the enzymatic activity of hexameric acetate, 0.1 mM, 48 h). Derivative data, including a data set from a crystal containing selenomethionine protein, were collected at 1.54 Å ATPases is customized to particular cellular tasks on a Rigaku rotating anode RU-300 equipped with an Raxis IV detector through structural differences in these auxiliary domains. and an X-stream cryo-cooler (MSC). Data indexing and reduction were carried out using the program DENZO/SCALEPACK [35]. Data trun- The DnaB N-terminal domain plays a central role in the cation and scaling between data sets were performed with the CCP4 program suite [36]. Electron-density maps were generated by multiple conformational changes of the enzyme: adjacent N-ter- isomorphous replacement (MIR) using the program SHARP [37]. The minal domains appear to coalesce into dimers when the positions of selenium atoms were determined from difference Fourier helicase switches from a sixfold to a threefold symmetri- maps using MIR phases from the other two derivatives as well as from cal form. We observe two copies of a DnaB N-terminal using difference Patterson maps between native and selenomethionine domain dimer in our crystals, providing a model for how data. Anomalous difference Patterson maps from the MAD data did show peaks at a subset of the appropriate positions, as was subse- these domains may pack in the threefold symmetric state quently verified using MIR methods. Selenium and thulium heavy-atom of the intact enzyme. positions were used to determine the noncrystallographic symmetry (NCS) operators relating the four molecules in the asymmetric unit, and NCS operators were refined using IMP [38]. Electron-density maps Materials and methods were improved by solvent flattening and NCS-averaging with the Protein production and crystallization program dm [39]. X-PLOR [40] was used for refinement, and The DnaB-III coding sequence (spanning residues 15–118 of the wild- anisotropic and bulk-solvent corrections were applied. type E. coli DnaB protein) was amplified by PCR from a plasmid con- taining the intact DnaB gene (CEB and JMB, unpublished observa- Accession numbers tions) and cloned into pET28b (Novagen). Five residues, MASHM (in The coordinates have been deposited in the Protein Data Bank with the single-letter amino acid code), were inserted at the N terminus during accession code 1b79. cloning. Protein expression was performed in BL21(DE3) pLysS cells by inducing at an A600 of 0.3 with 1 mM IPTG for 2.5 h at 37°C. Acknowledgements DnaB-III containing selenomethionine was expressed in BL21(DE3) The authors thank S Gamblin for advice on data collection and anisotropic cells as described [33] except that cells were induced at an A600 of 0.3 corrections, MD Nichols, M Botchan, J Keck and S Lynch for thoughtful dis- and harvested 3 h post-induction. In both cases, harvested cells were cussions and critical reading of the manuscript, C Ogata for assistance at resuspended at 1 g/ml in buffer A (50 mM Tris-HCl, pH 7.5; 1 mM Brookhaven beamline X4A, and the Harold G and Leila Y Mathers Charita- EDTA; 1 mM EGTA; 10% glycerol; 1 mM DTT; 1 mM phenyl-methyl- ble Foundation, Whitehead Institute and Berkeley MCB department for sulfonylfluoride (PMSF); 1 µM pepstatin-A; 1 µM leupeptin) and flash- funding assistance. frozen by dropwise pipetting into liquid nitrogen. DnaB-III was purified as follows: cells were thawed in a room temperature water bath, soni- References cated and centrifuged. 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