doi:10.1016/j.jmb.2008.10.059 J. Mol. Biol. (2009) 385, 368–380

Available online at www.sciencedirect.com

A Structural Basis for the Regulatory Inactivation of DnaA

Qingping Xu1†, Daniel McMullan2†, Polat Abdubek2†, Tamara Astakhova3†, Dennis Carlton4†, Connie Chen2†, Hsiu-Ju Chiu1†, Thomas Clayton4†, Debanu Das1†, Marc C. Deller4†, Lian Duan3†, Marc-Andre Elsliger4†, Julie Feuerhelm2†, Joanna Hale2†, Gye Won Han4†, Lukasz Jaroszewski3,5†, Kevin K. Jin1†, Hope A. Johnson4†, Heath E. Klock2†, Mark W. Knuth2†, Piotr Kozbial5†, S. Sri Krishna3,5†, Abhinav Kumar1†, David Marciano4†, Mitchell D. Miller1†, Andrew T. Morse3†, Edward Nigoghossian2†, Amanda Nopakun4†, Linda Okach2†, Silvya Oommachen1†, Jessica Paulsen2†, Christina Puckett2†, Ron Reyes1†, Christopher L. Rife1†, Natasha Sefcovic5†, Christine Trame1†, Henry van den Bedem1†, Dana Weekes5†, Keith O. Hodgson6†, John Wooley3†, Ashley M. Deacon1†, Adam Godzik3,5†, Scott A. Lesley2,4† and Ian A. Wilson4†⁎

1Stanford Synchrotron Regulatory inactivation of DnaA is dependent on Hda (homologous to Radiation Lightsource, Stanford DnaA), a protein homologous to the AAA+ (ATPases associated with University, Menlo Park, CA, diverse cellular activities) ATPase region of the replication initiator DnaA. USA When bound to the sliding clamp loaded onto duplex DNA, Hda can stimulate the transformation of active DnaA–ATP into inactive DnaA–ADP. 2Genomics Institute of the The crystal structure of Hda from Shewanella amazonensis SB2B at 1.75 Å Novartis Research Foundation, resolution reveals that Hda resembles typical AAA+ ATPases. The San Diego, CA, USA arrangement of the two subdomains in Hda (residues 1–174 and 175–241) 3Center for Research in differs dramatically from that of DnaA. A CDP molecule anchors the Hda Biological Systems, University domains in a conformation that promotes dimer formation. The Hda dimer of California, San Diego, adopts a novel oligomeric assembly for AAA+ proteins in which the La Jolla, CA, USA arginine finger, crucial for ATP hydrolysis, is fully exposed and available to – 4 hydrolyze DnaA ATP through a typical AAA+ type of mechanism. The The Scripps Research Institute, sliding clamp binding motifs at the N-terminus of each Hda monomer are La Jolla, CA, USA partially buried and combine to form an antiparallel β-sheet at the dimer 5Burnham Institute for Medical interface. The inaccessibility of the clamp binding motifs in the CDP-bound Research, La Jolla, CA, USA structure of Hda suggests that conformational changes are required for Hda 6 to form a functional complex with the clamp. Thus, the CDP-bound Hda Photon Science, SLAC dimer likely represents an inactive form of Hda. National Accelerator Laboratory, © 2008 Elsevier Ltd. All rights reserved. Menlo Park, CA, USA

*Corresponding author. E-mail address: [email protected]. † Joint Center for Structural Genomics, http://www.jcsg.org. Abbreviations used: Hda, homologous to DnaA; AAA+, ATPases associated with diverse cellular activities; oriC, chromosomal replication origin; DUE, DNA unwinding element; RIDA, regulatory inactivation of DnaA; MAD, multiwavelength anomalous dispersion; CDP, cytidine-5'-diphosphate.

0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved. Crystal Structure of Hda 369

Received 16 August 2008; received in revised form 18 October 2008; accepted 22 October 2008 Available online 30 October 2008 Edited by K. Morikawa Keywords: Hda; DnaA; RIDA; AAA+ ATPase; Structural Genomics

Introduction mote hydrolysis of DnaA–ATP to DnaA–ADP.11–13 As a result of RIDA, the level of DnaA–ATP in the In Escherichia coli, the initiation protein DnaA, cell, which peaks in vivo around the initiation of when complexed with ATP (DnaA–ATP), binds to replication, decreases rapidly after the start of the 9-bp DnaA boxes within the chromosomal replication.8 The Hda-mediated RIDA process is replication origin (oriC) to initiate DNA replica- an important mechanism for preventing overini- tion.1,2 At the beginning of initiation, 20–30 DnaA– tiation,14,15 and hda-deficient cells do in fact over- ATP proteins bind to the origin and oligomerize into initiate.9,16 Hda is a dimer in solution and binds to a large nucleoprotein complex, which facilitates the sliding clamp via its N-terminal sliding clamp melting of the adjacent AT-rich, 13-bp DNA unwind- binding motif.13,17 Besides being involved in RIDA, ing element (DUE).3 The initiation of chromosomal Hda may also have other cellular functions, since it DNA replication is tightly regulated to ensure only also interacts with the plasmid RK2 replication one replication per cell cycle.4,5 Three regulation initiation protein TrfA.10,18 Details of how and mechanisms have been identified. First, an oriC where RIDA occurs in the cell currently are not sequestration mechanism inactivates the newly clear. In vitro, the RIDA interaction of DnaA–Hda is replicated oriC through binding of the SeqA protein. dependent on the sliding clamp loaded onto double- SeqA has a higher affinity for hemimethylated GATC stranded DNA.12 In vivo, RIDA may occur on the sites within the oriC. These hemimethylated GATC postreplication sliding clamps that remain on the sites exist in newly synthesized oriC for about one- lagging strands after synthesis of Okazaki frag- third of a cell cycle after initiation until the oriC is ments.13 It has also been suggested that Hda binds fully methylated by the DNA adenine methyltrans- to the sliding clamp at the replication fork and that ferase. Second, a DnaA titration mechanism limits RIDA occurs at the fork.5 Another possibility is that the availability of the DnaA protein at the origin by Hda could interact with the DnaA–ATP origin diverting DnaA to other binding sites outside the complex after opening of the DUE.4,6,19 oriC region. For example, a single copy of the datA A typical Hda is about 250 residues in length. It is locus, located 470 kb downstream from oriC, could classified as an AAA+ (ATPases associated with di- bind an unusually large number of DnaA molecules. verse cellular activities) ATPase due to its sequence DnaA boxes are also present in the promoter regions homology to the AAA+ ATPase region of DnaA of many genes. Third, a mechanism known as RIDA (domains III and IIIb). A recent in vitro reconstitu- (regulatory inactivation of DnaA) negatively regu- tion of E. coli RIDA activity revealed that a con- lates DnaA by converting active DnaA–ATP into served arginine in the box VII motif of Hda is – inactive DnaA–ADP following the initiation.6 8 required to stimulate ATP hydrolysis in DnaA and DnaA–ADP cannot induce melting of the origin suggests that a conserved AAA+ type of interaction and thus is ineffective for replication initiation.1 takes place between Hda and DnaA during RIDA.13 The two essential components of the RIDA system Members of the AAA+ superfamily of ATPases are are the sliding clamp of DNA polymerase III (DnaN found in all kingdoms of living organisms. These or β-subunit) and Hda (homologous to DnaA, also ATPases act as motors or switches and control a wide – referred to as IdaB).9,10 When the sliding clamp is range of biological processes.20 24 Structural charac- loaded onto double-stranded DNA, Hda can pro- terization of AAA+ modules has revealed that they

Fig. 1. Structure of Hda from S. amazonensis. (a) Sequence alignment between Hda and DnaA (ATPase region) of E. coli and S. amazonensis. The Hda of S. amazonensis (Hda_sheam) is highly homologous to the Hda of E. coli (Hda_ecoli) with 48% sequence identity and to the DnaA–ATPase regions of E. coli (residues 107–365) and S. amazonensis (residues 95–355) with sequence identities of 24% and 21%, respectively. The secondary structure elements and sequence numbering of the S. amazonensis Hda structure are shown in the top row. The conserved Walker A, Walker B, sensor I, and sensor II motifs are marked in black with up, down, right, and left triangles in the bottom row, respectively. The arginine finger of box VII is indicated with a blue star. The sliding clamp binding motif is denoted with a green box. (b) Structure of an Hda monomer with bound CDP and magnesium. The Walker A (W-A, or P-loop), Walker B (W-B), sensor I (S-I), sensor II (S-II), sliding clamp binding, and box VII (arginine finger; Arg161) motifs are labeled corresponding to (a). (c) Comparison of Hda monomers (orange and green) with the DnaA III and IIIb domains (PDB ID 2hcb, gray). The Hda and DnaA are superimposed based on their respective NTPase domains (i.e., I and III). The axes of the sensor II helices of Hda (210–225) and DnaA (276–289) are shown as rods to highlight the significant difference in the domain Ib orientation. 370 Crystal Structure of Hda consist of two domains: an N-terminal P-loop shaped oligomeric assemblies, where one AAA+ NTPase homologous domain (the base domain) subunit inserts residues from a conserved motif (box and a smaller C-terminal helical bundle domain VII) into the ATP interaction site of its adjacent (the lid domain). The nucleotide binding site is subunit. ATP hydrolysis is enabled once the bipartite located at the domain interface (see Nucleotide nucleotide binding pocket is formed, and contact is binding site). AAA+ proteins generally form ring- made between a conserved arginine (“arginine

Fig. 1.eourutcrfHStdafomra)Sgqn.(eumeaseiciSlm.anwstbeoztaenHe daandDnaA(AToPner)oasgieanfd.TlEos.cheHiSm.anseozadaosHf(iSm.andshameo_szayhshg)ihliomoguolstotheHdao)wh4Hit(fdElo8%sica_e.cyatqeuneieiddtcnetotheDnaAdA–us1eTiosdPerne07365)a–souasrgs9esonids(ef53)w–shsrif2Elo4%a.ct(Siqm.aneuseiieodcznaetn.Tyd2hvmesleeueorncuieystd1%salcranptecnsetds,rqeunenecumngoibrehftesHSim.anseaueozrdesuatascrFig.hotwninthetoprow.ThecvdWeorns 1krAeal,W(krBelegendalonerI,a,ssdsonsaerIImsemfriotkdiearnbkwaclhutintsiepan,dhglebomriw,a.Tnytrgttrohdleefto onvniealefifb,rgiorwdwexVptcseat,rcnsihabdIiitIngcid.Tamuiesharil previousltpblngmdisdfidweniotthagtinbeeourutbcrfa)Sox.(nHt dam pageonomrwehbtioundC)DPandmumaginse.TheWkrA(elangcdamiWilpblA,sn-)go,Wd,a,onpiIrPdb)lkrB(Ie-onS;AerIalI(,ssgrdceefopxVsani)16)mWsoeiel)CrgtabcdgflnrIrI(ei,sso.(aro(BI(S-i)oam-InotsiparfHdamonomans(ogreaendgne)whtrtheDi naAIIabdnIdIIIomns(PDaiBID2.Thbay,gc)eHr daandDnaAaesmruipredbosdoveeNasiptntrrcsehTi.Te,IaPedhas)nea.dIeIosoxmeI.soenhcs(ftesifHaionerIIhsleda(2105)a–ndDnaA(27689)a–esrhoneiwentcrehedfnaanctdfonsrim.oidnIgfhstohttatesnboietgihlir Crystal Structure of Hda 371 finger”) from the box VII motif in one subunit and Table 1. Data collection and refinement statistics γ the -phosphate of the bound nucleotide in the λ λ adjacent monomer. ATP hydrolysis often results in 1 MADSe 2 MADSe intrasubunit and intersubunit conformational Data collection changes, which can be employed in many cellular Wavelength (Å) 0.9793 0.9184 24 Resolution range (Å) 29.6–1.75 29.5–1.75 events. The AAA+ ATPases are key components of No. of observations 204,590 204,972 DNA replication and repair complexes, such as the No. of unique 55,436 55,361 replication initiator DnaA.25 The opening of DUE by reflections DnaA does not involve ATP hydrolysis, since a Completeness (%) 99.7 (99.7)a 99.7 (100)a Mean I/σ (I) 9.1 (1.8)a 8.3 (1.6)a nonhydrolyzable ATP analog can functionally a a R on I (%) 0.10 (0.64) 0.11 (0.81) replace DnaA–ATP. 1 Thus, it appears that ATP sym hydrolysis in DnaA is employed in its inactivation. Model and refinement statistics – λ The recently reported structures of inactive DnaA Resolution range (Å) 29.6 1.75 Data set used 1 26,27 19 in refinement MADSe with ADP, active DnaA with an ATP analog, b N the DNA binding domain of DnaA complexed with No. of reflections (total) 55,402 Cutoff |F| 0 28 – 29 criteria DNA, the archaeal Orc1 DNA complex, and the No. of reflections (test) 2813 Rcryst 0.166 – 30 b heterodimeric Orc1 DNA complex have greatly Completeness (% total) 99.5 Rfree 0.201 enhanced our understanding of the role of AAA+ proteins in the initiation of replication.4 Here, we Stereochemical parameters Restraints (rms observed) present the crystal structure of Hda from Shewanella Bond angle (°) 1.47 amazonensis, the essential component of RIDA. S. Bond length (Å) 0.014 amazonensis is likely to share a similar RIDA system Average isotropic 24.1 2 with E. coli based on the similarity in the replication B-value (Å ) 31 ESU based on Rfree (Å) 0.10 origins and the presence of highly homologous Protein residues/atoms 461/3682 DnaA and Hda proteins (sequence identities of 81% Ligand and water 575 and 48%, respectively; Fig. 1a). Thus, the crystal molecules structure of Hda presented here provides an ESU indicates estimated overall coordinate error. excellent framework for understanding the corre- ∑ −〈 〉 ∑ Rsym = |Ii Ii |/ |Ii|, where Ii is the scaled intensity of the ith 〈 〉 sponding experimental results in E. coli and offers measurement and Ii is the mean intensity for that reflection. ∑ − ∑ valuable insights into the regulation of DnaA, as Rcryst = ||Fobs| |Fcalc||/ |Fobs|, where Fcalc and Fobs are the well as interactions between Hda, the sliding clamp, calculated and observed structure factor amplitudes, respectively. and DnaA. Rfree =as with Rcryst but for 5.1% of the total reflections chosen at random and omitted from refinement. a Highest-resolution shell values (1.80–1.75) are given in parentheses. Results and Discussion b Typically, the number of unique reflections used in refinement is less than the total number that was integrated and scaled. Reflections are excluded due to systematic absences, Overall structure negative intensities, and rounding errors in the resolution limits and cell parameters. The structure of Hda from S. amazonensis,com- plexed with cytidine-5'-diphosphate (CDP) and mag- nesium (Fig. 1a and b), was determined to 1.75 Å loop), Walker B, and sensor I motifs. An extended N- resolution by multiwavelength anomalous disper- β sion (MAD) with selenomethionine-labeled protein terminal loop contains a short strand ( 1) that has previously been shown to bind to the sliding (Table 1). The structure was refined to Rcryst and Rfree 13 – values of 16.6% and 20.1%, respectively. The model clamp. The C-terminal domain Ib (residues 175 241, the lid domain) is a short four-helix bundle displays good geometry with 99.3% favorable main- α –α chain torsion angles (no Ramachandran outliers) and ( 5 8) that contains the sensor II motif. The two domains of Hda are similar to domain III (rmsd of 98.4% favorable side-chain rotamers according to α 32 1.57 Å for 138 C ) and domain IIIb (rmsd of 1.56 Å MOLPROBITY. The final model contains a dimer α ‡ (A12–241, B11–241) in which each monomer is for 48 C ) of DnaA [Protein Data Bank (PDB) IDs complexed with a CDP and a magnesium ion. In 2hcb and l8q] when compared separately. The most addition, 571 solvent molecules (1 thiocyanate, 14 significant difference between domain I of Hda and domain III of DnaA is the nature of the insertion ethylene glycols, 1 sodium, 1 chloride, and 554 water β β “ ” 19 molecules) were modeled. The main chain for between 3 and 4 (the steric wedge in DnaA ) – – – (Fig. 1c). This region often varies among different residues A0 11, B0 10, and A32 33 and the side 21,22 chains for residues A12, A36, A134, A150, B11–12, AAA+ ATPases and is used to classify them. In – Hda, this insertion is shorter than most others and B81, B102 103, B136, B192, B221, and B229 were η η η contains three 310 helices ( 1, 2, and 3) (Fig. 1a). disordered and were not included in the final model. η –η The Hda monomer (Fig. 1b) displays a molecular Helices 2 3 are equivalent to the second helix of a architecture typical of AAA+ ATPases.22,23 The N- terminal domain I (residues 1–174, the base domain) has a RecA-like fold containing the Walker A (the P- ‡ http://www.pdb.org/ 372 Crystal Structure of Hda prototypical AAA+ base domain.22 The η1 of Hda is crystal. The bulk of the dimer interface is formed shorter than the equivalent helix of DnaA. The Hda from interactions between the two base domains. insertion η1–η3 is structurally similar to the clamp These interactions are clustered around β1 and η1. loaders, such as the clamp loader delta subunit.33 The positions of the two N-terminal extended loops However, Hda does not possess the corresponding (11–21) are swapped, with the N-terminal loop from long η3–β4 loop found in the clamp loader, which is one subunit making extensive contacts with the β3, critical in binding the sliding clamp. Since the β3–β4 η1, and η2 from the second subunit. Together, these insertion resides next to the clamp binding motif N-terminal loops form a short antiparallel β-sheet (β1), it could play a role in binding the sliding that contributes 25% of the buried surface area to clamp. the dimer interface. Additional dimer interactions The orientation between the base and lid domains are between the lid domain of one monomer and varies significantly between DnaA and Hda. A the base domain of the second monomer. Specifi- superposition of the base domains of DnaA (PDB ID cally, the N-terminal portion of α3 of the base 2hcb) and Hda reveals that the lid domain of Hda is domain is docked between α7 and α8 of the lid rotated a further 47° toward its base domain around domain from the other subunit. the principal axis of the bound nucleotide (Fig. 1c). The Hda homodimer represents a novel mode of AAA+ oligomerization. AAA+ proteins frequently Hda dimer form hexameric rings in a “head-to-tail” manner such that the arginine finger from one subunit can The crystallographic asymmetric unit contains a interact with the nucleotide of the adjacent one. homodimer (Fig. 2). The two monomers are similar However, the two monomers in the Hda dimer are α to each other with an rmsd of 0.99 Å for all C arranged in a “head-to-head” manner. As a result, atoms, with the most significant differences in the the dimerization of Hda places the two nucleotide orientation of the two C-terminal helices, α7 and α8 binding sites close to each other, while the two (Fig. 1c). The Hda dimer resembles a flat rhombic corresponding arginine fingers are located far apart prism, with dimensions of 79 Å×62 Å×43 Å, in on the perimeter (Fig. 2). which the two N-terminal base domains are aligned The transcription activator NtrC1 is an example of along the short axis, while the two C-terminal lid an AAA+ protein that forms a dimer in its inactive domains are aligned along the long axis of the form through a swapped N-terminal linker when its rhombus. The dimer buries a surface area of 2045 Å2 receiver domain is unphosphorylated.34 However, from each monomer (Fig. 2). Molecular masses of the NtrC1 dimer differs significantly from the Hda 52,480 and 54,030 Da were determined by two dimer. The noncrystallographic 2-fold axis in the independent runs of analytical size-exclusion chro- NtrC1 dimer is parallel with the linker region matography in combination with static light scatter- consisting of two long, antiparallel helices. The ing. Because an Hda monomer has a calculated NtrC1 dimer does not involve the base domains of molecular mass of 27,059.1 Da (mass determined by the AAA+ regions. In Hda, the noncrystallographic liquid chromatography/mass spectrometry was 2-fold axis is perpendicular to the antiparallel β- 27,060.8 Da), size-exclusion chromatography with sheet in the N-terminal region. In contrast to the static light scattering suggested that it forms a critical role of the NtrC1 linker in its dimerization, dimer in solution, consistent with its packing in the the N-terminus of Hda does not appear to be

Fig. 2. The Hda dimer. (a) The two monomers are shown in orange and green. The residues that are involved in dimer formation are shown in blue in one monomer. The two arginine fingers (Arg161) in the dimer are shown as red sticks. The bound nucleotides and magnesium ions are shown in sticks and spheres, respectively. (b) The surface representation shows the opposite side of the dimer in (a) (left). The right panel shows the Hda dimer colored by sequence conservation. The most conserved residues are shown in magenta, while the least conserved residues are in cyan. Crystal Structure of Hda 373 essential for dimerization, since HdaΔN26 can still (Fig. 3a). The possibility of ADP (or GDP) was dimerize.13 excluded due to poor fit to the electron density, lack of favorable hydrogen bonds to stabilize the adeno- Nucleotide binding site sine base, and steric clashes with the protein. Bound CDP is surprising since ADP or ATP was expected Well-defined electron density at the nucleotide based on similarity to DnaA and other AAA+ binding site identified a bound CDP that presumably proteins. However, CTP has previously been was obtained during protein expression in E. coli shown to bind to ATPases. For example, the E. coli

Fig. 3. Nucleotide binding site of Hda. (a) CDP (shown in sticks) with the experimental density contoured at 2.5σ. The map was calculated after density modification improvement of the initial MAD phases. The conserved magnesium ion (pink), coordinated by water molecules (red), and Thr65 (not shown) stabilize the β-phosphate of the CDP. (b). Interaction between the protein and the cytidine base and sugar of CDP. (c) Comparison of motifs Walker A and sensor II of Hda (orange) and DnaA (gray). The corresponding residues for DnaA (PDB ID 2hcb) are shown in parentheses. (d) Sequence conservation pattern of the P-loop region of putative DnaA and Hda proteins. The DnaA group of sequences (785 sequences) were selected based on the presence of both a C-terminal DNA binding motif and an ATPase domain. The putative Hda sequences (94 sequences) were selected by full-length homology to E. coli Hda and of length between 210 and 270 residues. 374 Crystal Structure of Hda

DnaA–CTP complex was effective in opening the structurally known P-loops have lysines at this posi- DUE (but not effective in subsequent steps of the tion. Mutational studies on other AAA+ ATPases initiation process).35 The conformation of the CDP is have indicated that the conserved lysine in the very similar to the nucleotides ADP and AMP–PCP Walker A motif is essential. For example, Lys-to-Arg bound to DnaA.19,26 The cytidine base is stabilized mutants of E. coli RuvB and DnaC are defective in through hydrophobic stacking interactions and three both ATP binding and hydrolysis.37,38 The require- hydrogen bonds with the protein, along with two ment of a conserved lysine becomes more apparent water-mediated hydrogen contacts (Fig. 3b). The when the Walker A motif is analyzed in all pertinent binding site clearly favors CDP, and replacement of bacterial sequences currently in Pfam PF00308.39 cytidine by uracil would result in the loss of two The sequences fall into two groups: one with a C- hydrogen bonds. These hydrogen-bond interactions terminal DNA binding domain (likely DnaA pro- involve the main chain of Tyr30 and the side chain of teins) and one that has the prototypical DnaA AAA+ Arg185. Since Arg185 is the only side chain involved module only (likely Hda proteins). In the first group, in hydrogen-bonding interaction with the base, it for which ATP is required, the conserved lysine is could play a role in specifying the substrate. Arg185 invariant (Fig. 3d). In the second group, both the and the cytidine binding pocket are conserved in E. Walker A motif and the lysine position show greater coli and highly conserved in other putative Hda sequence variability, as do the Walker B and sensor I proteins. Thus, it seems likely that other Hda pro- motifs. The loss of the strictly conserved lysine teins can also bind CDP. suggests that the γ-phosphate of NTP is less The conserved motifs Walker A, Walker B, sensor important for Hda function. Structurally, an overlay I, and sensor II found in AAA+ proteins are all of AMP–PCP of DnaA onto the CDP site of Hda present in S. amazonensis Hda. The position of the indicates that the guanidinium group of Arg64 sensor II motif of Hda is significantly different from would clash with the γ-phosphate (Fig. 3c) and that of DnaA due to the change in orientation of the would therefore disrupt NTP binding. lid domain Ib (Figs. 1c and 3c). The conserved Hda has retained its ability to bind NDP as the arginine residue in the sensor II motif (e.g., Arg277 residues involved in binding ribose (His65) and α- in Aquifex aeolicus DnaA) typically interacts with the or β-phosphate (Gly63, Arg210, Arg64) are invariant γ-phosphate of ATP.23 However, the corresponding or highly conserved. A possible role of the CDP is to residue in Hda (Arg210) interacts with Asp24 of the promote dimer formation and maintain its confor- base domain I, as well as the α-phosphate of CDP mation. The smaller cytidine base may facilitate (Fig. 3c). Hda has a variant (58-GPVKSGRT-65) of greater plasticity between the two domains com- the Walker A consensus sequence (GXXGXGK[ST]). pared to adenosine. Lys61 physically occupies the same space as the As ATP/ADP is found in all other AAA+ struc- DnaA box VIII (sensor II) Arg277. The Arg64 side tures and the nucleotide binding pockets are highly chain interacts with the β-phosphate of CDP conserved in AAA+ proteins,22 the possibility that (Fig. 3c). The conformation of Arg64 is identical to Hda can also bind ADP cannot be excluded from the Lys64Arg mutant of RuvB from Thermotoga this study. If Hda were to bind ADP like DnaA,26,27 maritima.36 it is likely that ADP may affect the conformation of Hda locally or globally. Due to the larger size of the NTP hydrolysis and possible roles of adenosine base, the region between 28 and 34 of nucleotides Hda must move outward to accommodate it (Fig. 4). Changes in this region could propagate to nearby Hda shares many of the structural features found regions, such as the linker region between domains I in DnaA and other members of the AAA+ ATPase and Ib, the first helix of domain Ib (α5), and the superfamily, including a conserved nucleotide bind- clamp binding motif region. Thus, any association of ing site. However, no experimental evidence is avai- ADP with Hda could disrupt interactions between lable to suggest that it has NTPase activity. It has domains I and Ib in Hda, and potentially alter the been proposed that Hda could form a typical AAA+ dimerization state or the conformation of the clamp head-to-tail dimer.13 Such a model is not consistent binding region. with the crystal structure determined here. The homodimer of Hda precludes a mechanism through The sliding clamp binding motif and the clamp a canonical arginine finger type of interaction interaction between two Hda monomers. The CDP binding site is buried within the dimer interface (Fig. 2), and, Hda can form a stable functional complex with despite a few solvent channels nearby, access to CDP the sliding clamp in vitro with a stoichiometry of 12,13 is limited. No space is available to form a bipartite Hda2:clamp or, less likely, Hda4:clamp. Previous complex similar to other AAA+ ATPases. Significant studies have shown that a conserved hexapeptide structural changes, such as dimer dissociation, motif, QL[SP]LPL, at the N-terminus of E. coli Hda is would be required to achieve NTP hydrolysis. essential for binding the sliding clamp.13,17 This It seems likely that Hda has lost its ability to motif is also widely conserved in other clamp tightly bind or efficiently hydrolyze NTP. The pre- binding proteins.40 sence of Arg64 in the Walker A motif appears to be Hda from S. amazonensis possesses a similar detrimental to NTP binding and hydrolysis. All conserved sequence motif (13-QLSLPV-18) at its N- Crystal Structure of Hda 375

Fig. 4. Structural comparisons of the regions around the cytidine/ adenosine bases in Hda–CDP (PDB ID 3bos, orange) and DnaA–ADP (PDB ID 1l8q, gray). The super- imposition is obtained by overlap- ping the base domains of Hda and DnaA. terminus (Fig. 1a and b). In the Hda dimer, the ted that the Hda clamp binding motif will adopt a clamp binding motifs of each subunit form a short, similar conformation when bound to the clamp. antiparallel β-sheet at the noncrystallographic 2-fold However, the 2-fold symmetric clamp binding axis of the dimer. Mutational studies on this motifs of the Hda dimer are partially buried and motif13,17 have suggested that Gln13, Leu16, and have an unusual conformation (Fig. 5). Clearly, they Val18 (equivalent to Gln21, Leu24, and Leu26 of E. cannot bind the clamp in a canonical manner in their coli) are critical for binding the sliding clamp. current conformation without significant steric Interestingly, the side chains of these residues are clashes. As a result, the CDP-bound dimer of Hda buried in the dimer interface. The Gln13 from one observed in the crystal structure is likely unable to subunit makes contact with the backbone of the bind the clamp and thus is likely inactive in RIDA, adjacent subunit through two hydrogen bonds (Fig. unless the hydrophobic patch of Hda binds the 5a). Leu14, Ser15, and Pro17 in this motif are solvent sliding clamp in an unconventional manner. exposed and, along with conserved residues Leu97, Hda must then adopt a different conformation in Leu101, and Phe104, located at the β3–β4 insertion order to bind to the clamp in a canonical manner. (η2 and η3), and Phe83 from β3, form a hydrophobic This alternative structure could involve dissociation patch of dimensions 25 Å×22 Å, with a central ridge of the clamp binding motifs from the rest of the and a shallow cavity on either side (Fig. 5b). dimer to an extended conformation, or the Hda Several characterized clamp binding motifs adopt dimer itself may dissociate and interact with the extended conformations and bind to a conserved clamp in a monomeric form (Fig. 6b). Dissociation of location between the second and third domains of Hda clamp binding motifs (or the dimer) may occur – the β-subunit (Fig. 6a).33,40 42 The clamp binding spontaneously in solution. However, deuterium motif of Hda is also likely equivalent to other clamp exchange mass spectroscopy experiments have binding motifs and interacts with the same binding indicated that the clamp binding motifs of the site on the clamp.17,43 The clamp binding motif of Hda–CDP dimer are not flexible in solution (data Hda (QL[SP]LPL) is similar in sequence to the Pol IV not shown), consistent with the crystal structure. clamp binding motif (QLVLGL);41 thus, it is expec- Thus, it appears that additional factors are needed to

Fig. 5. The clamp binding motifs in the Hda dimer. (a) Close-up view of the conserved residues including the clamp binding motif. The orientation of the dimer is the same as the Hda dimer shown in ribbons in Fig. 2. (b) Surface representation of the hydrophobic pocket. 376 Crystal Structure of Hda

regulate the conformation of the clamp binding motifs of Hda, such as a conformational switch associated with binding of different nucleotides (e.g., ADP and CDP; see above) and a weak intrinsic NTPase activity. The physiological role of the inactive Hda dimer is currently unclear; it may offer additional means to regulate Hda activity so that it only becomes active when associated with the sliding clamp. Since the clamp binding motif of Hda is located at its N-terminus rather than at the C-terminus as in Pol IV,41 the Hda molecule will likely be located close to the middle domain of the β-subunit (Fig. 6a). It is not clear whether the functional Hda–clamp complex for RIDA involves an Hda monomer or an Hda dimer at each site of the β-subunit (Fig. 6b). Although the conserved site on the clamp generally interacts with a monomer,33,41 Hda was previously shown to interact with the clamp as a dimer.11,13 However, a recent study suggested a possible mono- meric interaction between Hda and the clamp.12 Further biochemical experiments and the complex structure of Hda and the sliding clamp will help resolve such questions.

Arginine finger and Hda–DnaA interaction

In order to utilize the conserved mode of ATP hydrolysis in AAA+ proteins, it is expected that DnaA and Hda form a heterodimeric complex during the RIDA reaction. The arginine finger, Fig. 6. Proposed model for interaction between Hda Arg168, of E. coli Hda is critical for the deactivation and the sliding clamp. (a) The clamp binding motifs of Pol of ATP–DnaA,13 whereas the corresponding Arg161 IV (little finger domain, red) and the clamp loader (δ- of S. amazonensis Hda is located near the middle of a subunit, green) interact with the clamp (cyan and blue) in short helix α4 (box VII). The arginine fingers are a conserved fashion. The clamp binding motif of Pol IV – fully exposed to solvent. (residues 346 351) is also shown in a stick model. (b) The Residues in the immediate surrounding of Arg161 clamp binding motifs of Hda (or the whole Hda dimer) dissociate and one of the motifs binds the clamp in a are also highly conserved among Hda, as well as conserved fashion. DnaA (Figs. 1a and 7). Arg161 is preceded by an acidic (D/E) residue (Asp157) and a smaller polar (S/T) residue (Ser160), followed by a hydrophobic residue (Trp164). Additionally, two other residues,

Fig. 7. Arginine fingers of Hda. The superimposed conformations of the two arginine fingers (Arg161) in the dimer and surrounding conserved residues of Hda (orange and green) are shown. The positional equivalent residues of DnaA (PDB ID 2hcb, gray) are also shown. The corresponding residue numbers for DnaA are given in parentheses. Crystal Structure of Hda 377

Phe126 and Asn130, from the neighboring α3 (the α3 (120–135) and α4 (156–166). Helix α3 is close to a central helix) are also highly conserved. Asn130 groove on the surface of domain III, and α4 makes hydrogen bonds with Arg161 in one of the Hda contacts with domain IIIb (Fig. 8a). subunits, and both are located on the same helical This model is consistent with known AAA+ surface and are fully exposed to solvent. Arg161 and interactions for ATP hydrolysis. The γ-phosphate Trp164 are in different conformations in the two of ATP sits next to the strictly conserved Arg161 Hda monomers, indicating that these residues are and Asp157 (Fig. 8b); therefore, both these residues flexible. likely play a catalytic role in ATP hydrolysis. An In contrast to DnaA, which is present in all acidic residue, corresponding to Asp157, is con- bacteria, Hda is predominantly found in proteobac- served in many AAA+ modules, such as glutamate teria, which suggests that Hda originally evolved in Pol III delta subunit and aspartate in NSF-D1.20 from DnaA to acquire its regulatory function. Due to Asp157 is close to the Walker B motif of DnaA the high level of similarity in both sequence and and located at the gate of the binding pocket structure in the arginine finger regions (i.e., Phe126, where the γ-phosphate of the ATP would contact Asn130, Glu134, Ser160, Arg161, Trp164, and the solvent. Thus, a possible role for Asp157 is to Gly165) of both DnaA and Hda (Figs. 1a and 7), it help the Walker B motif stabilize a nucleophilic has been proposed that Hda may interact with DnaA water, which would interact with the γ-phosphate. in a similar fashion to the DnaA self-assembly.4,19 Asp157 is not, however, conserved in DnaA (Figs. Structural studies on A. aeolicus DnaA with ADP and 1a and 4) and may explain why DnaA–ATP in the an ATP analog have illustrated a rigid-body move- origin of replication assembly does not efficiently ment in the two AAA+ subdomains between the ATP self-hydrolyze. and ADP states.19,26 Although there is no Hda In the transition from DnaA–ATP to DnaA–ADP, homolog and likely no Hda-dependent RIDA in A. the wedge opening, where Hda binds, will narrow aeolicus, the structural changes of DnaA associated through interdomain movement between the III and with different nucleotide states and the mode of IIIb domains of DnaA.19,26 It is plausible that the DnaA self-assembly are likely to be generally conformational changes induced by ATP hydrolysis applicable to DnaA in other bacteria due to the in DnaA could drive the dissociation of Hda and highly conserved AAA+ region (37% sequence iden- DnaA–ADP. The released Hda could then be tity between A. aeolicus and S. amazonensis).27 We recycled for the next reaction. The cellular concen- therefore made use of these DnaA models in order to tration of Hda (∼50 dimers/cell) is much less than gain further insights into how the base domain and that of DnaA (500–2000 molecules/cell);13 thus, the arginine finger of Hda interact with DnaA. reuse of the Hda is important in RIDA. The Hda dimer seems to be compatible with the mechanism suggested for the DnaA self-assembly Possible association of Hda with the membrane structure.4,19 A geometrically plausible model can be built by replacing a DnaA molecule with an Hda The replicatively active DnaA–ATP is regenerated dimer (or monomer) at the “ATP end” (the side from DnaA–ADP by acidic phospholipids in the where the γ-phosphate of the ATP bound to DnaA is presence of oriC and ATP.44 A region near the N- accessible19) of the DnaA self-assembly through terminus of the bridging helix (residues 349–383) of superimposition of the base domains of DnaA and E. coli DnaA, between the lid domain (IIIb) and the Hda. Extensive contacts are made through domain DNA binding domain (IV), is critical in membrane- III of DnaA in the DnaA filament self-assembly.19 mediated nucleotide release.4,44 Hda may also The corresponding Hda secondary structure ele- interact with the membrane since the T7 epitope- ments that make contact with the DnaA–ATP site are tagged form of Hda was associated with the inner

Fig. 8. Hypothetical models for DnaA–Hda interaction. (a) Putative interaction between the base domain of Hda (shown in ribbon representation) and DnaA. Domains III, IIIb, and IV of DnaA are shown as surfaces and in gray, magenta, and cyan, respectively. (b) The bipartite active site for ATP hydrolysis is formed by the conserved residues in the DnaA–Hda (white/green) heterodimeric complex shown in (a). 378 Crystal Structure of Hda membrane by immunoblot analysis.10 Interestingly, Crystallization and data collection the lipid binding region of DnaA corresponds to a highly conserved region of Hda (residues 225–241; The crystallization reagent contained 0.2 M sodium Fig. 1a). The α8 helix of Hda is equivalent to the thiocyanate and 20% (w/v) PEG (polyethylene glycol) bridging helix of DnaA, although shorter in length. 3350 at pH 6.9. Ethylene glycol was added as a cryo- Additionally, Arg328 of E. coli DnaA, whose muta- protectant to a final concentration of 10% (v/v). MAD data 45 were collected at SSRL on beamline 11-1 at wavelengths tion affects membrane binding in DnaA, is also λ corresponding to the peak ( 1) and high-energy remote highly conserved in Hda and located in the same λ ( 2) of a selenium MAD experiment. The data sets were region (adjacent to the side chain of Lys236 of Hda). collected to 1.75 Å at 100 K using a Mar CCD 325 detector Thus, it is possible that Hda could interact with the (MarUSA). membrane in a similar fashion to DnaA. Structure determination and refinement

Materials and Methods The crystals were indexed in orthorhombic space group P212121 with unit cell dimensions a=55.04 Å, Protein production b=64.03 Å, and c=153.64 Å. Data processing and structure solution were carried using an automatic structure solution pipeline XSOLVE developed at the Joint Center The gene encoding a putative Hda from S. amazonensis for Structure Genomics. The MAD data were integrated SB2B (GenBank YP_927791,119775051 gi: 119775051) was and reduced using Mosflm49 and then scaled with the amplified by PCR (polymerase chain reaction) from program SCALA50 of the CCP4 suite.51 Phasing was genomic DNA using PfuTurbo DNA polymerase (Strata- performed with SHELXD52 and autoSHARP.53 The auto- gene) and primers corresponding to the predicted 5′ and mated model building was performed with ARP/wARP.54 3′ ends. The PCR product was cloned into plasmid Refinement was carried out using REFMAC555 inter- pSpeedET, which encodes an expression and purification spersed with manual building using COOT.56 The model tag followed by a tobacco etch virus protease cleavage site geometry was analyzed with MOLPROBITY32 to assess (MGSDKIHHHHHHENLYFQG) at the amino terminus of the Ramachandran plot, side-chain rotamers, hydrogen the protein. The cloning junctions were confirmed by bonds, steric clashes, and van der Waals contacts. Data DNA sequencing. Protein expression was performed in a and refinement statistics are summarized in Table 1. selenomethionine-containing medium using the E. coli strain GeneHogs (Invitrogen). At the end of fermentation, lysozyme was added to the culture to a final concentration Accession code of 250 μg/mL, and the cells were harvested. After one freeze–thaw cycle, the cells were homogenized in lysis Coordinates and structure factors have been deposited buffer [50 mM Hepes, pH 8.0, 50 mM NaCl, 10 mM in the PDB under accession code 3bos. imidazole, and 1 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP)] and passed through a Microflui- dizer (Microfluidics). The lysate was clarified by centrifu- gation at 32,500g for 30 min and loaded onto nickel- chelating resin (GE Healthcare) preequilibrated with lysis Acknowledgements buffer. The resin was washed with wash buffer [50 mM Hepes, pH 8.0, 300 mM NaCl, 40 mM imidazole, 10% (v/v) The project was sponsored by the National glycerol, and 1 mM TCEP], and the protein was eluted with Institute of General Medical Sciences Protein Struc- elution buffer [20 mM Hepes, pH 8.0, 300 mM imidazole, 10% (v/v) glycerol, and 1 mM TCEP]. The eluate was ture Initiative (P50 GM62411, U54 GM074898). buffer exchanged with Hepes crystallization buffer Portions of this research were carried out at the [20 mM Hepes, pH 8.0, 200 mM NaCl, 40 mM imidazole, SSRL. SSRL is a national user facility operated by and 1 mM TCEP] and treated with 1 mg of tobacco etch Stanford University on behalf of the U.S. Department virus protease per 10 mg of eluted protein. The digested of Energy Office of Basic Energy Sciences. The SSRL protein was passed over nickel-chelating resin (GE Structural Molecular Biology Program is supported Healthcare) preequilibrated with Hepes crystallization by the Department of Energy Office of Biological and buffer, and the resin was washed with the same buffer. Environmental Research and by the National Insti- The flow-through and wash fractions were combined and tutes of Health (National Center for Research concentrated for crystallization assays to 14 mg/mL by Resources, Biomedical Technology Program, and centrifugal ultrafiltration (Millipore). Hda was crystal- lized using the nanodroplet vapor-diffusion method46 the National Institute of General Medical Sciences). with standard JCSG crystallization protocols.47 Initial The contents of this work are solely the responsibility screening for diffraction was carried out using the of the authors and do not necessarily represent the Stanford Automated Mounting system48 at the Stanford official views of the National Institute of General Synchrotron Radiation Laboratory (SSRL, Menlo Park, Medical Sciences or the National Institutes of Health. CA). To determine its oligomeric state, we analyzed S. We greatly appreciate the very helpful comments of amazonensis Hda using a 0.8× 30-cm Shodex PROTEIN Dr. James Berger and his group. KW-803 column coupled with miniDAWN static light scattering and Optilab differential refractive index detec- tors (Wyatt Technology). The mobile phase consisted of References 20 mM Tris, pH 7.4, and 150 mM NaCl. The molecular mass was calculated using ASTRA 5.1.5 software (Wyatt 1. Sekimizu, K., Bramhill, D. & Kornberg, A. (1987). ATP Technology). activates dnaA protein in initiating replication of Crystal Structure of Hda 379

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