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ÿþdoi:10.1016/j.jmb.2010.08.022 J. Mál. Biál. (2010) 403, 24 39 Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb Structural Analysis of the Interactions Between Hsp70 Chaperones and the Yeast DNA Replication Protein Orc4p María Moreno-del Álamo1, Alicia Sánchez-Gorostiaga1, Ana M. Serrano1, Alicia Prieto2, Jorge Cuéllar3, Jaime Martín-Benito3, José M. Valpuesta3 and Rafael Giraldo1N 1 Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas  CSIC, C/ Ramiro de Maeztu, 9, E-28040 Madrid, Spain 2 Department of Environmental Biology, Centro de Investigaciones Biológicas  CSIC, C/ Ramiro de Maeztu, 9, E-28040 Madrid, Spain 3 Department of Macromolecular Structures, Centro Nacional de Biotecnología  CSIC, C/ Darwin, 3, E-28049 Madrid, Spain Received 4 May 2010; Hsp70 chaperones, besides their role in assisting protein folding, are key received in revised form modulators of protein disaggregation, being consistently found as 29 July 2010; components of most macromolecular assemblies isolated in proteome accepted 11 August 2010 wide affinity purifications. A wealth of structural information has been Available online recently acquired on Hsp70s complexed with Hsp40 and NEF co factors 21 August 2010 and with small hydrophobic target peptides. However, knowledge of how Hsp70s recognize large protein substrates is still limited. Earlier, we Edited by F. Schmid reported that homologue Hsp70 chaperones (DnaK in Escherichia coli and Ssa1 4p/Ssb1 2p in Saccharomyces cerevisiae) bind strongly, both in vitro and Keywords: in vivo, to the AAA+ domain in the Orc4p subunit of yeast origin DNA replication; recognition complex (ORC). ScORC is the paradigm for eukaryotic DNA Hsp70 chaperones; replication initiators and consists of six distinct protein subunits (ScOrc1p macromolecular assemblies; ScOrc 6p). Here, we report that a hydrophobic sequence (™L4) in the initiator origin recognition complex; specific motif (™SM) in Orc4p is the main target for DnaK/Hsp70. The three yeast dimensional electron microscopy reconstruction of a stable Orc4p2 DnaK complex suggests that the C terminal substrate binding domain in the chaperone clamps the AAA+ ™L4 motif in one Orc4p molecule, with the substrate binding domain lid subdomain wedging apart the other Orc4p subunit. Pairwise co expression in E. coli shows that Orc4p interacts with Orc1/2/5p. Mutation of ™L4 selectively disrupts Orc4p interaction with Orc2p. Allelic substitution of ORC4 by mutants in each residue of ™L4 results in lethal (™184A) or thermosensitive (L185A and L186A) initiation defective *Corresponding author. E mail address: rgiraldo@cib.csic.es. Present address: A. Sánchez Gorostiaga, Department of Microbial Biotechnology, Centro Nacional de Biotecnología  CS™C, C/ Darwin, 3, E 28049 Madrid, Spain. Abbreviations used: AAA+, ATPases associated with various cellular activities; ARS, autonomous replication sequences; DSG, disuccinimidyl glutarate; EM, electron microscopy; ™L4, ™le Leu Leu Leu Leu; ™MAC, immobilized metal ion affinity chromatography; ™SM, initiator specific motif; MS, mass spectrometry; MALD™ TOF, matrix assisted laser desorption/ionization time of flight; NBD, nucleotide binding domain; NEF, nucleotide exchange factor; ORC, origin recognition complex; SBD, substrate binding domain; WH, winged helix; WT, wild type. 0022 2836/$  see front matter © 2010 Elsevier Ltd. All rights reserved. Dissecting Orc4-Orc2/Hsp70 Interactiáns 25 phenotypes in vivo. The interplay between Hsp70 chaperones and the Orc4p ™L4 motif might have an adaptor role in the sequential, stoichiometric assembly of ScORC subunits. © 2010 Elsevier Ltd. All rights reserved. replication initiator monomers.25 The action of Introduction Hsp70s on DNA replication proteins was then Protein chaperones are macromolecular machines extended to the assembly of functional viral repli that convert the energy derived from ATP binding cases in eukaryotes.26 28 ™n spite of this background and hydrolysis into inter domain movements, which record, once it was found that a significant are transduced to the bound protein substrates proportion of the interactions described for the and results in their folding, assembly, disassembly/ proteome of Saccharomyces cerevisiae engaged disaggregation or proteolytic degradation.1 3 Hsp70s molecular chaperones, they were left aside as are a phylogenetically ubiquitous class of chaper non specific,29,30 although this does not necessarily ones that are composed of two specialized domains, mean that interaction with chaperones is func allosterically coupled through an intermediate tionally irrelevant. linker: an N terminal, actin like, ATP/nucleotide Earlier, we found that dimers of the S. cerevisiae (Sc) binding domain (NBD)4 and a C terminal substrate Orc4p protein (one of the six subunits (Orc1 6) of binding domain (SBD).5 The latter is composed of the origin recognition complex (ORC), the initiator a ² sandwich, peptide ligand subdomain and an of chromosomal DNA replication in eukaryotes),31,32 ± helical lid subdomain. While Hsp70s hold the co purified in E. coli tightly bound to DnaK, the central functional role, they act in concert with bacterial Hsp70 chaperone.33 This heterologous two types of protein co factors that (i) pre target interaction was likely to be significant, because suitable substrates, stimulating ATP hydrolysis at immunoprecipitation of the Hsp70 chaperones Hsp70 NBD (Hsp40s or J domain proteins);6 and (DnaK homologues) in yeast also pulled down (ii) foster the interchange of ADP by ATP upon ScOrc4p. The target for DnaK was found to be the nucleotide hydrolysis (nucleotide exchange factors, N terminal domain in ScOrc4p.33 Structural studies NEFs).7 9 The functional cycle of Hsp70s10 15 starts on proteins such as bacterial DnaA34,35 and archaeal with closure of the lid subdomain in the SBD on a Orc1 3,36 39 led to the conclusion that ORC type bound hydrophobic peptide stretch, which togeth initiators are composed of an N terminal AAA+ er with a Hsp40 co factor triggers ATP hydrolysis domain and a C terminal winged helix (WH) in the NBD and the relevant structural transac domain. The former is responsible for the ATP tions in the substrate. This is followed by dependent remodeling of ORC upon binding to displacement of the residual ADP by an NEF origin (ARS) DNA,40 which is led primarily by the and opening of the SBD lid upon allosteric WH domain38 in ScOrc1,2,4,5p.41,42 ™n the original binding of a new ATP molecule at NBD, which report on Hsp70 ScOrc4p binding, we noted a releases the protein substrate and closes the cycle. structural similarity between the C terminal domain The limiting, slow step is closure of SBD on the in ScOrc4p and the N terminal domain (WH1) of the substrate, whereas this is in quick binding pPS10 plasmid initiator RepA.33 This was confirmed equilibrium/exchange in the ATP form of the by the crystal structure of RepA WH143 and extrap chaperone. Mapping the interactions between olated to other ORC subunits, on the basis of their Hsp70s and large protein substrates with real common ancestry with archaeal ORC proteins.44 molecular detail has been made possible recently Furthermore, the structural similarities between through the combination of hydrogen/deuterium plasmid RepA and archaeal/eukaryal ORC proteins exchange and mass spectrometry,16 although a 3D extend beyond the WH fold to encompass additional structure at atomic resolution is still missing. ± helical elements in RepA dimers, which undergo After the initial association of Hsp70 chaperones conformational activation into ² strands to become with the cellular response to heat stress17 and the replication competent RepA monomers.43,25 Hsp70s proposal for a role in protein disaggregation,18 ScOrc4p binding appeared to mirror DnaK RepA pioneering biochemical studies were carried out in interactions and we speculated on a possible role for the DNA replication field. The role of the Escherichia Hsp70s in modulating the assembly of ORC coli Hsp70 Hsp40 NEF triad (DnaK DnaJ GrpE) in subunits.33 modulating the assembly of the bacteriophage » Considerable effort has been devoted to unra replisome was revealed,19 21 which was followed by veling the interactions between ORC subunits by studies on their action in plasmid replication.22 24 ™n means of indirect in vivo assays, such as the the latter case, Hsp70 chaperones are involved two hybrid,45,46 co expression studies in insect specifically in the conformational remodeling of cells using baculoviruses,41,47 50 or co translation RepA dimeric repressors to become active as DNA in vitro,51 often followed by immunoprecipitation. 26 Dissecting Orc4-Orc2/Hsp70 Interactiáns The emergent picture is not unique; differences are a WT ™SM ™L4 motif in ScOrc4p, where it replaces evident depending on the biological source of the Hsp70. In vivo studies of point mutations in this proteins and the experimental approach chosen, but motif show that it is essential for DNA replication it seems clear that Orc2,3,4,5 would constitute the in S. cerevisiae, ™184A being lethal and L185/186A core of the complex with Orc1,6 at a peripheral thermosensitive ORC4 alleles. The thermosensitive location. Sequential assembly of subunits has been mutants exhibit, after a shift to non permissive found for human ORC, in which binding of the temperature in G1 synchronized cultures, fewer stable Orc2,3,5 core to Orc4 and Orc1 is ATP viable initiations and thus a prolonged S phase and dependent.50 ™n ScORC, the Orc1p and Orc5p a final arrest at the beginning of the next cycle. This subunits bind ATP, but only Orc1p hydrolyzes phenotype is compatible with a defective assembly it.52 54 Orc4p contributes to a functional nucleotide of the pre replicative complex. Our data provide cycle55 and, in higher eukaryotes, binds ATP.50 support for a scenario in which Hsp70 chaperones Another factor essential for DNA replication, Cdc6, would act as modulators, through the recognition shares these DNA and ATP binding activities with of a conserved stretch in the pivotal ™SM, of subunit their ORC paralogues in building an active pre stoichiometry in the sequential assembly of ORC. replicative complex.42 Orc6p is structurally unre lated to the other subunits in the complex42 and, as Orc3p, does not bind to ARS DNA.41 ™n spite of this Results and Discussion wealth of information, little is known about the molecular details of the intersubunit contacts in ORC. The crystal structures of oligomeric DnaA34 Proteomic identification of the target for Hsp70 and hetero dimeric archaeal Orc1 338 point to two in ScOrc4p ± helices located between the ATP binding Walker A and B motifs, a signature for the replication The structural principles for protein recognition initiator family among all AAA+ proteins.56,57 Such by Hsp70 chaperones10 are the same across the ± helices constitute an initiator specific motif (™SM) evolutionary tree, reflecting the extreme conserva that has been proposed to modulate the assembly tion of the residues responsible for target recogni of ORC subunits into an open ring/superhelix tion at the SBD in both prokaryotic and eukaryotic that would partially encircle DNA.58 Recent Hsp70s.62,63 However, there is a single Hsp70 in electron microscopy (EM) 3D reconstructions have E. coli (DnaK) but 14 paralogues in S. cerevisiae, sketched the arrangement of subunits in yeast59 which are functionally organized into networks and Drosophila60 ORC. specialized in protecting proteins against stress or We report here that multiple yeast Hsp70 in assisting their folding.64 Up to nine yeast Hsp70s chaperones (Ssa1 4p and Ssb1/2p) bind to ScOrc4p (Ssa1p 4p, Ssb1p and  2p, Sse1p and  2p and Ssz1p) in vivo, coincident with earlier results from whole have a cytoplasmic/nuclear location and several of cell analysis.29,61 Using a stable, heterologous them have been isolated consistently in complex complex between E. coli DnaK and ScOrc4p, we with each ScORC subunit in proteome wide identify, by combining cross linking, proteolysis analysis.29,61 Actually, our own proteomic analysis and mass spectrometry (MS), the target for Hsp70 of the proteins co purifying with histidine tagged chaperones in ScOrc4p: a hydrophobic sequence ScOrc4p in S. cerevisiae confirms that Ssa1p and  2p (™L4: ™LLLL188) that it is part of the ™SM motif in and Ssb1p and  2p Hps70 chaperones are physically 184 the AAA+ domain.38 We show the 3D reconstruc bound to such bait (Supplementary Data Fig. S1). ™t tion of the ScOrc4p2 DnaK complex obtained by follows that the ScOrc4p Hsp70 complexes have a single particle EM methods. Docking of the model heterogeneous chaperone composition, making it for dimeric ScOrc4p and the atomic structure of the difficult to identify their contacting interfaces two domains of DnaK is consistent with the SBD in unambiguously. ™n this work, therefore, we have the latter interacting with ™L4 in one of the ScOrc4p characterized the tight ScOrc4p DnaK complex, monomers. This defines a DnaK ScOrc4p interface which can be purified in large quantities upon compatible with that found by cross linking and expression of the ScORC subunit in E. coli.33 MS studies. This structure provides insight into The ScOrc4p DnaK complex was cross linked how Hsp70s dissociate compact protein assemblies. in vitro with dissucinimidyl glutarate (DSG), a We have developed vectors for co expressing as bifunctional reagent specific for free amino groups, bipartite operons in E. coli ScOrc4p (either wild before resolving the cross linked species from the type (WT) or a mutant in the sequence targeted by unreacted proteins by SDS PAGE (Fig. 1a). Up to six Hsp70: ™L4 ’! A5) followed by each of the other discrete oligomeric bands with a slower electropho ScORC subunits (ScOrc1p,  2p,  3p and 5p) or retic mobility than those of the protein monomers ScCdc6p. We have mapped the interactions estab were visualized. We focused our study on the fourth lished by ScOrc4p in the binary complexes, of these bands because its mass (120 140 kDa) was showing that ScOrc2p assembly is dependent on compatible with homo/hetero dimers of the two Dissecting Orc4-Orc2/Hsp70 Interactiáns 27 Fig. 1. Dissection of proximal interfaces in ScOrc4p DnaK. (a) SDS PAGE of DSG treated ScOrc4p DnaK complexes. Arrows point to the cross linked, low mobility bands. Asterisks (N ) mark the homo/hetero dimeric complexes relevant for this work. (b) MALD™ TOF mass spectra of the trypsin digested bands labeled with open arrows in a. Experimental masses are depicted on each peak, together with their residue coordinates in ScOrc4p (blue) or DnaK (red). Peaks labeled in green were identified as arising from DSG cross linking. (c) The sequences of the four DSG cross linked peptide pairs that engage DnaK (red) and/or the ScOrc4p AAA+ (blue) or WH (cyan) domains (Fig. 2). component proteins of the complex. After trypsin (Fig. 1b). Most of the tryptic peptides could be digestion in the gel, the resulting peptides were assigned unambiguously to ScOrc4p or DnaK, analyzed by MALD™ TOF mass spectrometry whereas four orphan peaks were identified as 28 Dissecting Orc4-Orc2/Hsp70 Interactiáns peptides paired through a DSG link (Fig. 1c). gel filtration column (see below, Fig. 5), compatible Because of the monomeric nature of Hsp70s, the with a ScOrc4p2 DnaK hetero trimeric complex. DSG1 peak likely arises from an intermolecular This additional purification step reduced the hetero bridge between two neighbor DnaK molecules geneity found in the oligomers initially isolated bound to a ScOrc4p oligomer (for such oligomeric by means of Ni2+ immobilized metal ion affinity assemblies, see Fig. 5c below). ™nterestingly, one of chromatography (™MAC). the DnaK peptides involved (489DKNSGK494) is The 3D reconstruction obtained from the complex located at the entrance of the binding pocket in the revealed a bi lobed, figure of eight structure with a C terminal SBD of the chaperone.5 Because small additional mass breaking an apparent 2 fold ScOrc4p:DnaK stoichiometry approaches 2:1 in the symmetry (Fig. 3 and Supplementary Data Fig. S5). complexes33 (Figs. 1, 3 and 5), DSG2 would The region corresponding to the observed extra correspond to an intermolecular cross link between density allowed the fitting of a single copy of each of the AAA+ and WH domains from each protein the NBD and the SBD of a Hsp70 chaperone. Two chain in ScOrc4p dimers.33 This is compatible with a copies of the atomic model of ScOrc4p (Fig. 2a) were head to tail arrangement for the two ScOrc4p sub manually docked into the reconstructed volume units in the complex. The two most relevant peaks (Fig. 3), with their best fit obtained when the two are DSG3 and DSG4, because they correspond to ScOrc4p molecules were placed in a head to tail ScOrc4p DnaK adducts. The former was identified orientation. This arrangement of ScOrc4p subunits is as a cross link between the gatekeeper peptide of compatible with the cross linking results (DSG2; Fig. DnaK and a WH sequence (418M™KA™NSR425) that, 1) and with the crystal packing found for two in a model for ScOrc4p based on its homologue orthologue archaeal ORC proteins solved in the Orc1/Cdc6 of the archaea Pyrobaculum aerophilum36 absence of ligand DNA.36,37 However, it differs from (Fig. 2a), is structurally equivalent to the C terminus the side by side association of AAA+ domains of the second ± helix in the WH1 domain of typical for this protein family when assembled as pPS10 RepA.43 ™t is significant that, for the homo hexameric rings57 or spirals,58 or as DNA bound logue initiators P1 RepA65 and F RepE,66 such a dimers.38 This suggests an active role for Hsp70 region in WH1 had been identified as a major target chaperones in generating ScOrc4p monomers suit for DnaK. However, we did not co purify a com able for their subsequent assembly into functional plex between the isolated ScOrc4p WH domain and ScORC hetero hexamers. DnaK in an earlier study.33 This fact could indicate With the uncertainty intrinsic to the low resolution low stability or a high off rate (Fig. 2b) for the (22 Å; Materials and Methods) of the reconstructed WH chaperone complex, which can be detected only volume for the ScOrc4p2 DnaK complex (Fig. 3), the after covalent cross linking of the two proteins. The docked model has the ™L4 stretch in one of the two DSG4 peak corresponds to an adduct between a AAA+ domains sandwiched in a canonical way C terminal peptide in DnaK (635DKK637), also close to between the ± helical (lid) and the ² sheet sub the substrate binding pocket, and a sequence in the domains in the SBD of DnaK.5,10,68 The lid appears AAA+ domain of ScOrc4p (184™LLLLDSTTKTR195) to act on the ScOrc4p dimer as a wedge, separating predicted to be solvent exposed in an ± helix the previously interacting AAA+ and WH domains. (Fig. 2a). Obviously, both domains still interact at the We explored the ScOrc4p sequence with an opposite edge of the head to tail oriented ScOrc4p algorithm designed for the detection of potential dimer: if this were also bound by another molecule DnaK binding sites in proteins (hydrophobic of the chaperone, the ScOrc4p2 DnaK complex stretches flanked by basic residues).67 We found would be dissociated immediately into two multiple target peptides for DnaK (Fig. 2b), of which ScOrc4p DnaK heterodimers. Because the latter that having the lowest predicted variation in free are not the most abundant purified species, it energy of binding (””G =  16.37 kJ/mol) corre follows that the levels of the Hsp70 chaperone sponds to FEK™LLLLDSTTK193: it overlaps with must be limiting due to an excess of the recombinant 181 the peptide found in the DSG4 peak (Fig. 1c), thus protein (a hypothesis confirmed below; see Fig. 5c). pointing to the ™L4 sequence as the major binding The docked model presented here fits quite well in site for Hsp70 in ScOrc4p. the volume of the ScOrc4p2 DnaK complex, while the remaining empty regions could be attributed to Three-dimensional electron microscopy the protein portions missing from the atomic model reconstruction of the Hsp70 (DnaK)-ScOrc4p of ScOrc4p (broken red lines in Fig. 2a) and to a complex detached conformation of the WH domain37 that becomes freed due to the binding of DnaK SBD to ™n order to characterize the interaction between the AAA+ domain of the neighbor ScOrc4p subunit. ScOrc4p and DnaK in more detail, EM studies The reconstructed ScOrc4p2 DnaK complex sug were done with negatively stained samples gests a general mechanism for Hsp70s chaperones in corresponding to the major peak eluting from a disassembling compact protein complexes and Dissecting Orc4-Orc2/Hsp70 Interactiáns 29 aggregates, fuelled by allosteric binding/hydrolysis one of the contacting interfaces; (ii) closure of the lid of ATP at the NBD: (i) the SBD would bind to any would introduce a wedge, breaking a few inter accessible hydrophobic sequence stretch found at molecular contacts, thus destabilizing the aggregate; Fig. 2 (legend on next page) 30 Dissecting Orc4-Orc2/Hsp70 Interactiáns and (iii) the bound protein substrate would be matography elution profiles for ScOrc4p ™L4/A5 finally pulled apart by a large interdomain motion. (Supplementary Data Fig. S2c) indicated a lower Our current structural analysis of the ScOrc4p2 tendency towards aggregation for the latter, as DnaK particles most likely corresponds to the stable expected from the more reduced hydrophobicity of ADP and substrate bound state in the chaperone the mutant. cycle.10,33 A key issue still under debate is the The expression vectors for ScOrc4p ™L4/A5 relation between both chaperone domains in sub were then engineered to clone, downstream of strate and nucleotide bound Hsp70s.11 15 ™n our ORC4, each one of the other ScORC core subunits model (Fig. 3), the two chaperone domains are (ORC1, -2, -3 and -5) or ScCdc6p (CDC6) as bipartite arranged in a nearly orthogonal orientation and operons (Supplementary Data Fig. S3a). Subunits wrapped around the tip of one of the two AAA+ were cloned with distinct N or C terminal peptide domains in the ScOrc4p dimer. Such an orientation, tags (Supplementary Data Fig. S3b) in order to placing the ™A subdomain in the NBD close to the allow for immunodetection without compromising ² sheet in the SBD, is in agreement with the recently their assembly.70 ™nduced bacterial cells were lysed solved NMR structure for the ADP and peptide and His10 tagged ScOrc4p ™L4 (or A5) present in bound state of DnaK.68 The ScOrc4p2 DnaK struc the soluble fraction was then purified by means of ture is the second solved of a complex between a Ni2+ ™MAC. The presence of each companion complete Hsp70 chaperone and a full length protein ScORC subunit and the balance with DnaK substrate, rather than bound to a small hydrophobic chaperone were determined by means of Western peptide, after the recently reported cryo EM recon blotting with specific antibodies (Fig. 4). ScOrc3p struction of a complex between bovine Hsc70 and a failed to co purify with ScOrc4p, independent of clathrin coat, also in the ADP bound state.69 At a the presence of the ™L4’! A5 mutation (Fig. 4d). similar resolution to that achieved here for the ScCdc6p was proteolyzed to a large extent (not ScOrc4p2 DnaK complex, a single Hsc70 molecule shown) and thus it barely co localized with ScOrc4p was found bound to just one of the three hydro (Fig. 4f). ScOrc2p was the only subunit clearly phobic peptide tails (C terminal exposed QLMT) showing a differential association with ScOrc4p converging at the vertex of each clathrin triskelion. dependent on the presence of a WT ™L4 motif ™n this case, Hsc70 binding seems to stabilize a (Fig. 4c). Because ScOrc4p was expressed to a very strained conformation of the triskelion, leading to high level in all constructs, in a large excess over disassembly of the coat.69 any other downstream subunit, this ensures that the mutations in ScOrc4p A5 severely disrupt A map for the interactions mediated by binding to ScOrc2p. The other subunits (ScOrc1p ScOrc4p-ISM-IL4 and  5p; Fig. 4b and e, respectively) interacted strongly with ScOrc4p, as reported for two hybrid The peptide target for Hsp70 in the AAA+ domain assays,46 independent of the presence of a WT or of ScOrc4p corresponds to the C terminal ± helix of mutant ™L4 motif. This clearly points to the ™SM, a motif described to contribute to DNA existence of additional/alternative interfaces to recognition and proposed to be involved in the ™L4 for contacting with other ScORC subunits. assembly of initiator oligomers.38 To study further By showing that ScOrc4p interacts with ScOrc2p the role of the ™SM ™L4 motif in ScORC assembly, we but it does not with ScOrc3p, our results solve mutated its five large hydrophobic residues to Ala an ambiguity in the current models for ORC (™L4 ’! A5) to impair Hsp70 binding to this sequence structure.59,60 (Fig. 2b, estimated ””G =4.11 kJ/mol). We then Regarding DnaK association with each binary expressed both variants of ScOrc4p (WT ™L4 and assembly, except for ScOrc4 1p (Fig. 4b), there is a mutant A5) in E. coli and purified (Supplementary net reduction in the level of bound chaperone when Data Fig. S2a, inset), checking by means of circular the ScOrc4p A5 mutant is present. This suggests a dichroism spectroscopy that the mutations had not role for Hsp70 chaperones in shielding solvent altered the structure or folding stability of the exposed hydrophobic motifs, such as ™L4, until they protein (Supplementary Data Fig. S2a and b). are engaged in specific contacts between subunits However, the inspection of the gel filtration chro during ORC assembly. Since there are several Fig. 2. Mapping DnaK (Hsp70) binding sites in ScOrc4p. (a) ScOrc4p structure modeled on its P. aerophilum homologue Orc1/Cdc6 (PDB ™D 1FNN).36 ™ts AAA+ (N terminal) and WH (C terminal) domains are colored green and orange, respectively. Structurally uncertain ScOrc4p regions are depicted in red and those having no equivalence in PaOrc1/Cdc6 have been removed (broken lines). The peptides identified through proteomics to be cross linked to DnaK (Fig. 1c) have their side chains displayed and are colored blue and cyan. (b) On the ScOrc4p sequence, blue boxes mark the highest affinity DnaK binding sites predicted,67 (all of them in the AAA+ domain) with the calculated free energy variation, ””G, on top. The A5 mutations (in red) replacing the ™L4 stretch cause a drastic increase in ””G. A sequence in the WH domain with a predicted low affinity, but yet found cross linked to DnaK (Fig. 1c), is also boxed (cyan). Fig. 3. The EM 3D reconstruction of the DnaK (Hsp70) ScOrc4p2 complex is compatible with the biochemically identified intermolecular contacts. (a) Several views of the volume of the ScOrc4p2 DnaK complex. The atomic models for the ScOrc4p dimer (subunits colored cyan and magenta) and DnaK are displayed fitted into the volume. The nucleotide (NBD) and substrate (SBD) binding domains in DnaK are in yellow and red, respectively. Blue spheres: the ™L4 motif in the AAA+ domain in one of the two ScOrc4p subunits (cyan). ™L4 residues seem to be gripped in between the two subdomains (² sandwich and ± helical lid) in DnaK SBD (red). (b) The 3D volume of the particle using the same colors as those used for the ScOrc4p subunits and the DnaK domains in a. The scale bar represents 50 Å. Dissecting Orc4-Orc2/Hsp70 Interactiáns 31 Fig. 4. Mapping subunit interactions made by ScOrc4p ™L4/A5. (a) Ni2+ ™MAC elution profiles for His10 tagged ScOrc4p ™L4 (WT, blue) and mutant A5 (red). (b f) Profiles for the same proteins when co expressed with each of the other ScORC subunits, or ScCdc6p. SDS PAGE of the loaded soluble lysate (L) and the fractions collected (™ ™™™) from the second, specific elution peak are also shown. Arrowheads point to ScOrc4p and asterisks (N ) mark the co purified subunits. Western blots for detection of the specific tags for each companion subunit ScOrc4p (anti/± His) and DnaK (anti/± DnaK) are displayed below the gels. 32 Dissecting Orc4-Orc2/Hsp70 Interactiáns Dissecting Orc4-Orc2/Hsp70 Interactiáns 33 potential binding sites for DnaK in ScOrc4p (Fig. oligomeric ScOrc4 ScOrc2p assemblies eluting 2b), it is not thus strange that the ™L4 ’! A5 with the void volume, fractions distributed across mutations do not completely abolish, but reduce the sizing column were identified as hetero tetra chaperone interaction. meric ScOrc4p2 ScOrc2p2 and hetero trimeric The complex between ScOrc4p and ScOrc2p was ScOrc4p2 DnaK complexes and, finally, a vast found to be the most sensitive to the mutations in excess of monomeric DnaK. ™t is noteworthy that the ™SM ™L4 motif (see above) and was studied in the chaperone was marginally present in the largest greater detail. The Ni2+ ™MAC purified complex, molecular mass assemblies. On the contrary, when including DnaK (Fig. 4c), was analyzed in terms of ScOrc4p was over expressed in the absence of any its hydrodynamic stability and subunit stoichiom other ScORC subunit (Fig. 5a and c), it was found etry by means of gel filtration chromatography (Fig. in large assemblies with DnaK (Fig. 5b), titrating 5a) plus immunodetection of the proteins in the most of the available chaperone. The release of eluted fraction peaks (Fig. 5b). Besides large DnaK from the ScOrc4p2 ScOrc2p2 complex during chromatography probably reflects ScOrc2p pro moted weakening, to the point of dissociation due to the intrinsic dilution (by ca 10 fold) of the sample, of the interaction between the chaperone and ScOrc4p. An exhaustive proteome wide description of the interactions established by the whole set of chaper ones in S. cerevisiae has been published recently:61 ScOrc4p and ScOrc2p are the only ScORC subunits that share interactions with a module of four Hsp70 chaperones (Ssa1p, Ssa2p, Ssb1p and Sse1p) that seems to consistently function in a concerted way (supplementary table S2 in ref 61). This observation is fully compatible with the concept of an adaptor DnaK (Hsp70s) molecule being replaced by ScOrc2p in its complex with ScOrc4p. Mutations in ScOrc4p-ISM-IL4 lead to an initiation defective phenotype Once it was found that the ™SM ™L4 sequence in ScOrc4p, targeted by Hsp70s, is involved in binding to ScOrc2p, we surveyed the effect of the disrupting ™L4 ’! A5 mutations on yeast DNA replication in vivo. ™nS. cerevisiae haploid cells, we were unable to replace the genomic ORC4-WT by its allele orc4-A5, Fig. 5. ScOrc2p interchanges with Hsp70 chaperone in the complex with ScOrc4p. (a) Gel filtration elution profiles of the affinity co purified His10 ScOrc4p/DnaK (broken black plot; Fig. 4a) and His10 ScOrc4p/ScOrc2p/ DnaK (blue plot; Fig. 4c) assemblies. Arrows mark the elution positions (and masses, in kDa) of native protein standards (ferritin, catalase, aldolase, bovine serum albumin and ovoalbumin). (b) ScOrc4+2p run. The loaded sample (L), together with the fractions collected and molecular mass standards (ST) were then analyzed by SDS PAGE (top) and western blot (middle) with the antibodies indicated. Bars outline the eluted complexes (bottom). (c) Equivalent fractions from the ScOrc4 run were processed as decribed for b. The stable complex between a ScOrc4p dimer and a DnaK (Hsp70) molecule (ca 200 kDa) becomes labile upon co expression with ScOrc2p, resulting in ScOrc4+2p oligomeric species (V0) and a ScOrc4p2 ScOrc2p2 hetero tetramer (ca 270 kDa), thus generating a fraction of free DnaK monomers (70 kDa). 34 Dissecting Orc4-Orc2/Hsp70 Interactiáns Fig. 6. Mutation of the ™SM ™L4 motif in ScOrc4p results in defective replication in vivo. (a) Growth in YPAD agar of serial dilutions (5 ¼l, 105 101 cells/ml) of WT cells and the viable orc4 mutants, showing the thermosensitive phenotypes of orc4 L185/186A. (b) Flow cytometry of ORC4-WT or orc4-L185/186A yeast, after releasing G1 arrest and a simultaneous shift to 37 °C. Mutants show a prolonged S phase and impaired re initiation in the next cycle. (c) 2D neutral/neutral gels of Bgl™™ digested bulk DNA from ORC4-WT and orc4-L185/186A cells, carrying an ARS1 episome, grown as described for 32 b. Hybridization with a P labeled plasmid specific DNA fragment shows a reduced frequency for origin firing (arrows: bubble arc) in the mutants. an indication of its lethality. Therefore, we tried to mutations with such phenotype described so far in substitute each hydrophobic residue individually in ORC. We studied the cell cycles of ORC4-WT and the motif by Ala (Supplementary Data Fig. S6). We orc4-L185/186A mutant yeast, synchronized in G1, could not replace the first residue (™184) either, after releasing their arrest in combination with a underlining the essential role of the motif. On the shift to non permissive temperature (Fig. 6b). Both contrary, the last two leucines (L187 and L188) can ™SM ™L4 mutants exhibit a marked delay in their cell be mutated with no apparent effect on yeast growth. cycles, with a prolonged S phase and an apparent ™nterestingly, L185A and L186A resulted in thermo arrest in G2/M, when cells accumulate a 2C DNA sensitive growth (Fig. 6a), being among the very few content. They fail to efficiently enter into a new Dissecting Orc4-Orc2/Hsp70 Interactiáns 35 Biflex ™™™ MALD™ TOF mass spectrometer (Bruker) and cycle, as reported for other ScORC mutants.71 73 peptide masses were compared with those predicted Such a phenotype would be compatible with a (FindPept and PeptideMass ) from the tryptic digestion of reduced frequency of origin firing due to a defective ScOrc4p and/or DnaK, allowing for missing cleavages. assembly of ScORC, thus being most of the genome Orphan peaks were classified as potential cross linked passively replicated from a reduced set of active peptide pairs and these were identified by comparing their origins. ™n fact, analysis of the replication of experimental masses with those calculated for any pair of episomal ARS1 in vivo by means of 2D gel tryptic peptides including at least an internal undigested electrophoresis showed that this usually very active Lys residue plus the 98.1 Da link from the reacted DSG. origin is fired at a lower frequency (intensity of the bubble arc) in the orc4-L185/186A mutants than in EM and single particle reconstruction of ScOrc4p-DnaK ORC4-WT (Fig. 6c). The first and, to some extent, the complex fourth hydrophobic residues in ScOrc4p ™L4 are conserved across the whole family of Orc1 5/Cdc6 The ScOrc4p DnaK complex33 was further purified to proteins (Supplementary Data Fig. S4), being near homogeneity by passage through a gel filtration compatible with their location in our model in column (GE Healthcare Sephacryl S 200 HR, length 62 cm, diameter 2.6 cm) equilibrated at 4 °C in 0.5 M the packing core of ± helix 5. The functionally KCl, 0.02 M Hepes KOH (pH 7.1), 0.01 M MgCl2, 5% (v/v) relevant second and third residues in ™L4 are glycerol. Samples (1 ml) were injected into the top of the predicted to be solvent exposed: their variable column and eluted at a flow rate of 0.5 ml/min. A major nature would thus reflect the specific pairwise peak at H"200 kDa, the mass expected for a ScOrc4p2 interactions established, in a given species, by each DnaK complex, was collected, checked by SDS PAGE and subunit in ORC. stored at  70 °C. A few microliters of this preparation ™n summary, we have found a novel role for the were adsorbed onto glow discharged, carbon coated grids conserved ™SM stretch in the AAA+ domain of a and negatively stained with 1% (w/v) uranyl acetate. The eukaryotic ORC subunit (ScOrc4p), besides contrib grids were observed in a JEOL 1230 instrument operated uting to DNA binding:38 through recognition of the at 100 kV and micrographs were taken under low dose conditions at a magnification of 50,000×. The micrographs hydrophobic core (™L4) in that motif, Hsp70 chaper were digitized with a ZE™SS scanner to a final sampling ones could act as modulators of the ordered resolution of 2.8 Å/pixel. Particles were extracted from the assembly and stoichiometry of ORC subunits. Our micrographs using XM™PP software.74 A total of 6945 work is compatible with a scenario in which particles were classified in 16 classes using maximum chaperones, present in most of the complexes likelihood approaches75 and the best 12 classes in terms of characterized by proteomic analyses,29,30,61 might signal to noise ratio were used to generate a first volume be general co factors in the assembly of functional by a common lines approach. This initial model was macromolecular machines. filtered at very low resolution (50 Å) to provide a template and confronted with the data set using angular refinement methods implemented in EMAN.76 When the volume obtained after several iterations was stable, the 3298 Materials and Methods images that showed the best correlation coefficient were selected and subjected to a further refinement using the SP™DER package.77 The resolution of the final structure of Proteomic analysis of ScOrc4p-DnaK (Hsp70) the ScOrc4p2 DnaK complex (for an assessment of its interaction quality, see Supplementary Data Fig. S5) was estimated by Fourier shell correlation to be 22 Å (using the 0.3 ScOrc4p was expressed in E. coli and purified in cross correlation coefficient criteria; Supplementary Data complex with DnaK as described.33 Six 20 ¼l aliquots of Fig. S5b). The atomic structures of the nucleotide binding the complex (20 ¼M) in 0.2 M K2HPO4/KH2PO4 (pH 7) (PDB ™D 2V7Y) and substrate binding (PDB ™D 1DKX) were incubated with 0.6 ¼l of a 50 mM solution (in DMSO) domains of Hsp70 were docked manually, together with of disuccinimidyl glutarate (DSG; Pierce) for 30 min at two copies of an atomic model of ScOrc4p (Fig. 2a), into room temperature. Reactions were stopped with 2 ¼l of the 3D reconstruction of the complex using UCSF 1 M Tris HCl (pH 8) and then immediately analyzed by Chimera.78 ScOrc4p was modeled on its archaeal homo SDS PAGE (10% polyacrylamide gels) and staining with logue Pyrobaculm aerophilum Orc1/Cdc6 (PDB ™D 1FNN),36 Coomassie brilliant blue. Bands were excised, dehydrated by means of the Swiss Model server! ,79 starting from a with acetonitrile and then immersed in 100 ¼l of 25 mM pairwise sequence alignment of both proteins.44 Because NH4HCO3 (pH 8.9) with 0.02 unit of bovine trypsin and the yeast protein is larger that its archaeal counterpart, kept in ice for 45 min. Proteolysis in situ was achieved, sequence stretches in Orc4p aligning with gaps in after removing the excess of trypsin, by digestion PaCdc6/Orc1 (N39 D77, G159 G175, R195 R204 and overnight at 37 °C. Peptides were eluted by extraction N368 A398), due to their structural uncertainty, were with 0.5% (w/v) trifluoroacetic acid in 50% (v/v) deleted from the PDB file before docking. acetonitrile and then acetonitrile. Mass spectra of the peptides in the extracted fractions were acquired by mixing 0.5 ¼l of each sample with 0.1% trifluoroacetic acid in a saturated matrix solution of ± cyano 4 hydroxycyn http://www.expasy.org/tools/ namic acid in acetonitrile. Samples were analyzed in a ! http://swissmodel.expasy.org 36 Dissecting Orc4-Orc2/Hsp70 Interactiáns Mutagenesis of ORC4-IL4 and co-expression of alone) were affinity purified as described above, but using ScORC subunits in E. cáli a Hi Trap Chelating 5 ml cartridge and eluting the bound proteins in a single step with 0.5 M KCl, 0.4 M imidazole (pH 8). Peak fractions, containing the ScORC subunits and The expression vector pRGrectac ORC4 (Supplementary DnaK, were concentrated to 4 ml (Amicon, 50 K cutoff Data Fig. S3a) was used as the template for replacing the filter) and then 500 ¼l were injected into a Superdex 200 sequence coding for ™L4 in ScOrc4p by A5 (Supplementary (HR 10/30) column, coupled to an AKTA basic 10 FPLC, Data, Materials and Methods). The other ORC genes were at 0.4 ml/min in 1 M KCl, 0.025 M Hepes KOH (pH 7.5). PCR amplified and independently cloned into the vector ™dentical fractions from two independent column runs to form bipartite operons (Supplementary Data Fig. S3b). were pooled and proteins were then precipitated with Protein expression was performed in E. coli BL21 cells silica gel as described above. Pellets were suspended in carrying pR™L3, a pR™L (Stratagene) derivative with the 50 ¼l of SDS PAGE loading buffer and electrophoresis was genes of Lac™q repressor and T7 lysozyme cloned. Cells done in duplicate, with 25 ¼l samples, in 7.5% polyacryl were grown at 29 °C in 0.5 l of 2×TY medium, plus amide gels at 150 V for 1.75 h. One gel was stained with 0.1 mg/ml ampicillin, to A600 H"0.5, when expression was Coomassie brilliant blue and the other gel was Western achieved by adding ™PTG to 0.25 mM final concentration blotted as described above. for 3 h. Cells were harvested, washed with 0.9% (w/v) NaCl and resuspended in 10 ml of 1 M NaCl, 50 mM imidazole (pH 8), 0.5% (w/v) Brij 58, 1 mM p aminobenzamidine, Analysis of ORC4-IL4 mutants in vivá 10% (v/v) glycerol, plus one tablet of EDTA free protease inhibitors cocktail (Roche). Lysis was achieved A complete account of the relevant protocols is given in by freezing at  70 °C and then thawing to 4 °C. Soluble Supplementary Data, Materials and Methods. ™n summary, and insoluble fractions were separated by ultracentrifu point mutations in the ScOrc4 ™L4 motif were introduced gation (30,000 rpm, Beckman 60Ti, 1 h at 4 °C). into the ScORC4 gene by PCR, cloned in a vector tailored to allow for allelic replacement by homologous recombina Affinity purification of ScOrc4p-IL4/A5 and tion of the genomic ScORC4 in a yeast haploid strain immunodetection of co-purified ScORC subunits (Supplementary Data Fig. S6). After sequencing the viable clones, cell growth of the selected mutants at different temperatures was checked by serial dilutions on YPAD Samples of each soluble lysate (2× 0.5 ml) were injected agar (Fig. 6a). Yeast strains were synchronized by arresting in a Ni2+ activated Hi Trap Chelating 1 ml column set up in G1 with ± factor and then, upon releasing blockage at in an ÅKTA basic 10 FPLC (GE Healthcare) and equili non permissive temperature, cell cycles were analyzed by brated in 0.5 M NaCl, 0.05 M imidazole HCl (pH 8). After FACS (Fig. 6b). ™n parallel, firing of an ARS1 origin located washing the column with four volumes of the same buffer, in an episome was analyzed in each mutant strain by bound His10 Orc4p was eluted with an eight volumes means of neutral/neutral 2D gel electrophoresis, plus gradient to 0.5 M NaCl, 0.4 M imidazole HCl (pH 8). Southern hybridization with plasmid specific sequences Fractions of 0.5 ml were collected and proteins were (Fig. 6c). precipitated adding 2 ¼l of a suspension of StrataClean silica gel (Stratagene). Pellets obtained by centrifugation at 15,700g for 1 min at 4 °C were resuspended in 60 ¼l of SDS PAGE loading buffer and electrophoresis was done in triplicate, with 20 ¼l of each sample, in 10% polyacryl amide gels at 150 V for 2.5 h. One gel series was stained Acknowledgements with Coomassie brilliant blue and the other two were Western blotted to PVDF membranes and then blocked as We are grateful to the members of the oligonucle described.33 The dilutions for the distinct primary otide synthesis, DNA sequencing and flow cytome antibodies (incubation for 2 h) were as follows: murine anti VSV (monoclonal P5D4, Sigma), anti cMyc (ascites try facilities at C™B CS™C for their technical support. 9E10, Sigma), anti HA (monoclonal 12CA5, Roche), anti Thanks are due to E. Lanka for the antiserum against T7 Flag (monoclonal M2, Sigma) and rabbit anti HSV DnaK. We are indebted to C. Gancedo, C.L. Flores, (polyclonal, Sigma): 1/1000 (total lysates) or 1/300 (FPLC R. Bermejo and J.A. Tercero for helpful discussions fractions); anti His (murine monoclonal, Sigma): 1/20,000; on the experiments with yeast in vivo. This work has anti DnaK (rabbit polyclonal): 1/10,000. The secondary been financed by Spanish M™C™NN (grants antibodies (HRP conjugated anti mouse/rabbit ™gG, 1 h BFU2006 00494 and B™O2009 06952) and CAM incubation) were used at 1/10,000 dilution and then (GR/SAL/0651/2004) to R.G. and M™C™NN chemiluminiscence detection was achieved using the ECL (BFU2007 62382) and the EU ( 3D repertoire Plus reagent (GE Healthcare). ™f required, membranes LSHG CT 2005 512028) to J.M.V. were re hybridized (at most twice) after being stripped in 0.1 M 2 MeEtOH, 2% (w/v) SDS, 0.062 M Tris HCl (pH 6.8), at 65 °C for 45 min and then re blocked. 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