Structural Analysis of the Interactions Between
Hsp70 Chaperones and the Yeast DNA Replication
Protein Orc4p

María Moreno-del Álamo

1

, Alicia Sánchez-Gorostiaga

1

,

Ana M. Serrano

1

, Alicia Prieto

2

, Jorge Cuéllar

3

, Jaime Martín-Benito

3

,

José M. Valpuesta

3

and Rafael Giraldo

1

⁎

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;
received in revised form
29 July 2010;
accepted 11 August 2010
Available online
21 August 2010

Edited by F. Schmid

Keywords:
DNA replication;
Hsp70 chaperones;
macromolecular assemblies;
origin recognition complex;
yeast

Hsp70 chaperones, besides their role in assisting protein folding, are key
modulators of protein disaggregation, being consistently found as
components of most macromolecular assemblies isolated in proteome-
wide affinity purifications. A wealth of structural information has been
recently acquired on Hsp70s complexed with Hsp40 and NEF co-factors
and with small hydrophobic target peptides. However, knowledge of how
Hsp70s recognize large protein substrates is still limited. Earlier, we
reported that homologue Hsp70 chaperones (DnaK in Escherichia coli and
Ssa1-4p/Ssb1-2p in Saccharomyces cerevisiae) bind strongly, both in vitro and
in vivo, to the AAA+ domain in the Orc4p subunit of yeast origin
recognition complex (ORC). ScORC is the paradigm for eukaryotic DNA
replication initiators and consists of six distinct protein subunits (ScOrc1p

–

ScOrc 6p). Here, we report that a hydrophobic sequence (IL

4

) in the initiator

specific motif (ISM) in Orc4p is the main target for DnaK/Hsp70. The three-
dimensional electron microscopy reconstruction of a stable Orc4p

2

–DnaK

complex suggests that the C-terminal substrate-binding domain in the
chaperone clamps the AAA+ IL

4

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 IL

4

selectively disrupts Orc4p interaction with

Orc2p. Allelic substitution of ORC4 by mutants in each residue of IL

4

results

in lethal (I184A) or thermosensitive (L185A and L186A) initiation-defective

*Corresponding author. E-mail address:

[e-mail]

.

Present address: A. Sánchez-Gorostiaga, Department of Microbial Biotechnology, Centro Nacional de Biotecnología

–

CSIC, 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; IL

4

, Ile-Leu-Leu-Leu-Leu; IMAC, immobilized metal

ion affinity chromatography; ISM, initiator specific motif; MS, mass spectrometry; MALDI-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.

doi:

10.1016/j.jmb.2010.08.022

J. Mol. Biol. (2010) 403, 24–39

Contents lists available at

www.sciencedirect.com

Journal of Molecular Biology

j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b

0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.

phenotypes in vivo. The interplay between Hsp70 chaperones and the
Orc4p-IL

4

motif might have an adaptor role in the sequential, stoichiometric

assembly of ScORC subunits.

© 2010 Elsevier Ltd. All rights reserved.

Introduction

Protein chaperones are macromolecular machines

that convert the energy derived from ATP binding
and hydrolysis into inter-domain movements, which
are transduced to the bound protein substrates
and results in their folding, assembly, disassembly/
disaggregation or proteolytic degradation.

1

–3

Hsp70s

are a phylogenetically ubiquitous class of chaper-
ones that are composed of two specialized domains,
allosterically coupled through an intermediate
linker: an N-terminal, actin-like, ATP/nucleotide-
binding domain (NBD)

4

and a C-terminal substrate-

binding domain (SBD).

5

The latter is composed of

a

β-sandwich, peptide ligand subdomain and an

α-helical lid subdomain. While Hsp70s hold the
central functional role, they act in concert with
two types of protein co-factors that (i) pre-target
suitable substrates, stimulating ATP hydrolysis at
Hsp70-NBD (Hsp40s or J-domain proteins);

6

and

(ii) foster the interchange of ADP by ATP upon
nucleotide hydrolysis (nucleotide exchange factors,
NEFs).

7

–9

The functional cycle of Hsp70s

10-15

starts

with closure of the lid subdomain in the SBD on a
bound hydrophobic peptide stretch, which togeth-
er with a Hsp40 co-factor triggers ATP hydrolysis
in the NBD and the relevant structural transac-
tions in the substrate. This is followed by
displacement of the residual ADP by an NEF
and opening of the SBD lid upon allosteric
binding of a new ATP molecule at NBD, which
releases the protein substrate and closes the cycle.
The limiting, slow step is closure of SBD on the
substrate, whereas this is in quick binding
equilibrium/exchange in the ATP form of the
chaperone. Mapping the interactions between
Hsp70s and large protein substrates with real
molecular detail has been made possible recently
through the combination of hydrogen/deuterium
exchange and mass spectrometry,

16

although a 3D

structure at atomic resolution is still missing.

After the initial association of Hsp70 chaperones

with the cellular response to heat stress

17

and the

proposal for a role in protein disaggregation,

18

pioneering biochemical studies were carried out in
the DNA replication field. The role of the Escherichia
coli Hsp70

–Hsp40–NEF triad (DnaK-DnaJ-GrpE) in

modulating the assembly of the bacteriophage

λ

replisome was revealed,

19

–21

which was followed by

studies on their action in plasmid replication.

22

–24

In

the latter case, Hsp70 chaperones are involved
specifically in the conformational remodeling of
RepA dimeric repressors to become active as DNA

replication initiator monomers.

25

The action of

Hsp70s on DNA replication proteins was then
extended to the assembly of functional viral repli-
cases in eukaryotes.

26

–28

In spite of this background

record, once it was found that a significant
proportion of the interactions described for the
proteome of Saccharomyces cerevisiae engaged
molecular chaperones, they were left aside as
non-specific,

29,30

although this does not necessarily

mean that interaction with chaperones is func-
tionally irrelevant.

Earlier, we found that dimers of the S. cerevisiae (Sc)

Orc4p protein (one of the six subunits (Orc1

–6) of

the origin recognition complex (ORC), the initiator
of chromosomal DNA replication in eukaryotes),

31,32

co-purified in E. coli tightly bound to DnaK, the
bacterial Hsp70 chaperone.

33

This heterologous

interaction was likely to be significant, because
immunoprecipitation of the Hsp70 chaperones
(DnaK homologues) in yeast also pulled-down
ScOrc4p. The target for DnaK was found to be the
N-terminal domain in ScOrc4p.

33

Structural studies

on proteins such as bacterial DnaA

34,35

and archaeal

Orc1-3,

36

–39

led to the conclusion that ORC-type

initiators are composed of an N-terminal AAA+
domain and a C-terminal winged helix (WH)
domain. The former is responsible for the ATP-
dependent remodeling of ORC upon binding to
origin (ARS) DNA,

40

which is led primarily by the

WH domain

38

in ScOrc1,2,4,5p.

41,42

In the original

report on Hsp70-ScOrc4p binding, we noted a
structural similarity between the C-terminal domain
in ScOrc4p and the N-terminal domain (WH1) of the
pPS10 plasmid initiator RepA.

33

This was confirmed

by the crystal structure of RepA-WH1

43

and extrap-

olated to other ORC subunits, on the basis of their
common ancestry with archaeal ORC proteins.

44

Furthermore, the structural similarities between
plasmid RepA and archaeal/eukaryal ORC proteins
extend beyond the WH fold to encompass additional

α-helical elements in RepA dimers, which undergo
conformational activation into

β-strands to become

replication-competent RepA monomers.

43,25

Hsp70s-

ScOrc4p binding appeared to mirror DnaK

–RepA

interactions and we speculated on a possible role for
Hsp70s in modulating the assembly of ORC
subunits.

33

Considerable effort has been devoted to unra-

veling the interactions between ORC subunits by
means of indirect in vivo assays, such as the
two-hybrid,

45,46

co-expression studies in insect

cells using baculoviruses,

41,47

–50

or co-translation

in vitro,

51

often followed by immunoprecipitation.

25

Dissecting Orc4-Orc2/Hsp70 Interactions

The emergent picture is not unique; differences are
evident depending on the biological source of the
proteins and the experimental approach chosen, but
it seems clear that Orc2,3,4,5 would constitute the
core of the complex with Orc1,6 at a peripheral
location. Sequential assembly of subunits has been
found for human ORC, in which binding of the
stable Orc2,3,5 core to Orc4 and Orc1 is ATP-
dependent.

50

In ScORC, the Orc1p and Orc5p

subunits bind ATP, but only Orc1p hydrolyzes
it.

52

–54

Orc4p contributes to a functional nucleotide

cycle

55

and, in higher eukaryotes, binds ATP.

50

Another factor essential for DNA replication, Cdc6,
shares these DNA and ATP-binding activities with
their ORC paralogues in building an active pre-
replicative complex.

42

Orc6p is structurally unre-

lated to the other subunits in the complex

42

and, as

Orc3p, does not bind to ARS DNA.

41

In spite of this

wealth of information, little is known about the
molecular details of the intersubunit contacts in
ORC. The crystal structures of oligomeric DnaA

34

and hetero-dimeric archaeal Orc1-3

38

point to two

α-helices located between the ATP-binding Walker
A and B motifs, a signature for the replication
initiator family among all AAA+ proteins.

56,57

Such

α-helices constitute an initiator-specific motif (ISM)
that has been proposed to modulate the assembly
of ORC subunits into an open ring/superhelix
that would partially encircle DNA.

58

Recent

electron microscopy (EM) 3D reconstructions have
sketched the arrangement of subunits in yeast

59

and Drosophila

60

ORC.

We report here that multiple yeast Hsp70

chaperones (Ssa1-4p and Ssb1/2p) bind to ScOrc4p
in vivo, coincident with earlier results from whole
cell analysis.

29,61

Using a stable, heterologous

complex between E. coli DnaK and ScOrc4p, we
identify, by combining cross-linking, proteolysis
and mass spectrometry (MS), the target for Hsp70
chaperones in ScOrc4p: a hydrophobic sequence
(IL

4

:

184

ILLLL

188

) that it is part of the ISM motif in

the AAA+ domain.

38

We show the 3D reconstruc-

tion of the ScOrc4p

2

–DnaK complex obtained by

single-particle EM methods. Docking of the model
for dimeric ScOrc4p and the atomic structure of the
two domains of DnaK is consistent with the SBD in
the latter interacting with IL

4

in one of the ScOrc4p

monomers. This defines a DnaK

–ScOrc4p interface

compatible with that found by cross-linking and
MS studies. This structure provides insight into
how Hsp70s dissociate compact protein assemblies.
We have developed vectors for co-expressing as
bipartite operons in E. coli ScOrc4p (either wild
type (WT) or a mutant in the sequence targeted by
Hsp70: IL

4

→A

5

) followed by each of the other

ScORC subunits (ScOrc1p, -2p, -3p and 5p) or
ScCdc6p. We have mapped the interactions estab-
lished by ScOrc4p in the binary complexes,
showing that ScOrc2p assembly is dependent on

a WT ISM-IL

4

motif in ScOrc4p, where it replaces

Hsp70. In vivo studies of point mutations in this
motif show that it is essential for DNA replication
in S. cerevisiae, I184A being lethal and L185/186A
thermosensitive ORC4 alleles. The thermosensitive
mutants exhibit, after a shift to non-permissive
temperature in G1-synchronized cultures, fewer
viable initiations and thus a prolonged S-phase and
a final arrest at the beginning of the next cycle. This
phenotype is compatible with a defective assembly
of the pre-replicative complex. Our data provide
support for a scenario in which Hsp70 chaperones
would act as modulators, through the recognition
of a conserved stretch in the pivotal ISM, of subunit
stoichiometry in the sequential assembly of ORC.

Results and Discussion

Proteomic identification of the target for Hsp70
in ScOrc4p

The structural principles for protein recognition

by Hsp70 chaperones

10

are the same across the

evolutionary tree, reflecting the extreme conserva-
tion of the residues responsible for target recogni-
tion at the SBD in both prokaryotic and eukaryotic
Hsp70s.

62,63

However, there is a single Hsp70 in

E. coli (DnaK) but 14 paralogues in S. cerevisiae,
which are functionally organized into networks
specialized in protecting proteins against stress or
in assisting their folding.

64

Up to nine yeast Hsp70s

(Ssa1p

–4p, Ssb1p and -2p, Sse1p and -2p and Ssz1p)

have a cytoplasmic/nuclear location and several of
them have been isolated consistently in complex
with each ScORC subunit in proteome-wide
analysis.

29,61

Actually, our own proteomic analysis

of the proteins co-purifying with histidine-tagged
ScOrc4p in S. cerevisiae confirms that Ssa1p and -2p
and Ssb1p and -2p Hps70 chaperones are physically
bound to such bait (

Supplementary Data Fig. S1

). It

follows that the ScOrc4p

–Hsp70 complexes have a

heterogeneous chaperone composition, making it
difficult to identify their contacting interfaces
unambiguously. In this work, therefore, we have
characterized the tight ScOrc4p

–DnaK complex,

which can be purified in large quantities upon
expression of the ScORC subunit in E. coli.

33

The ScOrc4p-DnaK complex was cross-linked

in vitro with dissucinimidyl glutarate (DSG), a
bifunctional reagent specific for free amino groups,
before resolving the cross-linked species from the
unreacted proteins by SDS-PAGE (

Fig. 1

a). Up to six

discrete oligomeric bands with a slower electropho-
retic mobility than those of the protein monomers
were visualized. We focused our study on the fourth
of these bands because its mass (120

–140 kDa) was

compatible with homo/hetero-dimers of the two

26

Dissecting Orc4-Orc2/Hsp70 Interactions

component proteins of the complex. After trypsin
digestion in the gel, the resulting peptides were
analyzed by MALDI-TOF mass spectrometry

(

Fig. 1

b). Most of the tryptic peptides could be

assigned unambiguously to ScOrc4p or DnaK,
whereas four orphan peaks were identified as

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 (

⁎) mark the homo/hetero-dimeric complexes relevant

for this work. (b) MALDI-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

).

27

Dissecting Orc4-Orc2/Hsp70 Interactions

peptides paired through a DSG link (

Fig. 1

c).

Because of the monomeric nature of Hsp70s, the
DSG1 peak likely arises from an intermolecular
bridge between two neighbor DnaK molecules
bound to a ScOrc4p oligomer (for such oligomeric
assemblies, see

Fig. 5

c below). Interestingly, one of

the DnaK peptides involved (

489

DKNSGK

494

) is

located at the entrance of the binding pocket in the
C-terminal SBD of the chaperone.

5

Because

ScOrc4p:DnaK stoichiometry approaches 2:1 in the
complexes

33

(

Figs. 1

,

3 and 5

), DSG2 would

correspond to an intermolecular cross-link between
the AAA+ and WH domains from each protein
chain in ScOrc4p dimers.

33

This is compatible with a

head-to-tail arrangement for the two ScOrc4p sub-
units in the complex. The two most relevant peaks
are DSG3 and DSG4, because they correspond to
ScOrc4p-DnaK adducts. The former was identified
as a cross-link between the gatekeeper peptide of
DnaK and a WH sequence (

418

MIKAINSR

425

) that,

in a model for ScOrc4p based on its homologue
Orc1/Cdc6 of the archaea Pyrobaculum aerophilum

36

(

Fig. 2

a), is structurally equivalent to the C-terminus

of the second

α-helix in the WH1 domain of

pPS10-RepA.

43

It is significant that, for the homo-

logue initiators P1-RepA

65

and F-RepE,

66

such a

region in WH1 had been identified as a major target
for DnaK. However, we did not co-purify a com-
plex between the isolated ScOrc4p-WH domain and
DnaK in an earlier study.

33

This fact could indicate

low stability or a high off-rate (

Fig. 2

b) for the

WH

–chaperone complex, which can be detected only

after covalent cross-linking of the two proteins. The
DSG4 peak corresponds to an adduct between a
C-terminal peptide in DnaK (

635

DKK

637

), also close to

the substrate-binding pocket, and a sequence in the
AAA+ domain of ScOrc4p (

184

ILLLLDSTTKTR

195

)

predicted to be solvent-exposed in an

α-helix

(

Fig. 2

a).

We explored the ScOrc4p sequence with an

algorithm designed for the detection of potential
DnaK-binding sites in proteins (hydrophobic
stretches flanked by basic residues).

67

We found

multiple target peptides for DnaK (

Fig. 2

b), of which

that having the lowest predicted variation in free
energy of binding (

ΔΔG=–16.37 kJ/mol) corre-

sponds to

181

FEKILLLLDSTTK

193

: it overlaps with

the peptide found in the DSG4 peak (

Fig. 1

c), thus

pointing to the IL

4

sequence as the major binding

site for Hsp70 in ScOrc4p.

Three-dimensional electron microscopy
reconstruction of the Hsp70 (DnaK)-ScOrc4p
complex

In order to characterize the interaction between

ScOrc4p and DnaK in more detail, EM studies
were done with negatively stained samples
corresponding to the major peak eluting from a

gel-filtration column (see below,

Fig. 5

), compatible

with a ScOrc4p

2

–DnaK hetero-trimeric complex.

This additional purification step reduced the hetero-
geneity found in the oligomers initially isolated
by means of Ni

2+

-immobilized metal ion affinity

chromatography (IMAC).

The 3D reconstruction obtained from the complex

revealed a bi-lobed, figure-of-eight structure with a
small additional mass breaking an apparent 2-fold
symmetry (

Fig. 3

and

Supplementary Data Fig. S5

).

The region corresponding to the observed extra
density allowed the fitting of a single copy of each of
the NBD and the SBD of a Hsp70 chaperone. Two
copies of the atomic model of ScOrc4p (

Fig. 2

a) were

manually docked into the reconstructed volume
(

Fig. 3

), with their best fit obtained when the two

ScOrc4p molecules were placed in a head-to-tail
orientation. This arrangement of ScOrc4p subunits is
compatible with the cross-linking results (DSG2;

Fig.

1

) and with the crystal packing found for two

orthologue archaeal ORC proteins solved in the
absence of ligand DNA.

36,37

However, it differs from

the side-by-side association of AAA+ domains
typical for this protein family when assembled as
hexameric rings

57

or spirals,

58

or as DNA-bound

dimers.

38

This suggests an active role for Hsp70

chaperones in generating ScOrc4p monomers suit-
able for their subsequent assembly into functional
ScORC hetero-hexamers.

With the uncertainty intrinsic to the low resolution

(22 Å;

Materials and Methods

) of the reconstructed

volume for the ScOrc4p

2

-DnaK complex (

Fig. 3

), the

docked model has the IL

4

stretch in one of the two

AAA+ domains sandwiched in a canonical way
between the

α-helical (lid) and the β-sheet sub-

domains in the SBD of DnaK.

5,10,68

The lid appears

to act on the ScOrc4p dimer as a wedge, separating
the previously interacting AAA+ and WH domains.
Obviously, both domains still interact at the
opposite edge of the head-to-tail oriented ScOrc4p
dimer: if this were also bound by another molecule
of the chaperone, the ScOrc4p

2

–DnaK complex

would be dissociated immediately into two
ScOrc4p

–DnaK heterodimers. Because the latter

are not the most abundant purified species, it
follows that the levels of the Hsp70 chaperone
must be limiting due to an excess of the recombinant
protein (a hypothesis confirmed below; see

Fig. 5

c).

The docked model presented here fits quite well in

the volume of the ScOrc4p

2

–DnaK complex, while

the remaining empty regions could be attributed to
the protein portions missing from the atomic model
of ScOrc4p (broken red lines in

Fig. 2

a) and to a

detached conformation of the WH domain

37

that

becomes freed due to the binding of DnaK-SBD to
the AAA+ domain of the neighbor ScOrc4p subunit.
The reconstructed ScOrc4p

2

–DnaK complex sug-

gests a general mechanism for Hsp70s chaperones in
disassembling compact protein complexes and

28

Dissecting Orc4-Orc2/Hsp70 Interactions

aggregates, fuelled by allosteric binding/hydrolysis
of ATP at the NBD: (i) the SBD would bind to any
accessible hydrophobic sequence stretch found at

one of the contacting interfaces; (ii) closure of the lid
would introduce a wedge, breaking a few inter-
molecular contacts, thus destabilizing the aggregate;

Fig. 2 (legend on next page)

29

Dissecting Orc4-Orc2/Hsp70 Interactions

and (iii) the bound protein substrate would be
finally pulled apart by a large interdomain motion.

Our current structural analysis of the ScOrc4p

2

–

DnaK particles most likely corresponds to the stable
ADP and substrate-bound state in the chaperone
cycle.

10,33

A key issue still under debate is the

relation between both chaperone domains in sub-
strate and nucleotide-bound Hsp70s.

11

–15

In our

model (

Fig. 3

), the two chaperone domains are

arranged in a nearly orthogonal orientation and
wrapped around the tip of one of the two AAA+
domains in the ScOrc4p dimer. Such an orientation,
placing the IA subdomain in the NBD close to the

β-sheet in the SBD, is in agreement with the recently
solved NMR structure for the ADP and peptide-
bound state of DnaK.

68

The ScOrc4p

2

–DnaK struc-

ture is the second solved of a complex between a
complete Hsp70 chaperone and a full-length protein
substrate, rather than bound to a small hydrophobic
peptide, after the recently reported cryo-EM recon-
struction of a complex between bovine Hsc70 and a
clathrin coat, also in the ADP-bound state.

69

At a

similar resolution to that achieved here for the
ScOrc4p

2

–DnaK complex, a single Hsc70 molecule

was found bound to just one of the three hydro-
phobic peptide tails (C-terminal exposed QLMT)
converging at the vertex of each clathrin triskelion.
In this case, Hsc70 binding seems to stabilize a
strained conformation of the triskelion, leading to
disassembly of the coat.

69

A map for the interactions mediated by
ScOrc4p-ISM-IL

4

The peptide target for Hsp70 in the AAA+ domain

of ScOrc4p corresponds to the C-terminal

α-helix of

ISM, a motif described to contribute to DNA
recognition and proposed to be involved in the
assembly of initiator oligomers.

38

To study further

the role of the ISM-IL

4

motif in ScORC assembly, we

mutated its five large hydrophobic residues to Ala
(IL

4

→A

5

) to impair Hsp70 binding to this sequence

(

Fig. 2

b, estimated

ΔΔG=4.11 kJ/mol). We then

expressed both variants of ScOrc4p (WT-IL

4

and

mutant-A

5

) in E. coli and purified (

Supplementary

Data Fig. S2a

, inset), checking by means of circular

dichroism spectroscopy that the mutations had not
altered the structure or folding stability of the
protein (

Supplementary Data Fig. S2a and b

).

However, the inspection of the gel-filtration chro-

matography elution profiles for ScOrc4p-IL

4

/A

5

(

Supplementary Data Fig. S2c

) indicated a lower

tendency towards aggregation for the latter, as
expected from the more reduced hydrophobicity of
the mutant.

The expression vectors for ScOrc4p-IL

4

/A

5

were then engineered to clone, downstream of
ORC4, each one of the other ScORC core subunits
(ORC1, -2, -3 and -5) or ScCdc6p (CDC6) as bipartite
operons (

Supplementary Data Fig. S3a

). Subunits

were cloned with distinct N or C-terminal peptide
tags (

Supplementary Data Fig. S3b

) in order to

allow for immunodetection without compromising
their assembly.

70

Induced bacterial cells were lysed

and His10-tagged ScOrc4p-IL

4

(or A

5

) present in

the soluble fraction was then purified by means of
Ni

2+

-IMAC. The presence of each companion

ScORC subunit and the balance with DnaK
chaperone were determined by means of Western
blotting with specific antibodies (

Fig. 4

). ScOrc3p

failed to co-purify with ScOrc4p, independent of
the presence of the IL

4

→A

5

mutation (

Fig. 4

d).

ScCdc6p was proteolyzed to a large extent (not
shown) and thus it barely co-localized with ScOrc4p
(

Fig. 4

f). ScOrc2p was the only subunit clearly

showing a differential association with ScOrc4p
dependent on the presence of a WT-IL

4

motif

(

Fig. 4

c). Because ScOrc4p was expressed to a very

high level in all constructs, in a large excess over
any other downstream subunit, this ensures that
the mutations in ScOrc4p-A

5

severely disrupt

binding to ScOrc2p. The other subunits (ScOrc1p
and -5p;

Fig. 4

b and e, respectively) interacted

strongly with ScOrc4p, as reported for two-hybrid
assays,

46

independent of the presence of a WT or

mutant IL

4

motif. This clearly points to the

existence of additional/alternative interfaces to
IL

4

for contacting with other ScORC subunits.

By showing that ScOrc4p interacts with ScOrc2p
but it does not with ScOrc3p, our results solve
an ambiguity in the current models for ORC
structure.

59,60

Regarding DnaK association with each binary

assembly, except for ScOrc4-1p (

Fig. 4

b), there is a

net reduction in the level of bound chaperone when
the ScOrc4p-A

5

mutant is present. This suggests a

role for Hsp70 chaperones in shielding solvent-
exposed hydrophobic motifs, such as IL

4

, until they

are engaged in specific contacts between subunits
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 ID 1FNN).

36

Its 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. 1

c) 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,

ΔΔG, on top. The A

5

mutations (in red) replacing the IL

4

stretch cause a drastic increase in

ΔΔG. A sequence in

the WH domain with a predicted low affinity, but yet found cross-linked to DnaK (

Fig. 1

c), is also boxed (cyan).

30

Dissecting Orc4-Orc2/Hsp70 Interactions

Fig. 3. The EM 3D reconstruction of the DnaK (Hsp70)–ScOrc4p

2

complex is compatible with the biochemically identified intermolecular contacts. (a) Several views

of the volume of the ScOrc4p

2

–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 IL

4

motif in the AAA+ domain in

one of the two ScOrc4p subunits (cyan). IL

4

residues seem to be gripped in between the two subdomains (

β-sandwich and α-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.

31

Dissecting

Orc4-Orc2/Hsp70

Interactions

Fig. 4. Mapping subunit interactions made by ScOrc4p-IL

4

/A

5

. (a) Ni

2+

-IMAC elution profiles for His10-tagged ScOrc4p-IL

4

(WT, blue) and mutant A

5

(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 (I

–III) from the second, specific elution peak are also shown. Arrowheads point to ScOrc4p and asterisks (⁎) mark the co-purified subunits. Western blots for

detection of the specific tags for each companion subunit ScOrc4p (anti/

α-His) and DnaK (anti/α-DnaK) are displayed below the gels.

32

Dissecting

Orc4-Orc2/Hsp70

Interactions

potential binding sites for DnaK in ScOrc4p (

Fig.

2

b), it is not thus strange that the IL

4

→ A

5

mutations do not completely abolish, but reduce
chaperone interaction.

The complex between ScOrc4p and ScOrc2p was

found to be the most sensitive to the mutations in
the ISM-IL

4

motif (see above) and was studied in

greater detail. The Ni

2+

-IMAC-purified complex,

including DnaK (

Fig. 4

c), was analyzed in terms of

its hydrodynamic stability and subunit stoichiom-
etry by means of gel-filtration chromatography (

Fig.

5

a) plus immunodetection of the proteins in the

eluted fraction peaks (

Fig. 5

b). Besides large

oligomeric ScOrc4

–ScOrc2p assemblies eluting

with the void volume, fractions distributed across
the sizing column were identified as hetero-tetra-
meric ScOrc4p

2

–ScOrc2p

2

and hetero-trimeric

ScOrc4p

2

–DnaK complexes and, finally, a vast

excess of monomeric DnaK. It is noteworthy that
the chaperone was marginally present in the largest
molecular mass assemblies. On the contrary, when
ScOrc4p was over-expressed in the absence of any
other ScORC subunit (

Fig. 5

a and c), it was found

in large assemblies with DnaK (

Fig. 5

b), titrating

most of the available chaperone. The release of
DnaK from the ScOrc4p

2

–ScOrc2p

2

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-IL

4

lead to an

initiation defective phenotype

Once it was found that the ISM-IL

4

sequence in

ScOrc4p, targeted by Hsp70s, is involved in binding
to ScOrc2p, we surveyed the effect of the disrupting
IL

4

→A

5

mutations on yeast DNA replication in

vivo. In S. 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 (V

0

) and a ScOrc4p

2

-

ScOrc2p

2

hetero-tetramer (ca 270 kDa), thus generating a

fraction of free DnaK monomers (70 kDa).

33

Dissecting Orc4-Orc2/Hsp70 Interactions

an indication of its lethality. Therefore, we tried to
substitute each hydrophobic residue individually in
the motif by Ala (

Supplementary Data Fig. S6

). We

could not replace the first residue (I184) either,
underlining the essential role of the motif. On the
contrary, the last two leucines (L187 and L188) can
be mutated with no apparent effect on yeast growth.
Interestingly, L185A and L186A resulted in thermo-
sensitive growth (

Fig. 6

a), being among the very few

mutations with such phenotype described so far in
ORC. We studied the cell cycles of ORC4-WT and
orc4-L185/186A mutant yeast, synchronized in G1,
after releasing their arrest in combination with a
shift to non-permissive temperature (

Fig. 6

b). Both

ISM-IL

4

mutants exhibit a marked delay in their cell

cycles, with a prolonged S-phase and an apparent
arrest in G2/M, when cells accumulate a 2C DNA
content. They fail to efficiently enter into a new

Fig. 6. Mutation of the ISM-IL

4

motif in ScOrc4p results in defective replication in vivo. (a) Growth in YPAD-agar of

serial dilutions (5

μl, 10

5

–10

1

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 BglII-digested bulk DNA from ORC4-WT and orc4-L185/186A cells, carrying an ARS1 episome, grown as described for
b. Hybridization with a

32

P-labeled plasmid-specific DNA fragment shows a reduced frequency for origin firing (arrows:

bubble arc) in the mutants.

34

Dissecting Orc4-Orc2/Hsp70 Interactions

cycle, as reported for other ScORC mutants.

71

–73

Such a phenotype would be compatible with a
reduced frequency of origin firing due to a defective
assembly of ScORC, thus being most of the genome
passively replicated from a reduced set of active
origins. In fact, analysis of the replication of
episomal ARS1 in vivo by means of 2D gel
electrophoresis showed that this usually very active
origin is fired at a lower frequency (intensity of the
bubble arc) in the orc4-L185/186A mutants than in
ORC4-WT (

Fig. 6

c). The first and, to some extent, the

fourth hydrophobic residues in ScOrc4p-IL

4

are

conserved across the whole family of Orc1-5/Cdc6
proteins (

Supplementary Data Fig. S4

), being

compatible with their location in our model in
the packing core of

α-helix 5. The functionally

relevant second and third residues in IL

4

are

predicted to be solvent-exposed: their variable
nature would thus reflect the specific pairwise
interactions established, in a given species, by each
subunit in ORC.

In summary, we have found a novel role for the

conserved ISM stretch in the AAA+ domain of a
eukaryotic ORC subunit (ScOrc4p), besides contrib-
uting to DNA binding:

38

through recognition of the

hydrophobic core (IL

4

) in that motif, Hsp70 chaper-

ones could act as modulators of the ordered
assembly and stoichiometry of ORC subunits. Our
work is compatible with a scenario in which
chaperones, present in most of the complexes
characterized by proteomic analyses,

29,30,61

might

be general co-factors in the assembly of functional
macromolecular machines.

Materials and Methods

Proteomic analysis of ScOrc4p-DnaK (Hsp70)
interaction

ScOrc4p was expressed in E. coli and purified in

complex with DnaK as described.

33

Six 20

μl aliquots of

the complex (20

μM) in 0.2 M K

2

HPO

4

/KH

2

PO

4

(pH 7)

were incubated with 0.6

μl of a 50 mM solution (in DMSO)

of disuccinimidyl glutarate (DSG; Pierce) for 30 min at
room temperature. Reactions were stopped with 2

μl of

1 M Tris

–HCl (pH 8) and then immediately analyzed by

SDS-PAGE (10% polyacrylamide gels) and staining with
Coomassie brilliant blue. Bands were excised, dehydrated
with acetonitrile and then immersed in 100

μl of 25 mM

NH

4

HCO

3

(pH 8.9) with 0.02 unit of bovine trypsin and

kept in ice for 45 min. Proteolysis in situ was achieved,
after removing the excess of trypsin, by digestion
overnight at 37 °C. Peptides were eluted by extraction
with 0.5% (w/v) trifluoroacetic acid in 50% (v/v)
acetonitrile and then acetonitrile. Mass spectra of the
peptides in the extracted fractions were acquired by
mixing 0.5

μl of each sample with 0.1% trifluoroacetic acid

in a saturated matrix solution of

α-cyano-4-hydroxycyn-

namic acid in acetonitrile. Samples were analyzed in a

Biflex-III MALDI-TOF mass spectrometer (Bruker) and
peptide masses were compared with those predicted
(FindPept and PeptideMass

†

) from the tryptic digestion of

ScOrc4p and/or DnaK, allowing for missing cleavages.
Orphan peaks were classified as potential cross-linked
peptide pairs and these were identified by comparing their
experimental masses with those calculated for any pair of
tryptic peptides including at least an internal undigested
Lys residue plus the 98.1 Da link from the reacted DSG.

EM and single particle reconstruction of ScOrc4p-DnaK
complex

The ScOrc4p

–DnaK complex

33

was further purified to

near-homogeneity by passage through a gel-filtration
column (GE Healthcare Sephacryl S-200 HR, length
62 cm, diameter 2.6 cm) equilibrated at 4 °C in 0.5 M
KCl, 0.02 M Hepes

–KOH (pH 7.1), 0.01 M MgCl

2

, 5% (v/v)

glycerol. Samples (1 ml) were injected into the top of the
column and eluted at a flow rate of 0.5 ml/min. A major
peak at

≈200 kDa, the mass expected for a ScOrc4p

2

–

DnaK complex, was collected, checked by SDS-PAGE and
stored at

–70 °C. A few microliters of this preparation

were adsorbed onto glow-discharged, carbon-coated grids
and negatively stained with 1% (w/v) uranyl acetate. The
grids were observed in a JEOL 1230 instrument operated
at 100 kV and micrographs were taken under low-dose
conditions at a magnification of 50,000×. The micrographs
were digitized with a ZEISS scanner to a final sampling
resolution of 2.8 Å/pixel. Particles were extracted from the
micrographs using XMIPP software.

74

A total of 6945

particles were classified in 16 classes using maximum
likelihood approaches

75

and the best 12 classes in terms of

signal-to-noise ratio were used to generate a first volume
by a common lines approach. This initial model was
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
images that showed the best correlation coefficient were
selected and subjected to a further refinement using the
SPIDER package.

77

The resolution of the final structure of

the ScOrc4p

2

–DnaK complex (for an assessment of its

quality, see

Supplementary Data Fig. S5

) was estimated

by Fourier shell correlation to be 22 Å (using the 0.3
cross-correlation coefficient criteria;

Supplementary Data

Fig. S5b

). The atomic structures of the nucleotide-binding

(PDB ID 2V7Y) and substrate-binding (PDB ID 1DKX)
domains of Hsp70 were docked manually, together with
two copies of an atomic model of ScOrc4p (

Fig. 2

a), into

the 3D reconstruction of the complex using UCSF
Chimera.

78

ScOrc4p was modeled on its archaeal homo-

logue Pyrobaculm aerophilum Orc1/Cdc6 (PDB ID 1FNN),

36

by means of the Swiss-Model server

‡

,

79

starting from a

pairwise sequence alignment of both proteins.

44

Because

the yeast protein is larger that its archaeal counterpart,
sequence stretches in Orc4p aligning with gaps in
PaCdc6/Orc1 (N39

–D77, G159–G175, R195–R204 and

N368

–A398), due to their structural uncertainty, were

deleted from the PDB file before docking.

†

http://www.expasy.org/tools/

‡

http://swissmodel.expasy.org

35

Dissecting Orc4-Orc2/Hsp70 Interactions

Mutagenesis of

ORC4-IL

4

and co-expression of

ScORC subunits in

E. coli

The expression vector pRGrectac-ORC4 (

Supplementary

Data Fig. S3a

) was used as the template for replacing the

sequence coding for IL

4

in ScOrc4p by A

5

(

Supplementary

Data

,

Materials and Methods

). The other ORC genes were

PCR-amplified and independently cloned into the vector
to form bipartite operons (

Supplementary Data Fig. S3b

).

Protein expression was performed in E. coli BL21 cells
carrying pRIL3, a pRIL (Stratagene) derivative with the
genes of LacI

q

repressor and T7 lysozyme cloned. Cells

were grown at 29 °C in 0.5 l of 2 × TY medium, plus
0.1 mg/ml ampicillin, to A

600

≈0.5, when expression was

achieved by adding IPTG to 0.25 mM final concentration
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,
10% (v/v) glycerol, plus one tablet of EDTA-free
protease inhibitors cocktail (Roche). Lysis was achieved
by freezing at

–70 °C and then thawing to 4 °C. Soluble

and insoluble fractions were separated by ultracentrifu-
gation (30,000 rpm, Beckman 60Ti, 1 h at 4 °C).

Affinity purification of ScOrc4p-IL

4

/A

5

and

immunodetection of co-purified ScORC subunits

Samples of each soluble lysate (2 × 0.5 ml) were injected

in a Ni

2+

-activated Hi-Trap Chelating 1 ml column set-up

in an ÅKTA basic-10 FPLC (GE Healthcare) and equili-
brated in 0.5 M NaCl, 0.05 M imidazole-HCl (pH 8). After
washing the column with four volumes of the same buffer,
bound His10-Orc4p was eluted with an eight volumes
gradient to 0.5 M NaCl, 0.4 M imidazole-HCl (pH 8).
Fractions of 0.5 ml were collected and proteins were
precipitated adding 2

μl 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

μl of SDS-

PAGE loading buffer and electrophoresis was done in
triplicate, with 20

μl of each sample, in 10% polyacryl-

amide gels at 150 V for 2.5 h. One gel series was stained
with Coomassie brilliant blue and the other two were
Western-blotted to PVDF membranes and then blocked as
described.

33

The dilutions for the distinct primary

antibodies (incubation for 2 h) were as follows: murine
anti-VSV (monoclonal P5D4, Sigma), anti-cMyc (ascites
9E10, Sigma), anti-HA (monoclonal 12CA5, Roche), anti-
T7-Flag (monoclonal M2, Sigma) and rabbit anti-HSV
(polyclonal, Sigma): 1/1000 (total lysates) or 1/300 (FPLC
fractions); anti-His (murine monoclonal, Sigma): 1/20,000;
anti-DnaK (rabbit polyclonal): 1/10,000. The secondary
antibodies (HRP-conjugated anti-mouse/rabbit IgG, 1 h
incubation) were used at 1/10,000 dilution and then
chemiluminiscence detection was achieved using the ECL-
Plus reagent (GE Healthcare). If required, membranes
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.

Gel filtration of ScOrc4 + 2p/DnaK complexes

Soluble extracts from 2.5 l cultures of E. coli cells co-

expressing His10-ScOrc4p and ScOrc2p (or His10-ScOrc4p

alone) were affinity-purified as described above, but using
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
DnaK, were concentrated to 4 ml (Amicon, 50 K cutoff
filter) and then 500

μl were injected into a Superdex-200

(HR-10/30) column, coupled to an AKTA basic-10 FPLC,
at 0.4 ml/min in 1 M KCl, 0.025 M Hepes

–KOH (pH 7.5).

Identical fractions from two independent column runs
were pooled and proteins were then precipitated with
silica gel as described above. Pellets were suspended in
50

μl of SDS-PAGE loading buffer and electrophoresis was

done in duplicate, with 25

μl samples, in 7.5% polyacryl-

amide gels at 150 V for 1.75 h. One gel was stained with
Coomassie brilliant blue and the other gel was Western-
blotted as described above.

Analysis of

ORC4-IL

4

mutants

in vivo

A complete account of the relevant protocols is given in

Supplementary Data

,

Materials and Methods

. In summary,

point mutations in the ScOrc4-IL

4

motif were introduced

into the ScORC4 gene by PCR, cloned in a vector tailored to
allow for allelic replacement by homologous recombina-
tion of the genomic ScORC4 in a yeast haploid strain
(

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
agar (

Fig. 6

a). Yeast strains were synchronized by arresting

in G1 with

α-factor and then, upon releasing blockage at

non-permissive temperature, cell cycles were analyzed by
FACS (

Fig. 6

b). In parallel, firing of an ARS1 origin located

in an episome was analyzed in each mutant strain by
means of neutral/neutral 2D gel electrophoresis, plus
Southern hybridization with plasmid-specific sequences
(

Fig. 6

c).

Acknowledgements

We are grateful to the members of the oligonucle-

otide synthesis, DNA sequencing and flow cytome-
try facilities at CIB-CSIC for their technical support.
Thanks are due to E. Lanka for the antiserum against
DnaK. We are indebted to C. Gancedo, C.L. Flores,
R. Bermejo and J.A. Tercero for helpful discussions
on the experiments with yeast in vivo. This work has
been financed by Spanish MICINN (grants
BFU2006-00494 and BIO2009-06952) and CAM
(GR/SAL/0651/2004) to R.G. and MICINN
(BFU2007-62382) and the EU (

“3D repertoire”

LSHG-CT-2005-512028) to J.M.V.

Supplementary Data

Supplementary data associated with this article

can be found, in the online version, at

doi:10.1016/

j.jmb.2010.08.022

36

Dissecting Orc4-Orc2/Hsp70 Interactions

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