Eur. J. Biochem. 271, 439–449 (2004) FEBS 2004
doi:10.1046/j.1432-1033.2003.03944.x
Crystal structure of the chi:psi subassembly of the Escherichia coli
DNA polymerase clamp-loader complex
Jacqueline M. Gulbis1,*, Steven L. Kazmirski2,3, Jeff Finkelstein4, Zvi Kelman4,, Mike O’Donnell4
and John Kuriyan2,3
1
Laboratory of Molecular Biophysics and 4Laboratory of DNA Replication, Howard Hughes Medical Institute, The Rockefeller
University, New York, NY, USA; 2Department of Molecular and Cell Biology and of Chemistry, University of California,
Berkeley, CA, USA; 3Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
The chi (v) and psi (w) subunits of Escherichia coli DNA
polymerase III form a heterodimer that is associated with the
ATP-dependent clamp-loader machinery. In E. coli, the v:w
heterodimer serves as a bridge between the clamp-loader
complex and the single-stranded DNA-binding protein. We
determined the crystal structure of the v:w heterodimer at
2.1 Å resolution. Although neither v (147 residues) nor w
(137 residues) bind to nucleotides, the fold of each protein is
similar to the folds of mononucleotide-(v) or dinucleotide(w) binding proteins, without marked similarity to the
structures of the clamp-loader subunits. Genes encoding
v and w proteins are found to be readily identifiable in several bacterial genomes and sequence alignments showed that
residues at the v:w interface are highly conserved in both
proteins, suggesting that the heterodimeric interaction is of
functional significance. The conservation of surface-exposed
residues is restricted to the interfacial region and to just two
other regions in the v:w complex. One of the conserved
regions was found to be located on v, distal to the w interaction region, and we identified this as the binding site for a
C-terminal segment of the single-stranded DNA-binding
protein. The other region of sequence conservation is
localized to an N-terminal segment of w (26 residues) that is
disordered in the crystal structure. We speculate that w is
linked to the clamp-loader complex by this flexible, but
conserved, N-terminal segment, and that the v:w unit is
linked to the single-stranded DNA-binding protein via the
distal surface of v. The base of the clamp-loader complex has
an open C-shaped structure, and the shape of the v:w complex is suggestive of a loose docking within the crevice formed
by the open faces of the d and d¢ subunits of the clamp-loader.
The replication of genomic DNA appears to be carried out
in a fundamentally similar manner in prokaryotes, eukaryotes and archaebacteria [1]. In each case, the primary
replicase is distinguished from other DNA polymerases
by its ability to rapidly polymerize tens of thousands of
nucleotides without dissociating from the template. This
high level of processivity is conferred on the DNA
polymerase by ring-shaped sliding clamps [the b subunit
in bacteria, proliferating cell nuclear antigen (PCNA) in
eukaryotes and archaebacteria] that tether the DNA
polymerase to DNA [2]. The interaction between the
DNA polymerase and the sliding clamp enables the active
site of the polymerase to bind and release DNA rapidly
during its spiral progression along the template strand,
without actually dissociating from the template.
Each strand of DNA at the replication fork is copied by a
core DNA polymerase assembly that is attached to a sliding
clamp. Although a single sliding clamp may remain
attached to the polymerase during replication of the leading
strand, each Okazaki fragment that is generated on the
lagging strand requires a new clamp, and these must be
rapidly loaded onto newly primed sites. It appears that
proteins at the replication fork act in concert to rapidly and
repetitively cycle the lagging strand polymerase between
sliding clamps loaded at sites of Okazaki fragment synthesis
[3].
DNA polymerase III (Pol-III), an archetypal replicase,
from Escherichia coli, comprises several distinct subassemblies. Polymerase-exonuclease cores (the a and e subunits)
catalyze DNA synthesis and carry out proofreading, and
these are localized to the template by association of the a
subunit with the sliding clamp, b, which encircles DNA. The
b clamp is loaded onto DNA by a clamp-loading, ATPdependent machinery (the c or s complex), an integral part
of the Pol-III holoenzyme [4,5]. Cellular isolates of the
E. coli clamp-loader complex contain a mixture of proteins
with the following stoichiometry: (c/s)3 d1 d¢1 v1 w1 [4,6].
Correspondence to J. Kuriyan, 16 Barker Hall, University of
California, Berkeley, CA 94720-3202, USA.
Fax: + 510 643 0159, Tel.: + 510 643 0137,
E-mail: [email protected]
Abbreviations: PCNA, proliferating cell nuclear antigen; Pol-III, DNA
polymerase III; RFC, replication factor C; SIRAS, single isomorphous replacement and anomalous scattering; SSB, single-stranded
DNA-binding protein.
Present addresses: *Structural Biology Division, The Walter and Eliza
Hall Institute of Medical Research, 1G Royal Parade, Parkville,
Victoria 3050, Australia; University of Maryland Biotechnology
Institute, Center for Advanced Research in Biotechnology,
9600 Gudelsky Drive, Rockville, MD 20850, USA.
(Received 21 October 2003, revised 21 November 2003,
accepted 27 November 2003)
Keywords: clamp loader; DNA replication; processivity
factor; sliding clamp.
FEBS 2004
440 J. M. Gulbis et al. (Eur. J. Biochem. 271)
The major constituents, the c and s subunits, are ATPases
encoded by the DnaX gene [7,8]. Subunits c and s are
identical in sequence, except that the C-terminal region of
s is longer, and serves to interface with the polymerase
catalytic subunit (a) [9] and principal helicase (DnaB)
[10–12], thereby coupling replicase and primosome at the
fork [13,14]. The core structure of the c subunit, a protein
with an N-terminal RecA-like domain (domain I), flexibly
linked to helical domains II (middle) and III (C-terminal),
resembles that of the d and d¢ subunits [15] and of the
replication factor C (RFC) clamp-loader subunits of eukaryotes and archaebacteria [16].
Crystal structures of a functional E. coli clamp-loader
complex, cdd¢ (stoichiometry 3 : 1 : 1), and of a complex
between the b clamp and an isolated d subunit, have greatly
facilitated our understanding of the clamp loading mechanism [17,18]. The clamp-loader complex is pentameric, and
the subunits are arranged such that the major connections
occur between the five C-terminal domains, which form a
ring-shaped collar. It has been shown in vitro that loading of
the clamp onto DNA can be orchestrated by just these three
subunits (c, d and d¢) alone [19]. Structural considerations
suggest that conformational changes in the clamp-loader,
which occur in response to ATP binding at the c–c and c–d¢
interfaces, toggle the b-interacting element of d between
dormant and active conformations, and facilitate the
formation of multiple contacts between the b clamp and
the clamp-loader complex. The b clamp is stabilized in an
open conformation by this means, allowing the entry of
DNA. The smaller v and w subunits are not obligatory
participants in the clamp loading process.
The v and w subunits were first isolated from E. coli
extracts in association with the c subunit, and were shown
to bind to each other tightly [20]. The w subunit interacts
with c [21], specifically with domain III of the c subunit [22].
On this basis it was proposed that the sparingly soluble w
protein bridges between the v subunit and the c-ATPase
subunits in the c-complex [21]. Current evidence suggests
that the v:w subassembly plays an important role in the
processive synthesis of Okazaki fragments. Single-stranded
DNA-binding protein (SSB) binds to v in an interaction
that is strengthened nearly 1000-fold when SSB is also
bound to DNA [23,24]. SSB coats single-stranded DNA as
it unwinds, protecting it from nucleases and melting out
secondary structure, thereby circumventing barriers to
replication. The interaction between v and the clamp loader
stabilizes reconstituted DNA Pol-III holoenzyme at high
salt concentrations (up to 800 mM potassium) [25], and is
crucial for the rapid replication of the lagging strand. The
v subunit disrupts an otherwise stable contact between SSB
and primase at the replication fork [26]. This facilitates the
dissociation of primase from the newly synthesized RNA
primer, and primase is then free to be recycled to another
site. The b clamp is assembled onto the newly primed DNA
template concomitantly, in readiness for synthesis of the
next Okazaki fragment. This switching mechanism ensures
that priming and initiation of new fragments is coordinated
smoothly with the assembly of b clamps onto DNA.
We have determined the 3D structure of a 1 : 1 complex
of the E. coli v and w subunits in order to explore the
function of these two proteins. Subunits v and w interact to
form an elongated heterodimer, of comparable size to that
of the c, d, and d¢ protomers. Sequence comparisons
amongst 12 bacterial species, containing genes for both v
and w, provide some clues as to how the v:w subassembly
might interact with the clamp-loader complex and with SSB.
Experimental procedures
Crystallization
The v:w complex was reconstituted by combining the
individual component proteins, purified as previously
described [27], and separated from an excess of v by anion
exchange chromatography. The heterodimer was concentrated to 10 mgÆmL)1 after extensive dialysis against a
buffer comprising 20 mM Tris/HCl (pH 7.5), 4 mM dithiothreitol, 0.5 mM EDTA, 100 mM NaCl and 10% (v/v)
glycerol. Monoclinic crystals (spacegroup P21; a ¼ 64.4 Å,
b ¼ 65.7 Å, c ¼ 73.4 Å, b ¼ 116.2) were grown in
hanging drops at 4 C over a period of 7 days. One
microliter of the protein solution was mixed with 1 lL of a
reservoir solution containing 100 mM Hepes (pH 6.8), 25%
PEG 4000, 8% (v/v) glycerol and 8% (v/v) 2,5-methylpentanediol, prior to equilibration by vapour diffusion.
Crystals were directly mounted in nylon loops and flash
frozen at 100 K. Measurable diffraction extends beyond
2.1 Å on a laboratory detector system comprising an
R-Axis II image plate in conjunction with a Rigaku rotating
anode generator.
X-ray crystallography
The structure was solved by single isomorphous replacement with the inclusion of some multiple wavelength
anomalous diffraction data. An initial derivative dataset,
complete to 2.7 Å, was collected from a crystal soaked for
several days in a stabilizing solution supplemented with
1 lM ethyl mercury phosphate. The coordinates for four
mercury atoms were derived manually from a difference
Patterson map and verified using SHELXS-90 [28]. Multiple
wavelength data, to a resolution of 3.2 Å, were collected
from a similarly treated crystal on Beamline X25 at
Brookhaven National Synchotron Light Source. Monochromator positions, defining three discrete wavelengths
corresponding to the inflection point, peak, and a remote
high-energy point, were determined according to the X-ray
absorption fluorescence spectrum of the derivatized crystal.
Patterson maps, calculated using anomalous differences as
coefficients, confirmed the presence of the same four sites.
All images were processed using DENZO [29] and the
integrated intensities were scaled and merged using SCALEPACK [29].
Phasing and refinement
Initial attempts at structure determination by MAD phasing
were unsuccessful, owing to mediocre diffraction and a lack
of high quality dispersive difference data. Instead, a method
utilizing only the anomalous Df¢¢ data, measured at the
synchrotron and single isomorphous replacement and
anomalous scattering (SIRAS) phases obtained from
in-house native and derivative datasets, resulted in an
experimental map with clearly defined protein and solvent
FEBS 2004
E. coli DNA polymerase clamp-loader subassembly (Eur. J. Biochem. 271) 441
using CLUSTALX [38]. A sequence conservation score was
calculated for each amino acid position in the aligned
sequences, by pairwise comparisons between sequences for
each amino acid position. For each pairwise comparison,
the BLOSUM62 matrix [39] gives a substitution probability
score for the amino acid substitution. These scores are
summed for each amino acid position, divided by the
number of pairwise comparisons made and then scaled, so
that a score of 100% reflects absolute conservation. Using
this set of 12 w sequences, v sequences from the same
organisms were used to calculate the level of sequence
conservation for v and for other subunits of the clamploader complex (c, d, d¢).
regions. MLPHARE, from the CCP4 suite [30], was used to
refine heavy atom parameters and to generate phases.
Density modification procedures, including solvent flattening, histogram matching, and the derivation of phase
relationships using Sayre’s equation, were implemented
with SQUASH [31], and the improved phase information
yielded an interpretable electron density map. A partial
model of v was built into this density using O [32], and this
was used as a basis for positioning a second complex in the
asymmetric unit using AMORE [33]. Twofold noncrystallographic symmetry averaging of the experimental maps,
using the program RAVE [34], enhanced map quality
sufficiently to enable tracing of most structural elements
and assignment of the amino acid sequence. Iterative cycles
of building and refinement were required to place the
remainder of the structure.
Refinement was carried out by least-squares optimization
and simulated annealing procedures, using X-PLOR [35], and
by maximum likelihood methods, using CNS [36]. Strict
noncrystallographic symmetry constraints were released
in the final stages, and individual temperature factors were
refined for all nonhydrogen atoms. The final refined model
had no outliers in the Ramachandran diagram, and
contained 516 residues and 256 water molecules with refined
B-factors of less than 60 Å2. Twenty-six residues at the
N-terminus of each of the two crystallographically independent w molecules were omitted from the model because
of poor electron density in those regions. Side-chains were
not modeled beyond Ca for the following residues: (v1:
Arg92, Lys132, Arg135, Lys147); (v2: Arg92, Asp121,
Ser122, Lys132, Arg135, Lys147); (w1: Asp93, Glu94,
Arg135, Asn136); (w2: Gln26, Gln81, Gln123, His130,
Arg135). The atomic coordinates have been released in the
protein data bank with the access code 1EM8.
Results and discussion
Structure determination
Although v is well behaved as an isolated protein in
solution, w forms insoluble aggregates. w was therefore
purified under denaturing conditions and solubilized in the
presence of v [27]. The resulting 1 : 1 complex of v and w
is soluble and monodisperse in solution, as determined by
dynamic light scattering (data not shown). Monoclinic
crystals, containing two v:w complexes in the asymmetric
unit, diffract X-rays to 2 Å Bragg spacings on a rotating
anode X-ray source. The crystal structure of the v:w
complex was determined by SIRAS and refined using
data to Bragg spacings of 2.1 Å (Tables 1 and 2). Refinement against 27 852 reflections converged at a free R-value
of 0.265 and a conventional R-value of 0.229. The final
crystallographic model for each independent v:w heterodimer contained the complete sequence of v (147 residues)
except for the N-terminal methionine, and 110 of 137
residues of w (the N-terminal 26 resides of w were disordered
in both molecules in the asymmetric unit).
Sequence alignments and conservation scores
The E. coli amino acid sequence for w was used in BLAST
[37] to identify w sequences in other organisms. w Sequences
that were more than 80% identical to sequences already in
the set were excluded from further analysis. This search
resulted in the inclusion of 12 w sequences in an alignment
Structure of the v:w heterodimer
The v and w subunits formed an elongated heterodimer, in
agreement with predictions made on the basis of sedimentation equilibria [21,25] (Fig. 1A). The conformations of the
Table 1. Crystallographic structure determination. Rmerge ¼ R jIj hIi= R Ij ; Riso ¼ RjFP FPH =RjFP j.
j
j
Dataset
Sites
Resolution
(Å)
Reflections
(measured/unique)
Completeness (%)
(overall/outer shell)
Rmerge (%)
(overall/outer shell)
Riso (%)
(overall)
Native 1 Data
EMP (1.5418 Å)
EMP k1 ¼ 1.0093 Å
EMP k2 ¼ 0.9919 Å
EMP k3 ¼ 1.0062 Å
20.0–2.1
20.0–2.7
20.0–2.8
20.0–2.9
20.0–2.9
218
90
114
87
87
97.9/95.2
95.6/71.9
97.1/98.7
93.1/95.6
93.9/96.8
6.6/19.4
7.3/25.9
7.3/27.5
8.2/32.0
7.6/31.8
16.6
4
4
4
4
771/32
321/15
443/13
057/11
285/11
326
653
238
998
993
Table 2. Crystallographic structure refinement.
Refinement statistics
RMS deviations
Data
Resolution
(Å)
Rw
Rfree
No. of reflections
(all/working/free)
Bond lengths
(Å)
Bond angles
()
Native2
20.0–2.1
0.229
0.265
30 953/27 852/3101
0.01
3.2
442 J. M. Gulbis et al. (Eur. J. Biochem. 271)
FEBS 2004
Fig. 1. Structure of the v:w heterodimer. (A) Ribbon diagram of the v:w heterodimer crystal structure. The w subunit is colored cyan and sits on top
of the v subunit. The v subunit is colored green except for the stretch of residues that reside in the w-binding site, which have been colored red. (B)
An enlarged view of the contiguous loop region of v and how it interacts within the cleft of w. This loop region has high sequence similarity with a
DNA-dependent DNA polymerase from the bacteriophage PRD1. (C) A rotated view of the surface of w is shown. The w subunit has been rotated
to show the cleft between a1 and a4 that makes up the v-binding surface. Residues 61–66 of v are shown in green. The side-chain of Phe64 of v
inserts itself into a conserved hydrophobic pocket consisting of Val57, Leu121, Trp122 and Ile125. (D) A schematic diagram of the v:w heterodimer.
two crystallographically independent v:w complexes were
essentially identical, except for a small difference in tilt angle
between the v and w subunits. The root mean-square
deviation in the positions of the Ca atoms between the two
structures, calculated by superimposing 256 Ca atoms, was
0.83 Å.
Both v and w have central, parallel b-sheets that are
connected to a-helices (Fig. 1D); v resembles a classic
mononucleotide-binding fold, whilst w more closely typifies dinucleotide-binding proteins [40] (Fig. 2). The structures of both subunits were compared with structures in
the protein databank [41], using the DALI server [42].
FEBS 2004
E. coli DNA polymerase clamp-loader subassembly (Eur. J. Biochem. 271) 443
Fig. 2. Structural comparisons of w and v with
other proteins. (A) A side-by-side comparison
of w with the mismatch specific DNA uracyl
glycosylase, MUG [45]. Similar structural
features are colored yellow. (B) A side-by-side
comparison of the v DEAD box helicase,
PcrA [46].
Structural similarity between v and w was revealed to
proteins that are primarily nucleotide-binding proteins but,
like the d and d¢ subunits of the clamp-loader, neither v
nor w contain any of the functional elements required for
nucleotide binding. The topology of the w subunit
resembles that of the bacterial two-component signaling
protein, CheY [43], and the uracil DNA-glycosylases,
UDG [44] and MUG [45] (Fig. 2A).
The v subunit has a central b sheet with seven parallel
strands, which curve in a left-handed twist. There is
structural similarity between v and the non-ATP-binding
subdomain 2A of DEAD box helicases such as PcrA [46],
and Rep47 (Fig. 2B). There are only two significant
deviations in topology between v and subdomain 2A of
these proteins. One was observed at the w interface, where a
compact glycine-rich loop in v replaces a large insertion
(subdomain 2B) in PcrA and Rep. The other occurs where
an extended a helix, bordering the nucleotide-binding site
between subdomains 1A and 2A in the helicases, is
truncated in v to a short loop incorporating a single turn
of 310 helix. The functional significance of this structural
similarity between v and DEAD box helicases is unclear.
Two parallel helices on one side of a four stranded
b-sheet of w form the sides of an extended hydrophobic
crevice in the molecular surface. A single contiguous loop
region from v (residues 52–79) inserts snugly into this
cleft, placing Phe64 of v into the hydrophobic pocket on
w, and burying 1256 Å2 of surface area at the subunit
interface (Fig. 1B,C).
Interestingly, a DNA-dependent DNA polymerase from
the E. coli bacteriophage, PRD1, has high sequence similarity to this loop region alone of v, extending over 28
residues with 13 identities, suggesting that this DNA
polymerase might couple to the clamp-loader complex via
the w subunit. The functional significance of this interaction
is unclear, and no other proteins with significant sequence
similarity to v (or to w) were detected using a BLAST search
[37].
Sequence conservation in v and w
A query of the nonredundant protein sequence database
with the sequences of E. coli v and w, using BLAST and
PSI-BLAST, resulted in statistically significant matches that
were restricted to the genomes of certain bacteria
(Table 3). This sequence search identified more sequences
for v than for w (some bacterial genomes contained
identifiable sequences for v, but not for w, and the
genomes that contained both had only one instance of
each). We restricted our analysis to 12 v and w sequences
from genomes that contained clearly identifiable genes for
both proteins (Table 3). The presence of genes for v in
genomes that do not contain w was unexpected, because
the w-binding site appears to be conserved in these
FEBS 2004
444 J. M. Gulbis et al. (Eur. J. Biochem. 271)
Table 3. Sequences for v:w sequence comparison.a
w
v
Species
Accession no.
% Identity
to E. coli
Accession no.
% Identity
to E. coli
Escherichia coli
Shigella flexneri
Salmonella typhimurium
Yersinia pestis
Haemophilus influenzae Road
Vibrio parahaemolyticus
Pasteurella multocida
Haemophilus somnus
Vibrio vulnificus
Vibrio cholerae
Candidatus Blochmannia floridanus
Haemophilus ducreyi
Actinobacillus pleuropneumoniae serovar
Sequences for v aloneb
Shewanella oneidensis
Pseudomonas putida
Azotobacter vinelandii
Xylella fastidiosa Dixon
Xanthomonas campestris
Nitrosomonas europaea
Microbulbifer degradans
Bordetella pertussis
Magnetococcus sp. MC-1
Neisseria meningitidis
Burkholderia fungorum
Chromobacterium violaceum
Ralstonia metallidurans
Magnetospirillum magnetotacticum
Rhodopseudomonas palustris
Caulobacter crescentus
Rhodospirillum rubrum
Rhodobacter sphaeroides
Brucella melitensis
Novosphingobium aromaticivorans
16132190
30065610
16767798
16120761
16271986
28899216
15602824
32029392
27365077
15640676
33519585
33151459
32035477
100
99
78
59
32
31
34
27
29
31
24
36
25
15804851
30065454
16767721
16123590
16273306
28899419
15602687
23467212
27364859
15642498
33519517
33151849
32034863
100
99
95
72
49
39
50
50
38
41
43
54
54
24374933
26987715
23104211
22994951
21230123
30248459
23028461
33593407
22998318
15677419
22986355
34498368
22976686
23016368
22964979
16125938
22968178
22959552
17987543
23108152
39
33
30
32
34
24
27
25
31
30
26
26
19
21
19
21
21
24
20
25
a
b
Sequences in italics were not included in the sequence conservation calculations as they had higher than 80% sequence identity to E. coli w.
These sequences were not included in the sequence conservation calculations shown in Figs 3 and 4.
proteins. As discussed below, the sequence of w is not as
highly conserved as the v sequences, and it is possible that
the BLAST searches simply failed to identify w proteins that
have diverged greatly in sequence.
Residues located at the interface between v and w were
highly conserved in both proteins (Fig. 3). In w, the
hydrophobic pocket that binds v is situated between two
a helices (a1, residues 52–61 and a4, residues 118–126)
(Fig. 1C). Four hydrophobic residues in w that are within
this pocket have high conservation scores [Val57, 93.4%;
Leu121, 76.2%; Trp122, 100%; and Ile125, 87.1%) (see the
Experimental procedures for a definition of the conservation scores, which are based on the BLOSUM62 substitution
matrix) [39]. In v, the aromatic residue (Phe64) that is bound
within the pocket of w, is absolutely conserved (Fig. 1B,C).
Other interfacial residues that are highly conserved included
Trp57 of v (conservation score ¼ 100%) and Ala119 of w
(score ¼ 73.7%).
The surface of v also contains a highly conserved region
that is located distal to the region of interaction with w
(Fig. 4). This conserved surface region comprises an a helix
(a4, residues 124–135) and a b strand (b7, residue 139–143),
between which there is a cleft. Interestingly, helix a4 has
four absolutely conserved basic residues that are exposed
(Lys124, Arg128, Lys132 and Arg135). There is an
additional, absolutely conserved, arginine residue within
the helix (Arg130) that is buried and forms hydrogen bonds
with the main-chain oxygen atoms of Phe116 and the sidechain of a buried aspartic acid residue (Asp115).
Interaction between v and SSB
The interaction between v and SSB is mediated by residues
located within the very C-terminal region of SSB [23]. This
region of SSB includes a conserved sequence motif (173DDDIPF-178) in E. coli SSB, including three negatively
FEBS 2004
E. coli DNA polymerase clamp-loader subassembly (Eur. J. Biochem. 271) 445
Fig. 3. Sequence conservation in v and w. The conservation score using the BLOSUM62 substitution matrix (see the Experimental procedues) for each
residue in v and w was calculated for the 12 pairs of sequences shown in Table 3. The surfaces of v and w, shown in this figure, are colored according
to this conservation score. To the right, the binding surfaces of both proteins are shown. Both binding surfaces have been conserved in each protein.
On w, little surface conservation is observed outside the v-binding surface. In v, a large amount of surface area is conserved distal to the w-binding
site. This area is proposed to bind to single-stranded DNA-binding protein (SSB).
Fig. 4. Potential v:single-stranded DNA-binding protein (SSB) interaction. A region of v, with high sequence conservation, is shown (B). This surface
is suggested to bind to the negatively charged C-terminal tail of SSB. Absolutely conserved and positively charged residues, located within this
region, are shown on the left in a ribbon diagram in the same orientation (A). A schematic drawing of the inferred interaction between v and the
C-terminus consensus sequence of SSB is shown on the right (C).
charged residues [48]. A conditionally lethal E. coli mutant,
SSB-113, differs from the wild type SSB by a single amino
acid substitution in which the penultimate residue, Pro177,
is replaced with serine. Although SSB-113 binds singlestranded DNA as tightly as wild type SSB, it is unable to
interact with the v subunit [23]. Furthermore, removal of
the last 26 residues of SSB leads to the loss of interaction
between SSB and v [49]. This negatively charged motif could
potentially interact with the conserved and positively
charged surface region of v that is distal to the v:w interface
(Fig. 4).
The atomic coordinates of several SSB variants are listed
in the Protein Data Bank [50–52]. Unfortunately, none of
these structures include models for the acidic C-terminal
region that is responsible for binding v and, possibly,
primase. This region is not part of the DNA-binding
446 J. M. Gulbis et al. (Eur. J. Biochem. 271)
domain of SSB and it contains many Gly, Gln and Pro
residues, suggesting that it may not have a regular or defined
structure. The stabilization of this C-terminal region of
SSB by interaction with v might be responsible for the
stable interaction of single-stranded DNA, SSB and the
v:w complex.
The N-terminal segment of w is a possible linker
between v:w and the clamp-loader
In the crystals of v:w, the N-terminal 26 residues are
disordered and are not present in the structural model.
While disordered regions are not uncommon within crystal
structures, it is interesting that this region of w is highly
conserved in sequence (Fig. 5). Within the disordered
N-terminal region there are three absolutely conserved
hydrophobic residues (Ile14, Trp17 and Pro22). In contrast,
residues on the surface of the 3D model of w are not highly
conserved, except for those involved in the interaction with
v. This suggests to us that the conserved, but disordered,
N-terminal segment of w may have functional importance
for binding to the clamp-loader complex.
It is known that w interacts with domain III of c [22], thus
bridging the clamp-loader and v. To identify where the
N-terminal region of w might interact with the clamp-loader
complex, a sequence alignment was performed for each of
the clamp-loader subunits (d, d¢ and c), using sequences
from the same bacterial species that were used in the
alignment of the sequences of w and v. For d and d¢, there
was little evidence for conserved residues on the surface,
except for residues that are involved in binding to the c
subunit and the b clamp. As expected, the c subunit shows
a high degree of conservation in the first two domains that
make up the AAA+ ATPase portion of the subunit. The
third domain of the c subunit is its oligomerization domain,
and there is a high degree of sequence conservation in
regions that interacted to form the C-terminal collar of the
FEBS 2004
clamp-loader. Interestingly, the three c subunits also have a
conserved surface region inside the C-terminal collar, which
includes an exposed hydrophobic patch (Fig. 6). This
hydrophobic patch consists of Phe359 (absolutely conserved) and Leu327 (conservation score ¼ 71.2%), and
represents a potential interaction surface for w because it
does not appear to be involved directly in other interactions.
Examination of surface charge distributions and hydrophobicity on both the v:w heterodimer and the clamploader complex failed to reveal any obvious docking mode
for the v:w heterodimer onto the clamp-loader. The
structure of the clamp-loader complex is such that there is
a prominent gap in the C-shaped base of the structure,
between the d and d¢ subunits. It had been proposed that
this gap would close during one stage of the clamp loading
cycle [17], but recent fluorescence energy transfer measurements, made by our group on the c complex, indicated
that this gap stays open at all stages (E. Goedken,
M. Levitus, A. Johnson, C. Bustamante, M. O’Donnell &
J. Kuriyan, unpublished observation). Maintenance of the
open C-shape of the base of the clamp-loader complex is
also consistent with crystal structures determined recently
in our group, of a nucleotide-loaded c complex (S. L.
Kazmirski, M. Podobnik, T. F. Weitze, M. O’Donnell &
J. Kuriyan, unpublished observation) and a eukaryotic RFC
complex bound to PCNA (G. D. Bowman, M. O’Donnell
& J. Kuriyan, unpublished observation). Strikingly, in the
RFC–PCNA complex, an additional domain of the RFC-1
subunit is located in the gap corresponding to the d–d¢
opening in the c complex. These results suggest that this gap
does not close during the clamp loading cycle, and it is
possible that the v:w unit may be located in this region
(Fig. 6). The insertion of v:w into this prominent crevice on
the surface of the clamp-loader would explain why only one
v:w heterodimer is bound to one clamp-loader complex,
even though there are three c subunits (each with a potential
binding site for w) in the complex. The lack of sequence
Fig. 5. Conservation of sequences in the N-terminal segment of w. An alignment of the first 26 residues of w, from the list of sequences given in
Table 3, is shown. The alignment is colored according to the degree of sequence conservation. These 26 residues are disordered in the crystal
structure of the v:w complex, yet a high amount of conservation is observed. It is proposed that the this linker binds to the clamp-loader complex,
tethering the v:w heterodimer to the complex.
FEBS 2004
E. coli DNA polymerase clamp-loader subassembly (Eur. J. Biochem. 271) 447
Fig. 6. Potential clamp-loader:w interaction.
(A) Two views of the Escherichia coli clamploader complex are shown [17]. An exposed
hydrophobic region of the c subunit, which is
highly conserved but not involved in nucleotide binding or intersubunit interactions, is
indicated as a potential binding site for the
N-terminal disordered region of w. (B) On the
left are space filling structures of the E. coli
clamp-loader and v:w heterodimer, with different colors for different subunits: d (magenta), d¢ (orange), c1 (green), c2 (red), c3 (blue),
w (cyan), and v (dark green). A schematic
diagram showing a possible mode of interaction between the v:w heterodimer and the
clamp-loader complex is shown on the right.
The v:w heterodimer is believed to sit in the
gap between d and d¢, while the N terminus of
w interacts with the proposed binding region
of c inside the C-terminal collar of the clamploader complex.
conservation on the surfaces of the d and d¢ subunits
suggests that the docking of the v:w unit onto the clamploader may be loose, mediated primarily by the flexible
N-terminal segment of w. This general location for v, which
interacts with SSB, is consistent with the fact that the b
clamp will be opened in its vicinity, leading to the insertion
of DNA into the clamp at this site.
also lacking in eukaryotes and archaebacteria. The adapter
function imposes minimal sequence constraints on these
proteins, which could be replaced functionally by highly
divergent, but related, proteins, or even by completely
unrelated proteins.
Conclusions
We thank Lore Leighton for preparing the illustrations. We are grateful
to the members of the Kuriyan laboratory, and to Irina Bruck, Jerard
Hurwitz, Elena Conti, Marjetka Podobnik, David Jeruzalmi and
Declan Doyle, for assistance and insightful discussions. Lonnie Berman
and the staff of the National Light Source, Brookhaven National
Laboratory, willingly donated their time and expertise, for which we are
much indebted. J. M. G. holds a Wellcome Trust ISRF Fellowship.
This work was partially supported by grants from the NIH (GM 45547
to J. K., GM 38839 to M. O’D.).
We have presented the crystal structure of the v:w heterodimer, which, together, form an accessory factor for the
clamp loading process in E. coli and certain other bacteria.
Despite a clear structural similarity to nucleotide-binding
proteins, v and w are incapable of binding nucleotides.
Rather, the v:w complex functions as an adapter unit that
couples the clamp-loader complex to SSB. A conserved and
positively charged surface pocket on v is probably the
region that interacts with the C-terminal acidic region of
SSB. Structure-based sequence alignments suggest that w
may bind to the C-terminal collar domain of the c subunit
via its N-terminal segment, a region of 26 residues that is
highly conserved in sequence, but disordered in the crystal.
The loose docking of the v:w heterodimer onto the c
complex might explain why many bacterial genomes do not
contain readily identifiable v and w sequences, which are
Acknowledgements
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