Crystal structure of the chi:psi subassembly of the Escherichia coliDNA polymerase clamp-loader complex Jacqueline M.. Kazmirski2,3, Jeff Finkelstein4, Zvi Kelman4,, Mike O’Donnell4 and
Trang 1Crystal 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 and4Laboratory 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 A˚ 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
sev-eral 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 inter-action 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 com-plex is suggestive of a loose docking within the crevice formed
by the open faces of the d and d¢ subunits of the clamp-loader Keywords: clamp loader; DNA replication; processivity factor; sliding clamp
The replication of genomic DNA appears to be carried out
in a fundamentally similar manner in prokaryotes,
eukary-otes 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 subassem-blies 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, ATP-dependent machinery (the c or s complex), an integral part
of the Pol-III holoenzyme [4,5] Cellular isolates of the
E coliclamp-loader complex contain a mixture of proteins with the following stoichiometry: (c/s) d d¢ v w [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: kuriyan@uclink.berkeley.edu
Abbreviations: PCNA, proliferating cell nuclear antigen; Pol-III, DNA
polymerase III; RFC, replication factor C; SIRAS, single
isomorph-ous replacement and anomalisomorph-ous 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)
Trang 2The 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
euk-aryotes 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
mech-anism [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 mMpotassium) [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 concen-trated to 10 mgÆmL)1 after extensive dialysis against a buffer comprising 20 mMTris/HCl (pH 7.5), 4 mM dithio-threitol, 0.5 mM EDTA, 100 mM NaCl and 10% (v/v) glycerol Monoclinic crystals (spacegroup P21; a¼ 64.4 A˚,
b¼ 65.7 A˚, c ¼ 73.4 A˚, b ¼ 116.2) were grown in hanging drops at 4C over a period of 7 days One microliter of the protein solution was mixed with 1 lL of a reservoir solution containing 100 mMHepes (pH 6.8), 25% PEG 4000, 8% (v/v) glycerol and 8% (v/v) 2,5-methyl-pentanediol, 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 A˚ 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 replace-ment with the inclusion of some multiple wavelength anomalous diffraction data An initial derivative dataset, complete to 2.7 A˚, 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 usingSHELXS-90 [28] Multiple wavelength data, to a resolution of 3.2 A˚, were collected from a similarly treated crystal on Beamline X25 at Brookhaven National Synchotron Light Source Mono-chromator 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 SCALE-PACK[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
Trang 3regions.MLPHARE, from the CCP4 suite [30], was used to
refine heavy atom parameters and to generate phases
Density modification procedures, including solvent
flatten-ing, histogram matchflatten-ing, 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 usingAMORE [33] Twofold
noncrystallo-graphic 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, usingX-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 A˚2 Twenty-six residues at the
N-terminus of each of the two crystallographically
inde-pendent 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
Sequence alignments and conservation scores
The E coli amino acid sequence for w was used inBLAST
[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
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 clamp-loader complex (c, d, d¢)
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 A˚ 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 A˚ (Tables 1 and 2) Refine-ment 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 hetero-dimer 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)
Structure of the v:w heterodimer The v and w subunits formed an elongated heterodimer, in agreement with predictions made on the basis of sedimen-tation equilibria [21,25] (Fig 1A) The conformations of the
Table 1 Crystallographic structure determination R merge ¼ R
j jI j hIi = RjI j ; R iso ¼ RjF P F PH
=RjF P j.
Dataset Sites
Resolution (A˚)
Reflections (measured/unique)
Completeness (%) (overall/outer shell)
R merge (%) (overall/outer shell)
R iso (%) (overall) Native 1 Data 20.0–2.1 218 771/32 326 97.9/95.2 6.6/19.4 16.6 EMP (1.5418 A˚) 4 20.0–2.7 90 321/15 653 95.6/71.9 7.3/25.9
EMP k1 ¼ 1.0093 A˚ 4 20.0–2.8 114 443/13 238 97.1/98.7 7.3/27.5
EMP k2 ¼ 0.9919 A˚ 4 20.0–2.9 87 057/11 998 93.1/95.6 8.2/32.0
EMP k3 ¼ 1.0062 A˚ 4 20.0–2.9 87 285/11 993 93.9/96.8 7.6/31.8
Table 2 Crystallographic structure refinement.
Data
Resolution
No of reflections (all/working/free)
Bond lengths (A˚)
Bond angles () Native2 20.0–2.1 0.229 0.265 30 953/27 852/3101 0.01 3.2
Trang 4two 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 A˚
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 typi-fies dinucleotide-binding proteins [40] (Fig 2) The struc-tures of both subunits were compared with strucstruc-tures in the protein databank [41], using the server [42]
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.
Trang 5Structural 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 A˚2 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 simi-larity 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 aBLASTsearch [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
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].
Trang 6proteins As discussed below, the sequence of w is not as
highly conserved as the v sequences, and it is possible that
theBLASTsearches 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
conserva-tion scores, which are based on theBLOSUM62 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 score¼ 100%) and Ala119 of w
(score¼ 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 side-chain 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 (173-DDDIPF-178) in E coli SSB, including three negatively
Table 3 Sequences for v:w sequence comparison.a
Species
Accession no.
% Identity
to E coli Accession no.
% Identity
to E coli
Candidatus Blochmannia floridanus 33519585 24 33519517 43
Actinobacillus pleuropneumoniae serovar 32035477 25 32034863 54 Sequences for v aloneb
a
Sequences in italics were not included in the sequence conservation calculations as they had higher than 80% sequence identity to E coli w.
b
These sequences were not included in the sequence conservation calculations shown in Figs 3 and 4.
Trang 7charged 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
single-stranded 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
Fig 3 Sequence conservation in v and w The conservation score using the BLOSUM 62 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).
Trang 8domain 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
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 con-served) and Leu327 (conservation score¼ 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 hydro-phobicity on both the v:w heterodimer and the clamp-loader 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 meas-urements, 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.
Trang 9conservation on the surfaces of the d and d¢ subunits
suggests that the docking of the v:w unit onto the
clamp-loader 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
Conclusions
We have presented the crystal structure of the v:w
hetero-dimer, 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
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
Acknowledgements
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.).
References
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2 Jeruzalmi, D., O’Donnell, M & Kuriyan, J (2002) Clamp loaders and sliding clamps Curr Opin Struct Biol 12, 217–224.
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Fig 6 Potential clamp-loader:w interaction.
(A) Two views of the Escherichia coli
clamp-loader complex are shown [17] An exposed
hydrophobic region of the c subunit, which is
highly conserved but not involved in
nucleo-tide 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
dif-ferent colors for different subunits: d
(magen-ta), d¢ (orange), c1 (green), c2 (red), c3 (blue),
w (cyan), and v (dark green) A schematic
diagram showing a possible mode of
inter-action 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
clamp-loader complex.
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... heterodimer onto the clamp-loader The structure of the clamp-loader complex is such that there isa prominent gap in the C-shaped base of the structure, between the d and d¢ subunits It had... 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... site for w) in the complex The lack of sequence
Fig Conservation of sequences in the N-terminal segment of w An alignment of the first 26 residues of w, from the list of sequences