From the Krebs Institute for Biomolecular Research,
Department of Molecular Biology and Biotechnology, University of
Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom,
¶ Departamento de Biotecnología Microbiana, Centro
Nacional de Biotecnologia, CSIC, Campus Universidad Autonoma de Madrid,
Cantoblanco, 28049 Madrid,
Departamento de Biología
Molecular, Universidad Autónoma de Madrid, 28049 Madrid, Spain, and
Astbury Centre
for Structural Molecular Biology, School of Biology, University of
Leeds, Leeds LS2 9JT, United Kingdom
Received for publication, September 11, 2002, and in revised form, January 21, 2003
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ABSTRACT |
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The Bacillus subtilis SPP1
phage-encoded protein G39P is a loader and inhibitor of the
phage G40P replicative helicase involved in the initiation
of DNA replication. We have carried out a full x-ray crystallographic
and preliminary NMR analysis of G39P and functional studies
of the protein, including assays for helicase binding by a number of
truncated mutant forms, in an effort to improve our understanding of
how it both interacts with the helicase and with the phage replisome
organizer, G38P. Our structural analyses reveal that
G39P has a completely unexpected bipartite structure comprising a folded N-terminal domain and an essentially unfolded C-terminal domain. Although G39P has been shown to bind its
G40P target with a 6:6 stoichiometry, our crystal structure
and other biophysical characterization data reveal that the protein
probably exists predominantly as a monomer in solution. The
G39P protein is proteolytically sensitive, and our binding
assays show that the C-terminal domain is essential for helicase
interaction and that removal of just the 14 C-terminal residues
abolishes interaction with the helicase in vitro. We
propose a number of possible scenarios in which the flexibility of the
C-terminal domain of G39P and its proteolytic sensitivity
may have important roles for the function of G39P in
vivo that are consistent with other data on SPP1 phage DNA replication.
The initiation of DNA replication is a key step in the life cycle
of all cells and as such its careful and precise control is essential.
Studies of prokaryotic systems centered mainly on Escherichia
coli, and its extrachromosomal elements have identified the
following three key stages involved in DNA replication initiation: first, the recognition of the DNA replication origin and initial melting of the DNA strands; second, the recruitment of the replication machinery to the origin; and third, the remodeling of a replication complex to trigger the transition from a stable origin-bound complex to
a mobile replication machine (1).
Initiation of DNA replication in E. coli proceeds
via a sequence of events involving a replicon-specific recognition of
oriC by DnaA and the loading of the replicative helicase
DnaB by DnaC following the local melting of the DNA in an A + T-rich
region (1-3). Initiation of As the G39P protein exists in a variety of oligomeric forms
including monomers, hetero-oligomers with G40P (probably in
a 6:6 ratio of G39P:G40P-ATP), and heterodimers
with G38P, the protein must possess a fold capable of
allowing it to form a variety of specific interactions depending upon
its local environment and the exact stage of the DNA replication
process (4, 6). We have carried out a structural investigation of
G39P to get a better understanding of the way in which
G39P can interact specifically with both G38P and
G40P and thus act as a key component of the system. Thus we
present the first crystal structure and a preliminary NMR analysis of
G39P alongside functional analyses of deletion mutants. It
is the first structure of a prokaryotic helicase loader protein
involved in Crystal Structure Determination--
The details of wt
G39P and G39P112 variant constructs, their
overexpression, purification, and crystallization are described elsewhere.2 Following
purification, the wt protein was crystallized from ammonium phosphate,
and preliminary x-ray diffraction data were collected at room
temperature on home laboratory x-ray sources. These data suggested that
the crystals belong to space group P6122 (or
P6522) with cell dimensions a = b = 105.3 Å, c = 47.4 Å, and diffract
to ~3.5 Å. However, the reproducibility of these crystals was very
poor, and an analysis by mass spectrometry of freshly purified protein
and the crystals themselves revealed multiple fragments derived from
proteolytic cleavage at the C terminus of the protein. The proteolysis
terminated markedly after the final 14 residues of G39P had
been removed, and thus gene 39 was engineered to produce a
mutant construct that coded for a truncated protein comprising only the
first 112 of 126 residues of G39P. The G39P112
protein and selenomethionine incorporated form were prepared in a
similar manner to wild type protein but crystallized from ammonium
sulfate in space group P212121 with
cell dimensions a = 85.6 Å, b = 89.7 Å, c = 47.6 Å. The crystals have three monomers in
the asymmetric unit and an assumed solvent content of 47% based on a
VM (Matthews coefficient) of 2.3 Å3
Da
An electron density map was calculated at 3.0 Å and subsequently
improved by solvent flattening and histogram matching with the program
DM (17). This map was of good quality with readily identifiable regions
of secondary structure. Following a preliminary trace of the secondary
structure, non-crystallographic symmetry operators were determined for
the three monomers found in the asymmetric unit, and the map was
averaged and phase-extended to 2.4 Å using DM. The model fitted
to the resultant map was submitted to refinement using the program
REFMAC (18). Iterative cycles of phase combination of the partial
structure phases and those from the multiwavelength anomalous
dispersion experiment, model building, and refinement, which in the
latter stages was performed using individual isotropic B-factors,
translation, libration, and screw tensor parameterization (19),
and loose non-crystallographic symmetry restraints were used to
construct a model with good stereochemistry that accounted for residues
1-67 in each subunit. Maps that had not been solvent-flattened nor had
non-crystallographic symmetry operators applied were examined to check
for possible errors in the assignment of solvent boundary and
accidental protein density flattening or for use of inappropriate
restraints, but there was no indication that this had happened. The
positions of the six selenium atoms correlated with the locations of
the methionine residues in the N-terminal portions of each monomer. The
refinement statistics are presented in Table I.
NMR Analysis--
Protein samples at concentrations of 1-2
mM in 20 mM phosphate buffer, pH 6.5, and
temperatures ranging from 25 to 55 °C were used. Both one- and
two-dimensional 1H1H experiments were recorded
as described previously (20), using a Bruker DRX 500 spectrometer. Data
were processed using FELIX (Molecular Simulations Inc.).
Gel Filtration and Analytical Ultracentrifugation Analyses
Studies--
The G39P protein used in the gel filtration
experiments was prepared and analyzed as described elsewhere (6).
For protein cross-linking, pure G39P (6 µM)
was prepared in a phosphate buffer
(PO4H2Na/PO4HNa2, pH
7.5, 0.5 mM dithiothreitol, 5% glycerol) containing 50 mM NaCl and then incubated in the presence or absence of
glutaraldehyde (0-0.1%) for 30 min at room temperature. The reactions
were stopped by addition of stop buffer (50 mM Tris-HCl, pH
7.5, 400 mM glycine, 3% 2-mercaptoethanol, 2% SDS, 10%
glycerol) and loaded onto a 15% SDS-PAGE gel.
In the analytical ultracentrifugation sedimentation velocity analysis,
0.42-ml samples of protein at 1 mg ml Limited Proteolysis Assays--
For proteolytic studies, pure
wtG39P or N-terminal His-tagged variants (6 µM) were prepared in phosphate buffer
(PO4H2Na/PO4HNa2, pH
7.5, 0.5 mM dithiothreitol, 5% glycerol) containing 50 mM NaCl and 1 mM phenylmethylsulfonyl fluoride
and then incubated with proteinase K (62 ng/reaction) for increasing
time intervals (0.5, 1, 2, 5, and 10 min) at 37 °C. An aliquot of
the mixture was then removed, and the reaction was stopped by addition
of stop buffer (50 mM Tris-HCl, pH 7.5, 400 mM
glycine, 3% 2-mercaptoethanol, 2% SDS, 10% glycerol), before the
products were loaded onto a 15% SDS-PAGE gel. The signal was
quantified using a PhosphorImager. The 1-min proteinase K incubation
reaction mixture was dialyzed against water and subjected to
matrix-assisted laser desorption ionization/time of flight mass
spectrometry. The N-terminal His-tagged G39P variant was
incubated with proteinase K (62 ng/reaction) for 1 min at 37 °C. The
reaction mixture was loaded onto a Ni-NTA column, and the column was
washed in phosphate buffer containing 5 mM imidazole before
elution with phosphate buffer containing 250 mM imidazole
and subsequent analysis of the eluant on a 15% SDS-PAGE gel.
Deletion Mutant Assays--
The B. subtilis SPP1 wt
phage was routinely propagated in B. subtilis strain YB886
(supo) and the conditional lethal mutants SPP1sus53
and SPP1sus22 in BG295 (sup3) strain. Phage stocks had
titers of 1.0-5.0 × 1010 plaque-forming units/ml
when plated under permissive conditions. Reversion frequencies were not
higher than 10
For the affinity chromatography assay, gene 39 mutants were
constructed that encoded for His-tagged, truncated variants of the wt
protein. In separate experiments, each protein variant was loaded onto
a Ni-NTA-agarose column (2 µg of protein per 20 µl matrix).
G40P (2 µg) was then loaded onto the column, and the binding ability of the G39P variant was confirmed by elution
using imidazole (250 mM) followed by SDS-PAGE analysis.
Crystal Structure of G39P--
The x-ray crystallographic analysis
of G39P has revealed a completely unexpected bipartite
structure for the protein that is made even more striking given its
comparatively small size (126 residues in the wt protein). In the final
model fitted to a map at 2.4-Å resolution, residues 1-67 for each of
the three copies of the protein in the a.u. were present, and there was a total of 41 solvent molecules. There was no interpretable electron density for residues 68-112 at the C terminus of each subunit, and the
N-terminal domain was sufficient to make all the necessary crystal
packing contacts. The final model R-factor is 0.20 with a
corresponding value for Rfree of 0.23 and
strongly supports the proposal that all of the ordered scattering
matter has been reasonably modeled at this resolution.
Thus the non-complexed G39P protein in vitro
would appear to consist of two distinct domains as follows: a fully
folded 67-residue N-terminal domain, and a C-terminal domain that has
only limited fold (see NMR analysis below). Each of the three copies of
the G39P monomer in the a.u. has essentially the same fold
for the N-terminal domain that is composed of four helices. There are three
The G39P112 protein crystallized with three independent
monomers in the a.u., but they adopted an arrangement around a
non-crystallographic 61 screw axis parallel to the
crystallographic c axis (Fig.
2). When viewed along the direction of
the c axis, the disordered C-terminal domains are located on
the exterior of the helical arrays of monomers formed by the screw
axes. The cavities in the crystal lattice are clearly sufficient to
accommodate the C-terminal domains of the G39P112 variant as
supported by mass spectrometric analysis of the crystal used in the
structure determination that confirmed the presence of intact variant
(data not shown). However, it would seem that the extra bulk provided
by the 14 C-terminal residues of the intact wt protein necessitates an
alternative crystal packing arrangement that appears to be less stable
as judged by the noticeably poorer crystal reproducibility and
diffraction quality. The inter-monomer contacts are made predominantly
between residues immediately following helices
Calculations of the electrostatic surface potential of the folded
N-terminal domain of G39P reveals a somewhat negatively charged surface overall. However, there is a notable localized, highly
negative patch on the surface formed by residues at the N terminus of
helix
Molecular replacement attempts to determine the structure of the wt
G39P protein using the lower resolution data collected to
3.5 Å from the seemingly related P6122/P6522
crystal form are ongoing but have so far been unsuccessful.
Analysis of Internal Mobility--
In order to determine to what
extent the disorder observed for the C-terminal half of the molecule
reflected conformational heterogeneity in solution rather than disorder
within the crystal, the 1H-NMR behavior of G39P
was investigated. One- and two-dimensional 1H1H-TOCSY experiments were recorded on samples
of both wt G39P and G39P112 mutant protein forms
(Fig. 3). Spectra recorded at room temperature for both forms of the protein revealed two domains with
very different degrees of motion, as revealed by differential NMR
relaxation rates. For the C-terminal ~50 residues, i.e.
about half the size of G39P, NMR relaxation rates are slow
and are thus dominated by internal mobility far in excess of the
overall rotation of the protein. Resonances from these residues show
little chemical shift dispersion away from their random coil values,
indicative of conformational averaging, and high intensity cross-peaks
in TOCSY spectra, as illustrated by the correlations between the aromatic ring protons of Phe-76 and Tyr-80 in Fig. 3B. The
intensity of the primary amide cross-peaks of glutamines 84, 90, 104, and 107 and asparagines 99 and 110 is also apparent in Fig.
3B. Backbone amide proton resonances from the mobile
C-terminal domain, upon heating the sample, are severely attenuated in
intensity by solvent exchange, following saturation of the water
resonance (Fig. 3A). This demonstrates that there is weak or
no hydrogen bonding involving this part of the protein backbone other
than to solvent molecules.
The resonances from the N-terminal residues, at room temperature, have
far lower intensity than would be expected for a protein of around
13-15 kDa, indicative of a self-association process under the
conditions of the NMR experiments. Upon heating, resonances from this
region sharpen markedly, indicating the thermal dissociation of the
aggregate (Fig. 3A). In contrast to the resonances
corresponding to the mobile region, these resonances display the
chemical shift dispersion of a normally folded domain and include, for
example, the easily identifiable indole NH of Trp-33 located at the N
terminus of helix
The NMR experiments indicate that in solution the protein behaves as a
two-domain entity. One domain, corresponding to approximately the 67 N-terminal residues observed in the electron density map from the x-ray
experiment, exists in the fold determined above, although at room
temperature it is involved in a thermally labile, self-association
process. The second domain, corresponding to the remaining 60 C-terminal residues that are not observed in the electron density map,
has rapid internal motion and no well defined and stable fold involving
immobilized side chains. A detailed comparison of the spectra from the
intact wt and truncated forms of G39P revealed no major
difference between the two forms. Our findings strongly support the
idea that the disorder observed in the crystal structure for the
C-terminal region was not a result of the truncation of the protein nor
was it merely some form of crystal artifact but reflected an underlying
flexibility that may be closely related to the function of the protein.
Analysis of G39P Oligomeric State--
In order to examine further
the apparent difference between the oligomeric state of G39P
as observed in the crystal and that reported previously in solution
(6), the wt protein and the G39P112 mutant were subjected
both to gel filtration and analytical ultracentrifugation analyses.
The gel filtration studies suggested that under the conditions tested
(40 mM Tris-HCl, pH 8.0, containing 100 mM NaCl
at 4 and 25 °C) both the wt G39P and the
G39P112 mutant apparently exist largely as a dimer when in
the micromolar concentration range (Fig.
4) and as an equilibrium between dimer
and monomer when in the nanomolar concentration range (data not shown).
However, the NMR data above reveals a rapid equilibrium in solution
between dissociated and aggregated states of the protein and emphasizes the need for a more cautious interpretation of the results of gel
filtration experiments that are necessarily carried out over much
longer time scales. The observation of a species approximating to the
size of a dimer might actually arise from the rapid interchange between
the monomeric and aggregated forms of the protein and is further
complicated by an increase in the hydrodynamic radius arising from the
flexible C-terminal domain. Gel filtration of G39P samples
subjected to protein cross-linking in the micromolar concentration
range apparently revealed monomers, dimers, trimers, and higher order
oligomers, but interpretation of these results carries the same caveats
as described for the non-cross-linked gel filtration analysis with
respect to the rapid equilibration between the aggregated states of the
protein and its hydrodynamic radius.
In the analytical ultracentrifugation experiments, samples of the wt
G39P and G39P112 mutant proteins were subjected
to sedimentation velocity measurements under similar solvent conditions
to those used in the gel filtration experiment but at both acidic and
basic pH values (10 mM BisTris-HCl, pH 6.0, or Tris-HCl, pH
8.0, 160 mM KCl) and a protein concentration in the
micromolar range (Fig. 5). The average
mass calculated from both absorbance and interference scans of the
solute front was ~12.0 kDa for wt G39P and 12.4 kDa for
G39P112, which fits well with a monomer form. However, the plots of the rate of change of the concentration
(dc/dt) versus the sedimentation
coefficient S look slightly irregular for the wt G39P sample
at pH 8.0, and a better fit to a mixture of monomer and dimer can be
made, and it is reasonable to conclude that there is an equilibrium
between monomer and higher order oligomer forms favoring the monomeric
species under these conditions.
Limited Proteolysis Study of G39P--
An investigation of the
general susceptibility of G39P to proteolytic degradation
was performed using proteinase K and both wtG39P and an
N-terminal His-tagged variant. The analysis revealed a much more
pronounced sensitivity to proteolytic cleavage in the C-terminal half
of the protein that resulted in fragments corresponding to residues
1-79, 1-87, 1-90, 1-94, and 1-106 (Fig. 6). The identity of the fragments was
confirmed by the correspondence of the molecular weights determined by
mass spectrometry and by retention on a Ni-NTA column of equivalent
fragments (as assessed by SDS-PAGE) from the N-terminal His-tagged
variant.
Genetic and Biochemical Analysis of Deletion Mutants--
A series
of SPP1 gene 39 mutants was studied in complementation
assays, and their expressed protein products corresponding to fragments
of G39P were examined in vitro to test their
ability to interact with G40P-ATP
The gene 39 mutant, G39112, encoding the
truncated protein corresponding to residues 1-112, was established in
a plasmid-borne system, and this was used to test its ability to
complement the defect of the SPP1sus53 conditional lethal mutant. The
SPP1sus53 mutant allele has a suppressible mutation at the eighth codon of gene 39, and although a plasmid-borne wt 39 gene fully complements the defect of SPP1sus53, leading to a phage
yield indistinguishable from the titer of wt SPP1, the plasmid-borne
gene 39-112 mutant is unable to do so. This is consistent
with the fact that an SPP1 conditional lethal mutant with a
suppressible mutation at codon 103 of gene 39 (SPP1sus22)
has been isolated previously (4) and suggests that G39P112
is inactive as a loader and/or inhibitor of the G40P
helicase in vivo.
The full-length wt G39P protein is able to interact with
G40P-ATP Our structural analysis has shown that the G39P protein
has two domains: a stably folded 67-residue N-terminal domain and a
highly flexible and largely unfolded 59-residue C-terminal domain. This
correlates well with our biochemical observations that suggest a
bipartite nature for G39P in which the C-terminal domain has been implicated as the fragment of the protein responsible for the
interaction with the G40P helicase. The difference in the folding behavior of the two domains of the protein is striking and
unexpected and may also indicate some functional significance.
Multifunctional, multidomain proteins are common in biology, but
examples as small as the G39P protein are more rare. There is an increasing body of evidence to suggest that "natively
unfolded" proteins are quite common in vitro and possibly
in vivo (24) and that some can adopt more structured forms
only in the presence of partner or target molecules or other ligands
(25). Many of these "natively unfolded" proteins have been
implicated in disease states such as various forms of cancer,
Alzheimer's and Parkinson's diseases, and myotonic dystrophy (24),
and their unfolded nature has been linked often to their pathological
effects. The presence of ordered domains coupled to other largely
unfolded domains as observed for the N- and C-terminal domains in
G39P also has a precedent in other structural studies, and
in many of these cases a functional significance has been assigned to
the flexibility of the domains (26-28). Studies on the
Salmonella typhimurium regulatory protein FlgM suggest that
this protein is intrinsically unstructured when in dilute solution
in vitro (26). However, the C-terminal domain of FlgM is
observed to adopt a more structured form either when its partner
molecule, the RNA polymerase The flexibility of the C-terminal domain may also be intrinsic to the
ability of the protein to bind G38P and act as a linker in
the transfer of the G40P helicase onto its ssDNA target.
Upon transfer of the G40P-ATP to the DNA, the
G39P dissociates as a heterodimer with G38P (6).
The predicted pI of G38P is 9.0, and hence it is likely to
have a positive electrostatic surface consistent with its DNA binding
function, but this feature might also be important in the interaction
with G39P given its overall negative surface charge
distribution (see Fig. 2).
Our analyses of the oligomeric state of G39P when free in
solution suggest that it is most likely in a monomeric form at the sub-micromolar concentrations found in vivo but that it can
form higher order species or aggregates as the local concentration increases. The crystal structure implies that the oligomerization of
the monomers probably does not proceed via the initial formation of
2-fold rotationally symmetric dimers but rather the gradual building up
of larger species via growing chains of monomers that have 6-fold
symmetry potential. Indeed the primary function of the N-terminal
domain of G39P may be oligomerization for presentation of
the C-terminal domain to partner proteins, but we are currently investigating further possible roles in the interaction with
G38P.
Thus, this first crystal structure for a helicase loader/inhibitor
protein involved in
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-type DNA replication in many
different extrachromosomal elements follows a similar central scheme
but can differ in the requirements for host-encoded components and their remodeling (see Ref. 3). The most well characterized example of
initiation of DNA replication within a Gram-positive bacterial
environment is that of the Bacillus subtilis phage SPP1. Initiation of SPP1 replication requires the phage-encoded products of
genes 38, 39, and 40 (G38P,1
G39P, and G40P) in addition to the host DNA
polymerase III and DnaG primase (4). G38P (a monomer with a
predicted molecular mass of 29,997 Da) acts as a close functional
equivalent to DnaA (although the proteins share no sequence similarity)
and is the replisome organizer of the SPP1 system. The G38P
protein specifically interacts with its cognate site present in
multiple copies at the phage replication origins (oriL and
oriR). This interaction occurs in the absence of ATP and is
thought to induce the local unwinding of the adjacent A + T-rich
sequence present within oriL to initiate
-type DNA
replication (5-7). G40P is a DnaB-like helicase and as such
is a ring-shaped hexamer, capable of unwinding duplex DNA with a 5' to
3' polarity in a reaction fueled by nucleotide 5'-triphosphate
hydrolysis (6, 8). G39P is predominantly a monomer
(molecular mass 14,610 Da) when free in solution and forms a specific
interaction with ATP-activated G40P that inactivates the
ssDNA binding, ATPase, and unwinding activities of the helicase. Targeting of G40P by G39P to the
G38P-bound oriL then functions to activate
G40P upon delivery (6). It is believed that G40P, in the form of the G39P-G40P-ATP complex, is
delivered to G38P-bound oriL via the specific
protein-protein interaction of the helicase-bound G39P with
the G38P bound at oriL. These interactions result
in the formation of an unstable nucleoprotein
oriL-G38P·G39P·G40P-ATP intermediate, with subsequent release of
G38P/G39P heterodimers that leaves the
ATP-activated G40P complex to bind the melted origin region
(6). Uncomplexed G38P remains bound to oriL, and
the G40P helicase is free to interact with DnaG and the
subunit of both DNA polymerases and begin DNA unwinding (6, 9, 10). The
action of G39P protein is similar to that of the
bacteriophage
gene P helicase-loading protein, but P requires an
elaborate remodeling for freeing the helicase from the
helicase/helicase-loader complex (1, 3). Furthermore, the action of
G39P protein is quite distinct from that of the
bacteriophage T4 gene 59 helicase-loading protein whose
functions combine those of G38P and G39P as well as having a role in both replication and recombination (11, 12).
-type DNA replication that also functions as an
inhibitor of helicase function.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1. The SeMet G39P112 crystals were used in a
subsequent multiwavelength anomalous dispersion experiment in which
data were collected using a Mar Research 345 imaging plate scanner at
the European Synchrotron Radiation Facility on station BM30
using inverse beam geometry to collect Friedel pairs. The data for each
wavelength were processed individually and scaled in such a way as to
preserve anomalous signal using the HKL Suite of programs (13). The
data processing statistics are shown in Table
I. The positions of six selenium atoms
were found using the program SOLVE (14). These positions were then
refined, and initial phases were calculated in the program MLPHARE (15)
following the pseudo-MIR procedure (16). Phasing statistics are shown
in Table I.
Data collection, phasing, and refinement statistics
1 were centrifuged
in 1.20-cm path length, two-sector aluminum centerpiece cells with
sapphire windows in a four-place An-60 Ti analytical rotor running in a
Beckman Optima XL-I analytical ultracentrifuge at 50,000 rpm at
16 °C. Changes in solute concentration were detected by Rayleigh
interference and 280 nm absorbance scans. Results were analyzed by
g(s*) analysis (21) using the program DCDT+ version 1.13 (22).
5. SPP1 wt, SPP1sus53, and SPP1sus22 were
used to infect B. subtilis YB886 cells bearing plasmid-borne
gene 39 or gene 39-112, and manipulations
followed the standard procedures described for SPP1 (23).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices as follows:
A (residues 3-16);
B (residues
26-39);
C (residues 42-55), plus there is a very short
310 helix D (residues 62-65) comprising little more than
one turn. The helices can be described as two approximately parallel
pairs (
A/
C and
B/D) that cross at an angle of about 70°
(Fig. 1). The structure of the
bacteriophage T4 gene 59 helicase-loading protein also has two
-helical domains, but its N-terminal domain shows a strong structural similarity to the high mobility group family proteins (11),
which is not seen in the N-terminal domain of G39P.
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Fig. 1.
The sequence and crystal structure of
G39P. A, the amino acid sequence
of G39P with the secondary structure, the extent of the
ordered N-terminal domain observed in the crystal structure, and the
site of the C-terminal truncation are indicated, .
B, a portion of the refined protein model fitted to the
final 2Fo
Fc map, shown in
stereo. C, a stereo C
-backbone trace of the protein
with every 10th residue marked. D, a ribbon trace of a
monomer with the secondary structure elements labeled. Figure was
produced using the programs ALSCRIPT (35), BOBSCRIPT (36),
MOLSCRIPT (37), and Raster3D (38).
A and
C and those
preceding helices
B and D. The residues involved are both
polar and hydrophobic, and the interface includes two completely buried
water molecules. Pairwise superposition of the
-carbon positions of
each of the monomers gives root mean square deviation values of 0.2 to
0.3 Å. Approximately 1200 Å2 or 30% of the surface area
of each monomer is buried in the interfaces with other monomers in the
non-crystallographic 61 helix and a further ~10% on
average in contacts with other subunits in the crystal lattice. The
missing polypeptide chain in the electron density map, extending from
residue Lys-67, is directed toward a large cavity in the crystalline
lattice (Fig. 2) where it adopts a flexible, mainly unfolded state (see
NMR analysis below). Thus at least in the crystal lattice, the
G39P112 protein appears to exist as a monomer.
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Fig. 2.
The arrangement of monomers of
G39P112 in the crystal lattice and their electrostatic
surface potential. A, ribbon trace of the three
monomers in the a.u. labeled 1-3 and shown in red,
green, and blue. B, G39P112
monomers shown as ribbon trace and equivalent surface colored for
electrostatic potential (red 10 kcal
(mol·e)
1 and blue
10 kcal
(mol·e)
1). View is orthogonal to the
non-crystallographic 61 screw axis and shows two copies of
the a.u. (monomers labeled 1-3 and 1*, 2*, 3*).
C, the packing of adjacent helical repeats of
G39P112 monomers in the crystal lattice as viewed along the
non-crystallographic 61 screw axes, with the final residues
of the N-terminal domains marked C, indicating the locations
of the C-terminal domains. Figure was produced using the programs GRASP
(39), MOLSCRIPT (37), and Raster3D (38).
A and the N terminus and loop preceding helix
C that also
lies adjacent to the last observed residue in the map, Lys-67. The
distribution of charge is even more striking when one examines the
helical packing of the G39P monomers in the crystal that
reveals the helical array to have a very predominantly negatively
charged outer surface with the uncharged or positive surface mostly
buried in inter-monomer contacts or close to the helical axis (Fig. 2).
The unobserved, flexible C-terminal domain may modify the apparent
surface charge, but the calculated pI is 4.9 for the C-terminal 59 residues of G39P (the calculated pI for the N-terminal 67 residues is 5.2) and thus might suggest a generally negatively charged
surface. Apart from the slight imbalance in positively and negatively
charged residues that leads to the acidic pI, the C-terminal domain of
G39P does not show a particularly abnormal distribution of
residue type.
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Fig. 3.
NMR analysis of G39P.
A, the one-dimensional 1H NMR spectrum of
the G39P112 mutant protein is shown over a series of temperatures.
Resonances between 7.6 and 8.4 ppm principally arise from the backbone
amide protons of the mobile C-terminal domain. Their reduction in
intensity on increasing temperature reveals their lability to exchange
with saturated solvent protons. The resonances between 8.4 and 10.2 ppm
represent corresponding protons (and the indole NH proton of Trp-33 at
10.1 ppm) from the immobile N-domain. Their increase in intensity with
temperature reveals the thermal lability of a self-association process
involving this domain. B, the region of a
two-dimensional 1H1H-TOCSY spectrum (mixing
time 30 ms) containing correlations between the protons of the aromatic
rings of tyrosine and phenylalanine, illustrating the very high
intensity of the cross-peaks of the corresponding residues from the
mobile C-terminal domain, namely Phe-76 and Tyr-80. The relatively high
intensities of cross-peaks between primary amide protons of residues in
the mobile C-terminal domain can also be seen (upper left
and lower right).
B and found within the hydrophobic core of the
crystal structure of the N-terminal domain.
View larger version (17K):
[in a new window]
Fig. 4.
Gel filtration studies of
G39P. Superdex 200 HiLoad gel filtration column
chromatograms are shown of wtG39P (A) and
G39P112 (B). Experiments were performed in the
micromolar protein concentration range. Molecular weight standards are
shown, and the positions of their elution times are marked on the
chromatograms with the relevant number in a red
circle.
View larger version (23K):
[in a new window]
Fig. 5.
Analytical ultracentrifugation of
G39P. Plots of rate of change of the
concentration (dc/dt) versus
sedimentation coefficient S are shown. a,
wtG39P at pH 6; b, wtG39P at pH
8; c, G39P112 at pH 6; d,
G39P112 at pH 8. The experimentally derived plots are
presented (black) as well as the calculated plots based on
either a single species model for monomer (dark blue) or
dimer (green), or a two species model based on an
equilibrium between monomer and dimer (light blue).
View larger version (23K):
[in a new window]
Fig. 6.
Limited proteolysis of
wtG39P and His-tagged G39P. The
results of digestion of G39P with proteinase K are shown.
a, SDS-PAGE analysis of the products of digestion over
increasing time. Lanes 1-6 correspond to digestion of
wtG39P for 0 (lane 1), 0.5 (lane 2), 1 (lane 3), 2 (lane 4), 5 (lane 5), and
10 min (lane 6). Lanes 7 and 9 correspond to digestion of N-terminal His-tagged G39P for 1 and 0 min, respectively. Lane 8 corresponds to eluant from a
Ni-NTA column loaded with the products seen in lane 7,
i.e. following a 1-min digestion. b,
quantification of the results in lane 3 (1-min digestion of
G39P). c, matrix-assisted laser desorption
ionization/time of flight mass spectrometry analysis of G39P
digested for 1 min. The peak of 7,306 Da corresponds to the double
charge of full-length G39P. d, the residue
numbers for the predicted cleavage sites for bands/peaks
a-c are indicated by open arrowheads, and
G39P112 variant truncated at residue 112 is indicated by a
filled arrowhead. The N-terminal 67 residues seen in
the crystal structure are denoted in dark gray.
S and inhibit its
helicase, ATPase, or ssDNA binding activities (6). The results of these
studies can be analyzed in the light of the domain structure of
G39P revealed by this study.
S and to inhibit all three associated activities
(ssDNA binding, ATPase, and helicase activity (6)). Assays have been performed on fragments of G39P and have shown that the
G39P112 variant can neither exert a negative effect on
G40P activities nor compete out the wt protein from the
G39P-G40P-ATP complex (data not shown). A series
of G39P truncated variants with N termini deleted up to
residue 73 still show interaction with G40P-ATP as assessed
by affinity chromatography assay, but a variant consisting of the
N-terminal residues 1-68 does not (Fig.
7).
View larger version (19K):
[in a new window]
Fig. 7.
Binding of G40P by truncated
His-tagged G39P variants. A,
SDS-PAGE analysis of samples eluted from an Ni-NTA column
following affinity chromatography (see "Experimental Procedures").
The individual lanes show the results of using different
G39P constructs to capture G40P loaded on the
column. Lane 1, non-tagged wt G39P.
Lanes 2-7, N-terminal His-tagged proteins: lane
2, h39; lane 3, h 39N14; lane
4, h
39N22; lane 5, h
39N55; lane
6, h
39N73; lane 7, h
C69; and lane
8, C-terminal His-tagged protein
39C69h. The numbers
following the N or C indicate the extent of the truncation from the N
and C termini, respectively, and the constructs are shown schematically
in B.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-factor
28, is added
(26), when it is in vivo, or when in vitro
conditions are adjusted to match more closely those found in living
cells (29). Thus, it is possible that the C-terminal domain of
G39P may possess more structure under in vivo
conditions, even in the absence of partner molecules, than we have
observed in our experiments. However, it might also be the case that
the C-terminal domain of G39P is inherently flexible and has
little structure when not in a complex to enable the optimal
interaction of G39P with its G40P helicase
partner. This interaction has an apparent 6:6
G39P:G40P stoichiometry (6), and the flexibility
may be essential for the correct formation of a hexameric arrangement
of G39P on the surface of G40P that is still
accessible for interaction with origin bound G38P. Indeed
G39P may need to bind potentially to a number of monomer
forms of the G40P within the hexamer as these may vary
depending upon either the relative conformations of the helicase
subunits or the state of loading of ATP nucleotide or its hydrolyzed
products as observed for the T7 gene 4 helicase (30). Use of a largely
unfolded state to bind a variety of targets has been observed
previously (31), and the cyclin-dependent kinase inhibitor,
p21, has a completely unfolded native state that is suggested to
enhance its ability to bind multiple protein targets. Another possible
reason for maintaining a flexible C-terminal domain in G39P
could be to enable inactivation of the protein by rapid proteolytic
degradation (25). We have observed that removal of just the C-terminal
14 residues impairs G40P binding. Within the cell, random
unregulated DNA binding and unwinding by G40P-ATP would be
deleterious, and hence some control and targeting of its function is
required through the combined action of G38P and
G39P to ensure loading at the origin of replication.
However, once replication has commenced and the replication machinery
moved on from the origin, problems can arise from DNA damage, and the replication fork can stall with release of the replicative machinery and the requirement for subsequent reloading of these components after
damage repair. Recently, it has been shown that the loading of
G40P at any stalled replication fork by the SPP1
phage-encoded G35P protein can lead to replication fork
reactivation (32). At this point, binding of G40P by
G39P could be harmful, and thus the levels of
G39P might need to be kept low either through its interaction with other factors such as G38P or by its
degradation. Indeed, G39P accumulates very fast after phage
infection and reaches a plateau at minute 5, remains constant up to
minute 18, and goes to initial basal levels after minute 20, whereas
levels of G40P accumulate with similar kinetics to those of
G39P but remain constant until phage lysis (33). The reason
for the apparently abrupt stop in the proteolysis of G39P
after the removal of the 14 C-terminal residues under the experimental
conditions used prior to crystallization is still under investigation
as there is no obvious protease target site at this point. Our
proteolytic degradation experiments reveal that the extent of protease
sensitivity corresponds well with a more structured N-terminal domain
and a less structured C-terminal domain, although the presence of
substantial amounts of discrete fragments during the initial stages of
proteolysis may argue for some limited structure in the C-terminal
domain. Recent studies (34) on the structure of the E. coli
protein DnaC that loads the replicative helicase DnaB also suggest that
it is unusually flexible when free in solution. This prompts the
proposal that a high level of structural flexibility might be a
recurring theme in domains of loader proteins involved in
-type
replication that interact with replicative helicases.
-type DNA replication has revealed an
unexpected, highly plastic, bipartite structure that has developed to
fulfill multiple interaction functions and to ensure the critical loading of the replicative DNA helicase on the DNA origin of replication.
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ACKNOWLEDGEMENTS |
---|
We thank Michel Roth and the station staff on BM30 at the European Synchrotron Radiation Facility and the staff at the Central Laboratory of the Research Councils Daresbury Synchrotron Radiation Source laboratory.
![]() |
FOOTNOTES |
---|
* This work was supported in part by European Commission Grants BIO4-CT98-0106 and QLK2-CT-2000-00634 and an EMBL grant under the "Human Capital and Mobility" Programme. The Krebs Institute is a designated Biotechnology and Biological Sciences Research Council Biomolecular Sciences Centre and a member of the North of England Structural Biology Centre.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1NO1) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Present address: Dept. of Molecular Biophysics and Biochemistry, Yale University, Bass Center, Rm. 415, 266 Whitney Ave., New Haven, CT 06520-8114.
** Lister Institute Research Fellow.
§§ Royal Society Olga Kennard Fellow. To whom correspondence should be addressed. Tel.: 44-114-2222809; Fax: 44-114-2728697; E-mail: j.rafferty@sheffield.ac.uk.
Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M209300200
2 S. Bailey, S. E. Sedelnikova, P. Mesa, S. Ayora, J. C. Alonso, and J. B. Rafferty, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are:
GXP, gene
X product;
a.u., asymmetric unit;
ss, single-stranded;
wt, wild type;
BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol;
Ni-NTA, nickel-nitrilotriacetic acid;
ATPS, adenosine
5'-O-(thiotriphosphate).
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