(Received for publication, November 30, 1995; and in revised form, December 21, 1995)
From the
The direct evidence of dramatic conformational changes of the
DnaB hexamer, induced by nucleotide binding, and the presence of
multiple conformational states of the enzyme have been obtained by
using analytical sedimentation equilibrium, sedimentation velocity
studies, and the rigorous fluorescence titration technique. Equilibrium
sedimentation measurements show that in the presence of the ATP
nonhydrolyzable analog, AMP-PNP, the DnaB helicase fully preserves its
hexameric structure. However, in the presence of the saturating
concentration of AMP-PNP, the sedimentation coefficient of the hexamer
is s = 11.9 ±
0.2 compared to the sedimentation coefficient s
= 10.5 ±
0.2 of the free DnaB helicase hexamer. This large sedimentation
coefficient change indicates dramatic global conformational transitions
of the hexamer, encompassing all six subunits, upon binding
the ATP analog. In the presence of ADP, the sedimentation coefficient
is s
= 11.4 ±
0.2, indicating that the conformation of the ADP form of the hexamer is
different from the ATP form. The sedimentation coefficient of the
ternary complex DnaB-(AMP-PNP)-d
A(p
A)
, s
= 12.4, suggests
that the DnaB helicase undergoes further conformational changes upon
binding single-stranded DNA (ssDNA). The large global structural
changes correlate with the functional activities of the enzyme. In the
absence of the ATP analog, the hexamer exists in a ``closed''
conformation which has extremely low affinity toward ssDNA. Upon
binding the ATP analog, the DnaB hexamer transforms into a
``tense'' state which binds ssDNA with an affinity of
4
orders of magnitude higher than in the absence of the nucleotide. In
the presence of ADP, the DnaB hexamer assumes a ``relaxed''
conformation. The functional difference between these two conformations
is reflected in the much weaker allosteric effect of ADP on the ssDNA
binding with the affinity constant
3 orders of magnitude weaker
than in the presence of the ATP analog (tense state).
DnaB is the essential DNA replication protein in Escherichia
coli, originally identified based on its requirement for in
vitro X174 phage replication (Wickner et al., 1973;
McMacken et al., 1977). DnaB protein is the primary E.
coli replicative helicase, i.e. the factor responsible
for unwinding the DNA duplex in front of the replication fork. It is
the only helicase required to reconstitute DNA replication in vitro from the chromosomal origin of replication (oriC) (Kornberg and
Baker, 1992; LeBowitz and McMacken, 1986; Baker et al., 1987).
The enzyme is involved in both the initiation and elongation stages of
DNA replication (Matson and Kaiser-Rogers, 1990; Kornberg and Baker,
1992; Marians, 1992).
The native DnaB helicase forms a hexamer composed of six identical subunits (Reha-Krantz and Hurwitz, 1978; Bujalowski et al., 1994). Analytical sedimentation studies have shown that the DnaB helicase exists as a stable hexamer over a large protein concentration range with magnesium ions playing a crucial structural role in stabilizing the hexameric structure of the helicase. Hydrodynamic data indicate that six protomers aggregate with cyclic symmetry in which the protomer-protomer contacts are limited to only two neighboring subunits (Bujalowski et al., 1994).
Physiological functions of the DnaB helicase are related to the
ability of the protein to interact with ss- and dsDNA ()under the control of ATP binding and hydrolysis (Arai and
Kornberg, 1981a, 1981b; LeBowitz and McMacken, 1986). Studies of
nucleotide binding to the DnaB helicase have established that the
hexamer has six nucleotide binding sites, presumably one on each
protomer (Arai and Kornberg, 1981b; Bujalowski and Klonowska, 1993,
1994a, 1994b). The binding process is biphasic, resulting from the
negative cooperative interactions limited to neighboring subunits
(Bujalowski and Klonowska, 1993, 1994a, 1994b).
Interactions of the DnaB helicase with ssDNA and the structure of the formed complexes have only recently been quantitatively studied (Bujalowski and Jezewska, 1995). On the basis of thermodynamically rigorous fluorescence titrations, we have established that in the presence of the ATP nonhydrolyzable analog, AMP-PNP, stoichiometry of the DnaB hexamer complex with the polymer ssDNA (site size) is 20 ± 3 nucleotides. Binding studies performed with ssDNA oligomers have shown that the hexamer has only a single, strong ssDNA binding site. Moreover, photo-cross-linking experiments indicate that only a limited set of subunits, most probably only one, is engaged in the complex with the nucleic acid. These results indicate that long-range allosteric interactions occur on the level of the quaternary structure of the hexameric enzyme, leading to the selection of a limited set of subunits as a binding site for ssDNA. Such interactions require significant conformational changes of the hexamer, beyond the nearest neighboring protomers.
In this communication, we present the first direct
evidence that, in the presence of the ATP nonhydrolyzable analog,
AMP-PNP, the DnaB helicase undergoes global conformational transitions
which encompass all six subunits of the hexamer. The sedimentation
coefficient of the hexamer increases by 14% when compared to the
free hexamer. Binding of ADP to the DnaB helicase causes similar,
albeit smaller, global conformational changes. Thus, the nucleotide
binding and the ATP/ADP switch, which takes place after every ATP
hydrolysis step, are accompanied by significant conformational changes
of the entire DnaB hexamer. We report the first quantitative analysis
of the allosteric effect of nucleotide cofactors on the DnaB
helicase-ssDNA complex formation.
where c, t;ex2html_html_special_mark_amp;ngr;, and M are the concentration (absorption) at the bottom of the
cell, partial specific volume, and molecular weight of the protein,
respectively,
is the density of the solution,
is the
angular velocity, and b is the base line error term.
Equilibrium sedimentation profiles were fitted to with M and b as fitting parameters. The reported values of
the sedimentation coefficients were corrected to the standard
conditions, s
, for solvent density
and viscosity (Cantor and Schimmel, 1980).
where K is the intrinsic binding constant, n is the number of nucleotides covered by the protein in the complex
(site size), is the parameter characterizing cooperativity, and R = {[1 - (n +
1)
]
+ 4
(1 -
n
)}
. Binding of the DnaB helicase to the
polymer ssDNA has been analyzed using .
Figure 1:
DnaB helicase binding to ssDNA.
Fluorescence titrations of poly(dA) with the DnaB protein
monitored by the increase of the nucleic acid fluorescence in buffer T2
(pH 8.1, 10 °C) containing 50 mM NaCl, in the presence of
1 mM AMP-PNP (
), 1 mM ADP (
), and in the
absence of nucleotide cofactors (
), respectively. The nucleic
acid concentration is 2
10
M (nucleotide). Solid lines are computer fits of the
binding isotherms, using , with intrinsic binding constants K = 1.3
10
M
, 5.9
10
M
, and 60
10
M
for isotherms obtained in the
presence of AMP-PNP, ADP, and in the absence of nucleotides,
respectively. For the titration in the presence of AMP-PNP, the
cooperativity parameter
= 3.5 and
F
= 3.5. For titrations in the presence of ADP and the
absence of nucleotide cofactors, the value of the maximum increase of
the nucleic acid fluorescence,
F
, was
assumed to be 3.5, the same as determined for the titration in the
presence of AMP-PNP.
Figure 2:
a, sedimentation velocity absorption
profiles recorded at 292 nm of the DnaB hexamer in buffer T2 (pH 8.1,
10 °C) containing 50 mM NaCl and 5 10
M AMP-PNP. The concentration of the DnaB protein is 3
10
M (hexamer); 8-min intervals,
30,000 rpm. b, sedimentation equilibrium absorption profiles
of the DnaB helicase at two different wavelengths, 285 nm (
)
and 292 nm (
), in buffer T2 (pH 8.1, 10 °C) containing 50
mM NaCl and in the presence of 5
10
M AMP-PNP. Solid lines are nonlinear
least-squares fits to , with a single sedimenting species
having molecular weights of 303,000 (
) and 301,000 (
),
respectively. The DnaB protein concentration is 5.59
10
M (hexamer); 9,000
rpm.
It
should be pointed out that in our studies the error in the measurement
of the sedimentation coefficients, for each particular system, is a
standard deviation determined from 8 to 12 independent sedimentation
velocity experiments using two different protein concentrations. The
obtained value of s of the
DnaB-(AMP-PNP) complex is
14% higher than the sedimentation
coefficient of s
= 10.5
± 0.2 of the free DnaB hexamer (Bujalowski et al.,
1994). The magnitude of this increase of the sedimentation coefficient
can be realized by recalling that a simple dimerization of two hexamers
into a dodecamer would lead to an
58% increase of its
sedimentation coefficient (Cantor and Schimmel, 1980). To exclude the
possibility that the nucleotide binding affects the oligomeric state of
the DnaB hexamer, we performed sedimentation equilibrium measurements
of the DnaB helicase in the presence of saturating concentrations of
AMP-PNP. A set of DnaB protein concentration profiles (recorded at two
different wavelengths, 285 and 292 nm) as a function of the square of
the radius at sedimentation equilibrium, in buffer T2 (pH 8.1, 10
°C) containing 50 mM NaCl and 5
10
M AMP-PNP, are shown in Fig. 2b. The solid lines are nonlinear least-squares fits to a single
exponential function (). The excellent agreement between
the theoretical lines and the experimentally obtained concentration
profiles indicates that there is a single species in the solution. The
analysis of the concentration profiles provides the molecular weights
of 303,000 ± 10,000 and 301,000 ± 10,000 for scans
recorded at 285 and 292 nm, respectively. Comparison between the
obtained data and the known molecular weight of the DnaB hexamer
(313,590) shows that the DnaB helicase fully preserves its hexameric
structure in the presence of AMP-PNP (Bujalowski et al.,
1994). Therefore, since the six bound molecules of AMP-PNP per hexamer
constitute only
1% of the molecular weight of the protein, a large
increase of the sedimentation coefficient in the presence of AMP-PNP
indicates dramatic conformational changes of the entire hexamer induced
by the binding of the ATP analog (see ``Discussion'').
The
thermodynamic studies of the binding of the DnaB helicase to ssDNA
indicate that ADP also induces conformational transitions in the
enzyme; however, this leads to a modest increase of the binding
affinity toward the ssDNA (Fig. 1). Analogous sedimentation
velocity experiments (data not shown) of the DnaB hexamer in the
presence of the saturating concentration of ADP (5
10
M) show that the sedimentation
coefficient of the helicase is s
= 11.4 ± 0.2, a value significantly higher than the
sedimentation coefficient of the free enzyme. However, this value is
lower when compared to s
=
11.9 ± 0.2 obtained in the presence of the ATP analog, AMP-PNP,
and suggests conformational differences between these two complexes
(see ``Discussion'').
It is very interesting to determine
how the binding of the ssDNA affects the conformational transitions of
the DnaB hexamer induced by nucleotide binding. Because the etheno-
derivatives of the nucleic acids have significant absorption above 310
nm, where there is practically no contribution of the protein spectrum,
this allows us to monitor only one component, the nucleic acid, during
the sedimentation process. Sedimentation velocity profiles of the
mixture of the DnaB helicase and dA(p
A)
in the
1.25:1 molar excess of the enzyme over the nucleic acid are shown in Fig. 3. Recall, the 20-mer exactly spans the site size of the
DnaB helicase-ssDNA complex (Bujalowski and Jezewska, 1995). Because
the [DnaB] and [d
A(p
A)
] are
1/K
, all the nucleic acid should be complexed
with the helicase (Bujalowski and Jezewska, 1995). As a result, only a
single, well-defined boundary of the complex is observed throughout the
entire sedimentation process and has a sedimentation coefficient of s
= 12.4 ± 0.2. This
value is
17% higher than the sedimentation coefficient of the free
DnaB hexamer and
3 and
9% higher than the sedimentation
coefficient of the DnaB helicase, when saturated with the ATP analog or
ADP. This suggests that the binding of ssDNA, most probably, introduces
additional conformational changes in the ternary complex,
DnaB-(AMP-PNP)-d
A(p
A)
(see
``Discussion'').
Figure 3:
Sedimentation velocity profiles.
Sedimentation velocity absorption profiles recorded at 315 nm of the
mixture of dA(p
A)
-DnaB helicase at 1.25:1 molar
excess of the DnaB protein over the nucleic acid in buffer T2 (pH 8.1,
20 °C) containing 50 mM NaCl and 5
10
M AMP-PNP. The concentration of the DnaB protein is 5
10
M (hexamer), and the
concentration of the nucleic acid is 4
10
M (oligomer); 8-min intervals, 30,000
rpm.
Hydrodynamic properties,
including the sedimentation coefficient, are directly sensitive to the
global conformational properties of the macromolecules (Cantor and
Schimmel, 1980). Using analytical sedimentation velocity experiments
and the rigorous fluorescence titration technique, we present direct
evidence that the E. coli primary replicative helicase DnaB
protein undergoes dramatic conformational transitions induced by
binding of the ATP nonhydrolyzable analog, AMP-PNP, and ADP. The
sedimentation coefficient of the free DnaB hexamer increases from s = 10.5 to s
= 11.9 for the hexamer in
the presence of 5
10
M AMP-PNP and
to s
= 11.4 in the presence
of 5
10
M ADP. At these
concentrations of AMP-PNP and ADP, all six binding sites are saturated
with the nucleotide (Bujalowski and Klonowska, 1993).
The very large
increase of the sedimentation coefficient does not result from the
dimerization of the DnaB hexamer induced by nucleotide binding.
Equilibrium sedimentation measurements show that, in the presence of
the saturating concentration of AMP-PNP, the hexameric structure of the
enzyme is fully preserved (Fig. 2b). In the absence of
oligomerization, the sedimentation coefficient of the protein can
change as a result of changes of its partial specific volume or
frictional coefficient, or both. Partial specific volume of the ATP
molecule is t;ex2html_html_special_mark_amp;ngr; = 0.44 ml/g compared
with t;ex2html_html_special_mark_amp;ngr; = 0.732 ml/g of the DnaB
protein (Howlett and Schachman, 1977; Bujalowski et al.,
1994). However, the six bound nucleotide molecules constitute only
1% of the molecular weight of the hexamer and the trivial effect
of a lower t;ex2html_html_special_mark_amp;ngr; of the bound nucleotides on
the measured s
, would amount to
0.004; thus, it is negligible. Moreover, the additional data
suggest that the global conformational changes are predominantly
induced by the binding of only the first three nucleotide molecules to
the three high affinity binding sites of the hexamer. (
)Therefore, regardless of the nature of the observed
conformational transitions, the large increase of the sedimentation
coefficient indicates large global structural changes of the DnaB
hexamer, which are induced by ATP analog binding, and these structural
transitions lead to intrinsic changes of the partial specific
volume of the hexamer and/or its frictional coefficient. Although the
exact nature of the conformational transition induced by nucleotide
binding is still unknown, it should be mentioned that dynamic light
scattering data indicate that the diffusion coefficient of the hexamer
is increased in the presence of the saturating concentration of
AMP-PNP, indicating that the frictional coefficient of the hexamer is decreased, in agreement with the sedimentation data reported
here.
Binding of ADP to the DnaB helicase also induces
global conformational changes in the protein hexamer. The sedimentation
coefficient, s = 11.4, is
increased by
9% when compared to the free DnaB hexamer; however,
this value is smaller than the 11.9 obtained in the presence of
AMP-PNP. The difference is larger than the error of the determination
of s
(± 0.2) estimated using
multiple scans at different protein concentrations (see above). Thus,
the sedimentation data suggest that the conformation of the hexamer-ADP
complex differs from that of the free DnaB protein and is also
different from the DnaB helicase-(AMP-PNP) complex. As a result, the
affinity of the hexamer toward ssDNA drops by
3 orders of
magnitude when compared to the affinity of the DnaB-(AMP-PNP) complex
(see above). The difference between the effect of AMP-PNP and ADP does
not result from the weaker binding of ADP to the helicase. As we
determined previously, using a series of fluorescent nucleotide
analogs, the ADP and ADP analogs bind with higher affinity to the DnaB
helicase than the ATP analogs (Bujalowski and Klonowska, 1993, 1994a,
1994b). Thus, the allosteric effect of AMP-PNP on the DnaB global
conformation, which leads to the dramatically increased affinity of the
helicase to ssDNA as opposed to the dramatic drop in the affinity in
the presence of ADP, must result from the specific interactions of
-phosphate in the nucleotide binding site.
The sedimentation
coefficient of the DnaB-(AMP-PNP)-dA(p
A)
ternary
complex, s
= 12.4 ±
0.2, indicates that conformational changes induced by AMP-PNP binding
are preserved in the ternary complex. It should noted that the increase
of the sedimentation coefficient of the ternary complex exceeds the
value expected from the combined trivial effects of the different
partial specific volume of the nucleic acid and the higher molecular
weight of the DnaB hexamer-20-mer complex. The bound 20-mer constitutes
only an additional
2% of the molecular weight of the complex. The
trivial effect of the lower partial specific volume of the nucleic acid
molecule (t;ex2html_html_special_mark_amp;ngr; = 0.531 ml/g, Pearce et al.(1975)) and the increased molecular weight of the
complex would contribute
0.008 and
0.2 to the measured
sedimentation coefficient, respectively (Cantor and Schimmel, 1980).
Moreover, binding of the shorter 10-mer ssDNA fragment (data not shown)
causes the same increase of the sedimentation coefficient of the
hexamer (s
= 12.5 ±
0.2) as the 20-mer, although it has 10 less nucleotide residues than
the 20-mer and constitutes only
1% of the molecular weight of the
hexamer. If the trivial effects were mainly responsible for the
observed increase of s
of the
ternary complex, then the effect of the 10-mer should be half of the
one observed for the 20-mer, but this is not what is experimentally
observed. Thus, the increased value of s
of the ternary complex suggests that, most probably, the DnaB
helicase undergoes further conformational changes upon binding ssDNA,
although the obtained hydrodynamic data indicate that the major
conformational transition of the helicase is induced by AMP-PNP or ADP
binding.
The mechanism of the dsDNA unwinding by the replicative helicase and the mechanism of the enzyme translocation on the nucleic acid lattice is still unknown. In general, after breaking hydrogen bonds between the base pairs of the duplex DNA in the replication fork, the enzyme must be released from the formed ss nucleic acid and move, in an unidirectional translocation event, toward dsDNA (Hill and Tsuchiya, 1981). Our recent results show the existence of only a single, strong binding site and a very low stoichiometry of the DnaB-ssDNA complex. These results are not compatible with the models of hexameric helicase translocation along the nucleic acid lattice in which all six protomers and/or multiple binding sites are involved in ssDNA binding (Bujalowski and Jezewska, 1995).
The existence of different conformational states of the hexameric helicase described in this work strongly suggests that the mechanism of translocation and nucleic acid unwinding might rely on global, not local, conformational changes in the hexamer which are induced by the ATP/ADP switch and/or nucleic acid binding. Such conformational transitions were postulated as necessary elements of the helicase mechanism by Hill and Tsuchiya(1981). Thus, because the transition from the tense to the relaxed state of the helicase must accompany each ATP hydrolysis step, when the ATP/ADP switch takes place in the nucleotide binding site, this transition is most probably responsible for the partial release of the ssDNA from the nucleic acid binding site (Arai and Kornberg, 1981b). After the release of ADP, the global rearrangement of the protomers within the entire DnaB hexamer, from a closed conformation to a tense one upon rebinding ATP, would allow the enzyme to translocate and encompass the subsequent fragment of dsDNA, within the active site of the helicase.