(Received for publication, November 22, 1994; and in revised form, January 16, 1995)
From the
Sedimentation and high performance liquid chromatography studies
show that the functional DNA replication helicase of bacteriophage T4
(gp41) exists primarily as a dimer at physiological protein
concentrations, assembling from gp41 monomers with an association
constant of 10
M
.
Cryoelectron microscopy, analytical ultracentrifugation, and
protein-protein cross-linking studies demonstrate that the binding of
ATP or GTP drives the assembly of these dimers into monodisperse
hexameric complexes, which redissociate following depletion of the
purine nucleotide triphosphatase (PuTP) substrates by the
DNA-stimulated PuTPase activity of the helicase. The hexameric state of
gp41 can be stabilized for detailed study by the addition of the
nonhydrolyzable PuTP analogs ATP
S and GTP
S and is not
significantly affected by the presence of ADP, GDP, or single-stranded
or forked DNA template constructs, although some structural details of
the hexameric complex may be altered by DNA binding. Our results also
indicate that the active gp41 helicase exists as a hexagonal trimer of
asymmetric dimers, and that the hexamer is probably characterized by D3
symmetry. The assembly pathway of the gp41 helicase has been analyzed,
and its structure and properties compared with those of other helicases
involved in a variety of cellular processes. Functional implications of
such structural organization are also considered.
Studies of a variety of systems that catalyze the elongation
phase of DNA replication (Kornberg and Baker, 1992) have shown that
most can be described in terms of three functional components or
subassemblies. These are: (i) the central polymerase molecules, which
often carry a 3` 5` exonuclease (editing) activity in addition
to their template-directed 5`
3` polymerization function; (ii) a
set of accessory proteins that interact with the polymerases to form
``holoenzyme'' complexes that can synthesize leading strand
DNA processively and at physiological rates and fidelities; and (iii)
helicase-primase (``primosome'') complexes that open the
double-stranded DNA ahead of the overall replication complex, prime
(with RNA) the initiation of the discontinuously synthesized lagging
strands, and integrate the function of the holoenzymes involved in
leading and lagging strand synthesis with the overall movement of the
replication fork. The number of discrete protein subunits required to
operate and integrate each of these functional subassemblies varies
from one organism to another, but this general pattern of organization
seems to prevail from simple bacteriophages to complex eukaryotes.
In keeping with this organizational pattern, the elongation phase of
DNA replication in bacteriophage T4 also requires the coordinated
interaction of three such sets of components, which here consist of
seven discrete T4-coded protein species (for reviews, see Alberts
(1987), Cha and Alberts(1988), or Nossal(1994)). These are the
DNA-dependent DNA polymerase (gp43), ()which together with
three accessory proteins (gp44, gp45, and gp62), and a single-stranded
DNA-binding protein (gp32), form the processive holoenzyme. Efficient
DNA replication on double-stranded DNA templates also requires the
participation of a primosome subassembly, which in T4 contains helicase
(gp41) and primase (gp61) subunits and catalyzes the ATP or GTP-driven
unwinding of the double-stranded DNA template, as well as the synthesis
of the pentameric RNA primers required for coordinate lagging-strand
DNA synthesis. This relative parsimony of required components has made
the T4 system a useful paradigm system for mechanistic studies of DNA
replication.
The interactions of the polymerase and the accessory
proteins within the T4 holoenzyme are reasonably well understood (see
Young et al. (1992, 1994a) for recent reviews), but little is
yet known about the properties and function of the primosome at the
structural or mechanistic level. Many lines of evidence suggest that
gp41 and gp61 interact to form a primosome complex that provides both
primase and helicase activities. In studies in particular from the
Alberts and Nossal laboratories, gp41 and gp61 have been purified and
characterized as enzymes, and some aspects of their helicase and
primase functions have been described. The gp41 helicase exhibits a
single-stranded DNA-stimulated GTPase/ATPase activity and a 5`
3` DNA helicase activity that is driven by PuTP hydrolysis (Morris et al., 1979; Liu and Alberts, 1981a; Venkatesan et
al., 1982). Analysis of the dependence of the steady-state
kinetics of this DNA-stimulated PuTPase activity as a function of
single-stranded DNA length has also shown that PuTP hydrolysis drives
processive (and unidirectional) gp41 translocation along the DNA (Young et al., 1994b, 1994c; Liu and Alberts, 1981a).
The gp61 primase alone can catalyze limited, templatedependent RNA primer synthesis, with the dimers ppApC and ppGpC as major products (Hinton and Nossal, 1987; Nossal and Hinton, 1987; Cha and Alberts, 1990). However, the physiologically relevant primers produced by the T4 primosome are pentaribonucleotides (Kurosawa and Okazaki, 1979), and the dinucleotides made by gp61 primase alone are utilized only poorly by the T4 DNA polymerase as primers of lagging-strand DNA synthesis.
It is clear that the physiologically relevant properties and activities of gp41 and gp61 are only displayed when these two proteins are integrated as a primosome complex. Thus the primosome is required to unwind double-stranded DNA at high rates, to synthesize the physiologically relevant pentaribonucleotide RNA primers efficiently (Cha and Alberts, 1986; Hinton and Nossal, 1987; Liu and Alberts, 1980, 1981b), and to coordinate leading and lagging strand DNA synthesis at a DNA replication fork (Nossal and Hinton, 1987; Cha and Alberts, 1990).
As a step in developing a molecular picture of the proteins of the
T4 primosome, we present here a detailed study of the assembly and
properties of gp41 alone. We show that at physiological protein
concentrations this protein exists primarily as a dimer, although at
somewhat lower concentrations a dynamic monomer dimer
equilibrium can be demonstrated. On binding ATP or GTP substrates (or
their nonhydrolyzable analogs), these dimers assemble to a hexameric
complex that can interact with DNA models of the replication fork and
exhibit the DNA-stimulated PuTPase activity required for helicase
action. Cross-linking and cryoelectron microscopic studies suggest that
the hexamer is structured as a trimer of asymmetric dimers in the form
of a ring-shaped hexagon. Under ``Discussion,'' this complex
is compared with those of other (largely hexameric) helicases that have
recently been characterized, and the functional implications of this
assembly mode are considered.
Figure 1: Nucleotide sequences of the DNA replication fork constructs that were used as the helicase binding substrate in the protein-protein chemical cross-linking and the cryoelectron microscopic imaging experiments. A, a forked DNA template with a double-stranded DNA handle and two single-stranded DNA tines. The lengths of the two single-stranded tines on this forked DNA template (30 and 35 nucleotides for the 3` and 5` overhanging single-stranded sequences, respectively) were designed on the basis of the known requirements of the helicase (Venkatesan et al., 1982; Richardson and Nossal, 1989a; Hinton et al., 1987), and the two single stranded homo-oligonucleotides were used to minimize the formation of possible secondary structures. B, a forked DNA template that has been used in our laboratory for studies of the T4 DNA replication accessory proteins (Reddy et al., 1993).
The synthetic fork
constructs were assembled from the component strands by mixing
equimolar quantities of the partially complimentary oligonucleotides in
HKAE hybridization buffer, usually at 10-100 µM concentrations. Samples were incubated at 80 °C for 2-5
min to remove residual secondary structure and then slow-cooled to room
temperature over a period of 1-2 h to allow hybridization to
proceed. The formation of unique hybrid complexes was assayed and
verified by electrophoresis on a 10% (20:1 acrylamide:bisacrylamide)
nondenaturing polyacrylamide gel. Gels were run at low current (20 mA)
in TAE running buffer to prevent heating, and 5 mM MgCl was added to both the gel and the running buffer to stabilize the
hybridized complexes. Aliquots of the synthetic fork constructs were
stored frozen at -20 °C in HKAE buffer.
where M is the molar mass and v the
partial specific volume of the molecule, is the
density of the solvent, f is the frictional
coefficient, and N
is Avogadro's
number. For an unhydrated, spherical
macromolecule
where is the viscosity of the solution and R
the radius of the molecule. Hence, from ,
Therefore, for an unhydrated and spherical molecule,
when s is in Svedbergs and all other
factors are in cgs units.
In our calculations we have modeled the gp41 monomer as a hydrated spherical protein, using the empirical equation noted by van Holde (1975), which has been shown to be applicable to many globular proteins of average hydration
Here s is the sedimentation
coefficient (in Svedbergs) of the gp41 monomer at 20 °C in water.
The molar mass and partial specific volume for the gp41 protein were
calculated on the basis of the amino acid composition of the protein,
and were 53,554 g/mol and 0.735 cm
/g, respectively.
Sedimentation coefficients for the gp41 dimer and various hexamer forms were calculated using the hydrodynamic simulation method ofKirkwood(1954) and the equation of van Holde(1975):
where s is the sedimentation
coefficient predicted for a rigid structure containing n identical spherical beads, each with radius R
and sedimentation coefficient s
. R
is the
center-to-center distance between these beads. Therefore each value of
corresponds to a ratio of an intersubunit
distance to the subunit radius that has been derived on the basis of
the assumed geometry of the complex.
As a consequence the sedimentation coefficient for any complex can be calculated using:
Association constants (K) for the gp41 dimerization reaction
were estimated using:
Here f represents the fraction of
monomer present at each total gp41 concentration ([gp41]). The data were plotted as
f
/(1 - f
) versus (1/[gp41]), and K
was
calculated from the slope of the best straight line fitted to the data
with an intercept of zero.
Specimens were applied to EM grids coated with perforated carbon films, and were blotted and frozen as described elsewhere (Gogol et al., 1992). Sample preparation was carried out in a 4 °C coldroom to minimize evaporation prior to freezing. Solutions containing gp41 were maintained at 37 °C from the time of nucleotide addition until applied to the grids. Three or four samples were prepared from each solution within approximately 1 min, resulting in a standard incubation time (at 37 °C) in the presence of nucleotide triphosphates of 1.2-2 min between the freezing of the first and last grids. Grids were subsequently transferred and stored under liquid nitrogen until examined in the cryoelectron microscope.
The specimens were imaged
using a Philips EM400 electron microscope equipped with a low dose
operations kit (Philips Electronic Instruments, Mahwah, NJ) and a
custom-made double-bladed anticontaminator. Specimens were loaded in a
Gatan (Pleasanton, CA) cryotransfer apparatus and maintained at
temperatures below -160 °C at all times. Images were recorded
on Kodak SO163 film using minimal-dose (20 e
/A
) methods at a magnification
of 57,000. The magnification of the microscope was calibrated by
imaging thin negatively stained crystals of catalase at the same
objective lens current used to record the gp41 images. Images were
recorded at a defocus of 1 µm, which correctly represents spatial
information down to a resolution of approximately 20 A.
Image
alignment and averaging were performed with the SPIDER software package
(Frank et al., 1981). Areas of micrographs, selected for image
clarity and the presence of a suitable number of particles, were
digitized with an Eikonix 1412 digitizing camera at a magnification
that yielded individual 25 25-µm image elements on the
film, corresponding to 4.4
4.4-A areas on the specimen.
Digitized images were displayed on a VaxStation 3100 (Digital Equipment
Corp., Woburn, MA), and individual particles were selected from the
regions of thin ice that spanned the holes in the carbon support film.
The only criterion used to select molecules for image analysis was
that the chosen particles be fairly circular in shape and show no
obvious overlap with nearby molecules. Images were aligned by iterative
translational and rotational matching to a reference particle chosen
from the micrograph on the basis of its clarity and contrast.
Subsequent cycles of alignment used the averaged image as a reference.
Different starting images were used to test the dependence of the final
alignment and the resulting averaged image on the choice of initial
reference image, and the results of these parallel alignments were
compared visually. In most cases, alignments starting with different
reference images converged to visually identical averaged images after
the second cycle. For the most highly variable data set (the
nucleotide-gp41 complexes in the absence of DNA), a
``reference-free'' alignment procedure described by Penczek et al.(1992) was applied. This procedure reduced the
dependence of the final average on the selection of the initial
reference. For each set of images 10% of the aligned images were
discarded on the basis of perceived poor alignment (judged both
visually and by correlation coefficient versus the reference
image), and the remainder were averaged.
Figure 2:
Traces of sedimentation boundary scans
from XLA analytical ultracentrifugation runs. Samples contained 5
µM T4 gp41 protein in the absence (A) and the
presence (B) of 0.1 mM GTPS (see
``Materials and Methods'' for details of experimental
conditions). Intervals between scans in each experiment were
10
min.
Figure 3:
Integral distributions of the
sedimentation coefficients of samples along the respective sedimenting
boundaries: , g41p helicase in TAMK buffer;
, g41p
helicase in the presence of 0.1 mM GTP
S in TAMK
buffer.
These results show that both forms of gp41
are quite homogeneous at total gp41 (monomer) concentrations down to
about 0.5 µM, and that, under these conditions, both
samples sediment as fairly monodisperse species. However, as is
suggested by the trend toward lower values of s at the lower ends of both
sedimentation profiles in Fig. 3, the dominant states of
association present under both sets of conditions exist in equilibrium
with smaller species, and these smaller species start to make
appreciable contributions at the lower concentration ends of the
sedimentation profiles. This surmise will be confirmed by experiments
described below, in which we show (using protein-protein cross-linking
experiments carried out under the same conditions and HPLC experiments
at still lower protein concentrations), that gp41 in the absence of
PuTP
S actually exists as an equilibrium mixture of gp41 monomers
and dimers, with dimer representing the most abundant species at
protein concentrations >0.5 µM. We also use (see below)
protein cross-linking and cryoelectron microscopic methods to show that
the larger species (s
= 12.2
S) observed in the presence of 0.05-1 mM GTP
S
corresponds to gp41 hexamers, and suggest that these species are also,
at lower protein concentrations, in equilibrium with smaller states of
association on the assembly pathway (presumably dimers and tetramers).
In further confirmation of this latter assignment, velocity
sedimentation experiments were performed with purified gp41 hexamers
that had been cross-linked to completion in the presence of GTPS
and then run in the ultracentrifuge in the absence of nucleotides. The
sedimentation coefficient obtained for these cross-linked hexamers was
very similar to that measured with the non-cross-linked material in the
presence of 0.1 mM GTP
S (see Table 1); the slightly
higher sedimentation coefficient of the cross-linked species probably
reflects some molecular compaction resulting from cross-link formation.
Hydrodynamic modeling calculations based on these sedimentation
measurements (see below) also strongly support the conclusion that gp41
exists largely as dimers at micromolar protein concentrations, and that
these dimers associate to form hexamers in the presence of GTPS. (
)As we will show below, gp41 dimers can also associate to
form hexamers in the presence of the physiological substrates ATP or
GTP. However, the gp41 hexamers formed in the presence of these
hydrolyzable NTPs are short-lived and redissociate to the largely dimer
state after hydrolysis. Thus we conclude that PuTP
S can be used to
``freeze'' the otherwise transient PuTP-bound
(pre-hydrolysis) state of the helicase. The fact that the
non-hydrolyzable substrate analogs, ATP
S or GTP
S, can drive
the hexamerization of this helicase protein also indicates that only
binding of ATP or GTP to gp41 is required for hexamer formation, and
that hydrolysis of these substrates results in reversion to the largely
dimeric state.
Figure 4: HPLC molecular exclusion chromatograms for gp41 at three selected total protein concentrations. The amounts of gp41 injected into the columns were (respectively): a, 500 pmol; b, 1 nmol; c, 2 nmol. Positions of the dimer and monomer elution peaks are indicated by D and M, respectively. The corresponding elution times are approximately 23 and 25 min for dimer and monomer, respectively. The average total concentration of gp41 in each elution peak are noted.
Variation of the total protein concentration from 100 nM to micromolar concentrations significantly changed the distribution of protein between the two peaks, with the size of the peak corresponding to the larger species increasing with increasing protein concentration, and that of the peak corresponding to the smaller species decreasing in proportion. The larger species became undetectable when the total gp41 concentration in the column fell below 100 nM (data not shown). These results suggest that the larger species (earlier eluting peak) seen in these experiments corresponds to gp41 dimers, and that the smaller species corresponds to gp41 monomers.
The relative changes in the areas of the two peaks with change in
total protein concentration also permit us to estimate a monomer
dimer association constant. The data from a series of these experiments
are plotted in Fig. 5as the fraction of the monomer present versus total protein concentration. When this plot was
analyzed as described under ``Materials and Methods,'' a
dimerization constant (K
) of 0.7 (±
0.2)
10
M
was obtained.
We note that this value should be considered to be approximate, since
in addition to the experimental uncertainty contained in the standard
error given, conditions within gel filtration columns of this type may
not reflect true thermodynamic equilibrium. (
)However,
experiments involving injection of constant amounts of protein at
varying protein concentrations and volumes (e.g. 100-µl
injection of a 5 µM sample versus 25 µl of a
20 µM sample) showed that the gp41 protein does
equilibrate between monomers and dimers, since these procedures
produced indistinguishable elution profiles. These HPLC results clearly
demonstrate the existence of both gp41 monomers and dimers at these
total protein concentrations, and the value of K
of
10
M
obtained
from the HPLC data is in good agreement with an estimate of this
parameter based on protein-protein cross-linking and sedimentation
experiments (see below).
Figure 5:
Analysis of gp41 dimerization in the
absence of nucleotide triphosphate substrates. Data from HPLC molecular
exclusion chromatography were plotted using
f/(1 - f
)
as a function of the reciprocal of total gp41 protein concentration on
elution (see text).
Fig. 6A shows that cross-linking to completion at
low (200 nM) protein concentrations in the absence of PuTP (or
PuTPS) yielded primarily an intramolecularly cross-linked gp41
monomer, which migrated considerably faster in an SDS gel
electrophoresis (Fig. 6A) than did the noncross-linked
gp41 monomer (presumably due to the molecular compacting and
neutralization of positive charges of the protein after cross-linking).
In contrast, Fig. 6A also shows that in the presence of
GTP
S all of the gp41 present associates to form large cross-linked
complexes, resulting in a single high molecular weight electrophoresis
band. The average size of the complexes present in the high molecular
mass band was estimated at >300 kDa, but the exact size (and hence
the exact number of gp41 subunits in the complex) could not be
determined by this means since the discontinuous SDS-polyacrylamide gel
system of Laemmli(1971) used here could not resolve cross-linked gp41
complexes containing more than three subunits (although complexes
containing three or less gp41 subunits were advantageously separated by
this system).
Figure 6:
SDS-polyacrylamide gel electrophoresis to
analyze the products of protein-protein cross-linking of g41p by DSP in
the absence and presence of nucleotide triphosphates. A, 7.5%
(110:1) SDS-PAGE using the gel system of Laemmli (1971). Concentrations
of gp41 protein (in total monomers) and DSP in the experiments were 200
nM and 0.15% (w/v), respectively. Times for cross-linking (in
min) are indicated at the top of the gel. In the absence of GTPS,
the protein monomer was cross-linked intramolecularly and migrates
faster than the non-cross-linked protein control (ctrl) lane
(see text). B, 3% (30:0.8) SDS-PAGE using the phosphate-SDS
gel system of Weber and Osborn(1975) to analyze the gp41 association
states in the presence of GTP
S. Two sets of reaction conditions
were used to permit display of all the reaction intermediates. In lanes 1-3, 5 µM gp41 and 0.01% DSP (from a
0.5% DSP stock solution) were reacted for 1, 5, and 20 min,
respectively. In lane4, 5 µM gp41 and
0.1% DSP were reacted for 1 min.
To determine the state of association of the protein
under these conditions, and to permit estimation of the concentrations
of association intermediates, cross-linking experiments were performed
at higher protein and lower cross-linker concentrations so that the
number of subunits in the complex could be determined. We used the
phosphate-SDS gel electrophoresis system of Weber and Osborn(1975) to
separate and visualize the larger cross-linked species. The
cross-linked proteins do not migrate exactly according to the logarithm
of their molecular weights in this gel, but mixtures of proteins in the
size range of interest can be resolved into discrete intermediate bands (e.g. see Finger and Richardson(1982), who used this gel
system to study the oligomerization of E. coli Rho protein). Fig. 6B demonstrates the resolving power of this gel
system for cross-linked gp41 species and clearly shows the presence of
gp41 tetramers, pentamers, and hexamers. Most importantly, and
consistent with our quantitative sedimentation velocity results, Fig. 6B shows that the highest association state of
gp41 in solution in the presence of GTPS is indeed a gp41 hexamer.
These conclusions were confirmed by the experiments shown in Fig. 7, which were carried out under the same conditions used
for sedimentation and show more clearly a ladder of cross-linked
intermediates ranging up to hexamers in the presence of GTPS (Fig. 7B), while showing only dimers and monomers in
the absence of this nucleotide analog (Fig. 7A). Fig. 7B also shows that no higher species than hexamers
are seen even after extensive cross-linking. In addition, the relative
intensities of the bands of Fig. 7A show that the
system contains mostly dimers and relatively fewer monomers when gp41
was cross-linked to completion under these concentration conditions in
the absence of GTP
S,, in good agreement with the value of K
10
M
estimated for this equilibrium from HPLC data.
Figure 7:
Protein-protein cross-linking of gp41
helicase under the same conditions used for the analytical
ultracentrifugation experiments: in the absence of GTPS (A) and in the presence of 0.1 mM GTP
S (B). Both experiments were carried out by reacting 0.1% DSP
for 1, 5, and 20 min., respectively. The reaction products were
separated using 3% (30:0.8) phosphate-SDS-polyacrylamide gel
electrophoresis.
The hexameric
complex observed by cross-linking in the presence of GTPS clearly
represents a real structure in solution, since no cross-linked species
larger than dimer were seen in control experiments in which
cross-linking was carried out with gp41 in the absence of nucleotide (Fig. 7A). This shows that nonspecific
(``collisional'') cross-linking does not occur with this
reagent at the protein concentrations used in these experiments. On the
other hand, since cross-linking to completion preferentially traps the
largest associated species present in a dynamic equilibrium, it cannot
be determined from these results alone whether the protein exists
primarily as hexamers at these protein concentrations or whether (in
the presence of GTP
S) several different states of association of
gp41 coexist in equilibrium. However, the sedimentation results
presented above show that gp41, at total protein concentrations of
5 µM, forms a homogeneous (monodisperse) solution of
hexamers at GTP
S concentrations exceeding 100 µM.
Further examination of the cross-linking patterns of Fig. 7B shows that the amounts of cross-linked dimer
and tetramer species significantly exceed those of trimer and pentamer
in the late stages of cross-linking. We note that the cross-linking
experiments in Fig. 7B were carried out at the same
total gp41 and GTPS concentrations used in our analytical
ultracentrifugation experiments. As discussed above, we observed a
homogeneous 12.2 S complex in analytical ultracentrifugation
experiments containing PuTP
S, indicating that nearly all the gp41
molecules are present as hexamers. Thus, the cross-linking pattern we
observe in Fig. 7B featuring, in particular, the
intense dimer band, does not reflect the presence of significant
concentrations of this species in the solution. Instead we suspect that
this behavior reflects the existence of two different kinds of subunit
interfaces within the hexamer structure, resulting in different
cross-linking efficiencies at these interfaces. (
)This
cross-linking behavior is very reminiscent of the distribution of
cross-linked species obtained in similar experiments with the E.
coli transcription termination factor Rho, which also exists in
its functional form as a hexameric helicase formed by the association
of three asymmetric dimers (Seifried et al., 1991; Geiselmann et al., 1992a, 1992b). As with Rho, this finding suggests that
the assembly of gp41 subunits as a consequence of PuTP binding probably
follows primarily a monomer
dimer
tetramer
hexamer
pathway. (See ``Discussion'' for further comparative
consideration of these helicases.)
Finally, we have used cross-linking experiments to look for any additional effects on the association states of gp41 that might result from the addition of adding single-stranded or forked DNA templates, or nucleotide diphosphates (the product of gp41 ATPase/GTPase activity). We find (data not shown) that the presence of these molecules has little effect on the oligomerization equilibria of gp41 assembly.
Figure 8: Models and results of hydrodynamic modeling of the gp41 dimer and the various forms of gp41 hexamers, using the methods of Kirkwood(1954) and van Holde(1975). Calculated sedmentation coefficients for the various forms of gp41 complexes are indicated below their respective proposed conformations.
Our sedimentation experiments
yielded a sedimentation coefficient (s) of 5.7 S for gp41 in the absence
of GTP
S. This value of s
is very close
to the theoretical value for a dimer of this size (5.76 S), which is
consistent with our finding that gp41 forms dimers with an association
constant of about 10
M
. This
follows because our analytical ultracentrifugation experiments were
carried out at total gp41 concentrations of
5 µM.
Therefore the protein should be mostly in the dimer form, in
equilibrium with only minor amounts of monomer. This conclusion is also
supported by the observed integral distribution of sedimentation
coefficients, which slopes downward to about 4.5 S at protein
concentrations below 1 µM (see Fig. 3).
The
experimental sedimentation coefficient of 12.2 S for gp41 in the
presence of GTPS is best fit by a structure of type D shown in Fig. 8, although (since the subunits of real macromolecules
would be expected to interact over a surface rather than merely at
single points) a slightly compressed version of Structure C would also
fit this experimental parameter. Thus our sedimentation results and
calculations suggest that the arrangement of gp41 subunits in the
hexamer structure is likely to resemble C, with all six subunits lying
on a ring within one plane, or D, with all six subunits lying around a
ring with three subunits in an upper plane and three in a lower plane.
As will be shown below, the structure of hexameric gp41 as determined
by cryoelectron microscopy is in total accord with this conclusion.
In the
absence of purine nucleotide triphosphates or their analogs, images of
gp41 revealed no reproducible structures other than small dots, which
sometimes appeared as arcs (see arrows in Fig. 9A). These particles have an apparent diameter of
approximately 20 A, and although they could not be measured reliably
under the imaging conditions that we have used because structural
information at this spatial resolution is convoluted by the contrast
transfer function of the electron microscope (Erickson and Klug, 1971),
our results from sedimentation and HPLC studies (above) suggest that
these particles probably correspond to gp41 monomers and dimers.
Occasionally somewhat larger particles (50-60 A in diameter)
were observed, which might correspond to disordered aggregates of the
dots shown in Fig. 9A.
Figure 9: Cryoelectron micrographs of g41p helicase complexes formed: in the absence of nucleotide triphosphate (A), in the presence of ATP (B), and in the presence of both ATP and a DNA replication fork construct (C). The same magnification factors apply to all panels of this figure; the bar in panelA corresponds to 1000 A.
Addition of PuTP or
PuTPS to gp41-containing solutions resulted in the appearance of
the larger and much more easily visible particles corresponding to the circularrings shown in Fig. 9B.
These toroidal structures are approximately 100 A in diameter; much
larger than any of the smaller particles seen in the absence of
nucleotides. (Some of the latter type of particles are still
occasionally visible in the presence of PuTP
S, but at much reduced
numbers.) The rings formed in the presence of ATP (or GTP) and
ATP
S (or GTP
S) appear identical, and no such structures are
seen in the presence of ADP alone.
The structures formed with the nonhydrolyzable nucleotide triphosphate analogs seem to be indefinitely stable (i.e. at least for 20 min). However comparable structures induced by the addition of ATP had a much shorter lifetime, perhaps reflecting the depletion by gp41-catalyzed hydrolysis of the available PuTPs. To test this point solutions containing 5 µM gp41 were incubated with an initial concentration of 1 mM ATP, and specimens were prepared after 1-2 min, and again after 15-18 min. While numerous rings were present at the earlier time, none were visible at the 15-18 min, when nearly all of the available ATP had been hydrolyzed. The addition of another aliquot of 1 mM ATP to this same sample caused the reappearance of the ring structures (after an additional 1-2 min of incubation).
Addition of the miniature DNA forks to gp41 in the absence of
nucleotide triphosphates (or their PuTPS analogs) did not cause
the appearance of the rings or any other structures, and the images
obtained are indistinguishable from the small particles (dots) seen
with gp41 alone. In the presence of both the miniature DNA forks and
nucleotide triphosphates, rings similar to those seen with nucleosides
in the absence of DNA cofactors were abundant (Fig. 9C). In the presence of DNA, however, the
majority of the rings were very clearly hexagonal, with pronounced
vertices. An occasional hexagonal ring of this type was sometimes
present in the populations of rings obtained with gp41 containing
nucleotides in the absence of DNA (Fig. 9B), but these
were infrequent, whereas they seem to make up the majority of particles
in the specimens obtained in the presence of the DNA forks and ATP.
Substitution of the nonhydrolyzable thio-analogs for NTP yielded images
indistinguishable from those obtained in the presence of the NTP
substrates. Preliminary observations suggest that the observed change
in the appearance of gp41 hexamer (from roughly circular to distinctly
hexagonal) can also be brought about by single-stranded DNA
oligonucleotides in the presence of NTP, and thus may not require the
presence of a model DNA replication fork construct.
Computer-based
analysis of images (Fig. 10) of the ATP-induced gp41 structures,
in the presence and absence of DNA forks, confirms these direct visual
observations. In both cases six protein subunits can be clearly seen in
the ring-shaped complexes. Comparison between these images also
provides additional structural details and reveal subtle differences
between the two complexes. The structure of the gp41 hexamer in the
presence of the miniature DNA forks is clearly hexagonal, with all the
subunits clearly separated (Fig. 10B), while the
hexamer visualized in the absence of the DNA fork appears more
``doughnut-like'' (Fig. 10A). Also, the
structure formed by gp41 in the presence of the DNA fork appears
slightly larger in diameter (20%), suggesting that the protein
subunits are ``stretched out'' from the center of the
structure by DNA binding (Fig. 10B). In addition, the
center of the complexes seems to be less open (denser) when the DNA
fork is bound (Fig. 10, compare A and B).
Figure 10: Averaged image of the gp41 helicase hexamers in the presence of ATP (A) and both ATP and a miniature DNA replication fork (B). The images from the original cryoelectron micrographs were first digitized and then aligned (both translationally and rotationally) and computer-averaged (see text). Over 200 individual images were averaged in each case.
Figure 11: A schematic diagram of the association pathways used in the assembly of the gp41 helicase in the absence and in the presence of nucleotide triphosphate substrates.
However, on binding of ATP or GTP, these dimers are
driven to associate to a hexameric state, which redissociates to dimers
after depletion of the PuTP by the intrinsic PuTPase activity of the
gp41 helicase. The hexameric state can be stabilized and made
``permanent'' (and thus more easily amenable to structural
study) by binding the essentially nonhydrolyzable PuTP analogs (and
PuTPase inhibitors) ATPS and GTP
S to the nucleotide binding
sites of the gp41 molecules. Since PuTP hydrolysis, single-stranded DNA
translocation, and double-stranded DNA unwinding are concurrent
activities of the gp41 helicase, the gp41 hexamer formed upon binding
to ATP or GTP is likely to represent the active form of this helicase.
Protein-protein cross-linking studies and cryoelectron microscopy
have suggested that the assembly process of the ATP/GTP induced hexamer
follows a dimer tetramer
(open hexamer)
closed
hexamer pathway, as outlined in Fig. 11, while hydrodynamic
modeling calculations and cryoelectron microscopic observation suggest
that the end product of this pathway is a hexagonal structure of type C
or D, shown schematically in Fig. 8. The relationship between
the abundance of the gp41 hexamer species and total concentration of
gp41 subunits also suggests that PuTP (or PuTP
S) binding
strengthens both the head-to-head and the tail-to-tail interactions
within and between gp41 dimers. This change must be particularly large
for the interaction across the putative tail-to-tail interface that
assembles the gp41 dimers to hexamers, since the association constant
for this interface (K
) must increase
from <10
M
to >10
M
in order to drive hexamer formation
to completion at the experimental concentrations of gp41 protein used.
This means that PuTP binding must increase the free energy of
association between gp41 dimers from less than -6 kcal/mol to
more than -9 kcal/mol. The molecular basis of how this change in
binding free energy is achieved at the tail-to-tail interface has not
been defined.
Cross-linking studies have also shown that the ``active'' gp41 hexamer is neither further stabilized or destabilized by the binding of single-stranded DNA (or DNA ``fork'' constructs), although cryoEM observations suggest that the molecular structure of the hexamer may be altered somewhat by interaction with such DNA substrates (the hexamer is driven from a more doughnut-like to a more sharply hexagonal state on binding DNA; see ``Results''). Since DNA binding does greatly stimulate the intrinsic PuTPase activity of gp41, this functional alteration may be reflected in these minor structural changes we have observed with cryoEM.
However, it is tempting
to speculate that the similar structures of the active Rho and gp41
hexamers may reflect functional similarities as well. Rho is thought to
translocate unidirectionally (5` 3`) along single-stranded RNA
(Geiselmann et al., 1993; Steinmetz and Platt, 1994) and
steady-state kinetic analysis of its ATPase activity is consistent with
the notion that gp41 does translocate unidirectionally (5`
3`)
along single-stranded DNA (Young et al., 1994c). The details
of the more complex interactions of both of these proteins when working
within the functional transcription and replication complexes remain to
be elucidated, but the hexameric structure of these helicases may have
general mechanistic significance.
This speculation is strengthened
by the collection, in Table 2, of the properties of a variety of
prokaryotic and eukaryotic helicases that have subjected to some
structural investigation in recent years. This table represents an
updating of two shorter similar sets of data that were presented
earlier by Matson and Kaiser-Rogers(1990) and by Lohman(1993). As Table 2shows, these various helicases function within the
contexts of complex DNA replication, transcription, recombination, and
repair processes, and perhaps of RNA splicing processes as well, and
the details of how they operate may differ quite appreciably from one
to another. However the prevalence of hexamers among these helicases is
striking, and in the limited number of cases where actual structural
studies have been performed, the ring-shaped hexamer motif seen with
gp41 and Rho also seems to be present (see the studies of T7 gp4 by
Hingorani and Patel (1994), E. coli RuvB by Stasiak et
al.(1994), and E. coli DnaB by Bujalowski and
Klonowska(1993, 1994). It will be interesting to see whether common
functional mechanisms emerge as molecular studies of these helicases,
operating both alone and as parts of biologically relevant complexes,
are carried further. ()