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INTRODUCTION |
N-Ethylmaleimide-sensitive fusion protein
(NSF)1 is an ATPase that
plays an essential role in intracellular membrane trafficking (1).
Together with soluble NSF attachment proteins (SNAPs) NSF binds to a
group of membrane receptors known as SNAREs (SNAP receptors) which are
involved in membrane docking and/or fusion (2). SNAREs are anchored in
either donor ("vesicle," v-SNARE) or acceptor ("target,"
t-SNARE) membranes, and bind to each other to form stable oligomeric
complexes (3, 4). NSF and SNAP cooperate to disassemble these SNARE
complexes in a reaction that requires ATP hydrolysis; the disassembly
is a result of conformational changes that NSF induces in SNAPs and
SNAREs (3, 5). Disassembly of SNARE complexes enables individual SNAREs
to participate in a dynamic series of protein-protein interactions that
are essential for membrane trafficking. The timing of SNARE complex
disassembly, and hence of NSF's action relative to the docking and
fusion of two membranes has not been definitively established. In
different systems, NSF appears to act before membrane docking (6, 7), after membrane docking but before fusion (1, 8), or even after fusion
(9).
Nevertheless, it is apparent that inactivation of NSF, either by
treatment with N-ethylmaleimide or by mutation of residues critical for ATP binding and hydrolysis, leads to accumulation of SNARE
complexes and a block in membrane transport reactions (10, 11). To
understand the mechanistic details of how ATP hydrolysis-dependent conformational changes within NSF are
transduced to SNAPs and SNAREs, an accurate description of NSF's
oligomeric structure is essential.
Previous reports have concluded that NSF forms either a tetramer (10)
or a trimer (12) in solution, and that assembly of the oligomer is
essential for efficient activity in membrane transport reactions (12,
13). Each NSF subunit contains three primary domains: an amino-terminal
N-domain required for substrate binding, and two ATPase domains
referred to as D1 and D2 required for membrane transport activity and
oligomerization, respectively (12, 14, 15). Inactivation of one or more
subunits within a single NSF oligomer results in a nonfunctional
enzyme, indicating that the NSF oligomer exerts its function through a
cooperative mechanism (12).
Recent analyses of NSF's structure by electron microscopy demonstrate
that NSF is a hexagonal cylinder (16) similar in size to related
ATPases thought to be hexamers in solution (17-20, 34-36). In view of
these findings, we have reevaluated NSF's oligomeric structure using a
variety of quantitative analytical methods. The results unequivocally
show NSF to be a hexamer, held together by oligomerization of its D2
domains. This clearly places NSF among a group of hexameric ATPases
with a common mode of action. In many cases they trigger conformational
switches in their protein substrates, which in turn engage in
subsequent reactions.
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EXPERIMENTAL PROCEDURES |
Preparation of NSF--
NSF bearing an amino-terminal histidine
tag and a carboxyl-terminal Myc tag (His6-NSF-Myc) was
expressed in Escherichia coli and purified according to
published procedures (12, 16). In brief, a bacterial lysate containing
His6-NSF-Myc was incubated with
Ni2+-nitrilotriacetic acid agarose (Qiagen) and bound
proteins were eluted with imidazole. For sedimentation equilibrium and
sedimentation velocity experiments, samples were eluted with steps of
imidazole and further purified by size exclusion chromatography on a
Superose 6 column (Pharmacia Biotech Inc.) in the indicated final
buffers. Peak fractions were stored on ice and used within a few days. For electron microscopy, NSF was eluted with a linear 50-500
mM imidazole gradient. The single symmetric peak was
divided into two pools, one containing 90-95% of the NSF (~200-350
mM imidazole, "major peak") and the second containing
the trailing 5-10% of the NSF (~350-450 mM imidazole,
"trailing edge"). These pools were dialyzed against 20 mM HEPES at pH 7.0, 150 mM KCl, 1 mM MgCl2, 0.5 mM ATP, 1 mM DTT, and 15% (w/v) glycerol (buffer A) or 25 mM HEPES at pH 7.0, 100 mM KCl, 5% (w/v)
glycerol, 2 mM MgCl2, 0.5 mM ATP,
and 1 mM DTT (buffer B). In some instances His6-NSF-Myc was purified further on a Superose 6 column
run in buffer B. His6-NSF-Myc was either frozen in liquid
N2 (preparation 1 in Fig. 5A) or kept on ice
(preparation 2 in Fig. 5A) and shipped between laboratories
for EM analyses. NSF domain mutant D2 with a carboxyl-terminal
His6 tag was similarly prepared, as described previously
(16).
Sedimentation Equilibrium--
Equilibrium sedimentation
experiments on His6-NSF-Myc were performed in a Beckman
Optima XL-I ultracentrifuge using the interference optics system to
visualize the protein. Two sector cells equipped with sapphire windows
were used. Blank scans on the buffer were collected at all speeds prior
to sedimentation equilibrium experiments. Both blank and sample volumes
were 150 µl. Data were collected at 4 °C at speeds of 4700, 5700, 7000, and 8800 rpm on samples at three initial concentrations ranging
from 5 to 14 µM in buffer containing 20 mM
HEPES at pH 7.6, 150 mM KCl, 5 mM
MgCl2, 2 mM
-mercaptoethanol or 5 mM DTT, 1% (v/v) glycerol supplemented with either 1 or 5 mM ATP. Initial protein concentrations were determined by a Bradford protein assay with bovine serum albumin as a
standard. Scans were collected at 60 min intervals, and
sedimentation equilibrium was established using the MATCH
algorithm.
Equilibrium sedimentation data of NSF samples were initially analyzed
for average molecular weights in terms of a single, homogeneous species
according to
|
(Eq. 1)
|
where cr is the concentration of NSF at a
given radial position, cm is the concentration of
the enzyme at a reference position (e.g. the meniscus),
is the reduced molecular weight defined as,
= M(1

)
2/RT, M is
the molecular weight,
is the partial specific volume,
is the solvent density,
is the angular velocity,
= r2/2, r is the radial distance (cm)
from the center of rotation, rm is the radial
distance (cm) from the center of rotation to the meniscus, R
is the universal gas constant, T is the absolute temperature
(Kelvin), and base is a correction term for a non-zero base line. The
SEDNTERP program was used to calculate the partial specific volume
(0.7348) and the buffer density (1.01142 at 4 °C). The final
distribution of species was determined by global fitting of
sedimentation equilibrium data according to
|
(Eq. 2)
|
where
1 is the reduced molecular weight defined
above) calculated for the NSF monomer and
cm,3,
cm,6, and
cm,12 are the concentrations of the
trimeric, hexameric, and dodecameric species at the meniscus. All fits
were done by nonlinear least-squares analysis of the primary data using
the Windows 95 version of the NONLIN algorithm (21). The fit was optimized by minimization of the variance, and its quality was determined by examination of the residuals.
Equilibrium sedimentation experiments on the D2 fragment of NSF were
performed with a Beckman Optima XL-I ultracentrifuge using the
absorbance optics system to visualize the protein. Six sector cells
equipped with quartz windows were used. The sample volumes were 110 µl. Data were collected at 4 °C at speeds of 10,800, and 13,300 rpm on samples at three initial concentrations ranging from 8 to 20 µM in buffer containing 20 mM HEPES at pH 7.6, 150 mM NaCl, 1 mM MgCl2, 2 mM
-mercaptoethanol, 1% (v/v) glycerol supplemented
with 0.2 mM ATP. The best fit distribution of species was
described according to
|
(Eq. 3)
|
where
6 is the reduced molecular weight
calculated for the D2 hexamer and the other terms are as described
above.
Sedimentation Velocity--
Sedimentation velocity data were
collected at 4 °C at 24000 or 50000 rpm in 20 mM HEPES
at pH 7.6, 150 mM KCl, 5 mM MgCl2, 2 mM
-mercaptoethanol, or 5 mM DTT, 1%
(v/v) glycerol using interference optics to visualize the protein.
Individual runs were supplemented with 5 mM ADP, 0.5 mM ATP, or 5 mM ATP
S. The sample and buffer volumes were 450 µl. Initial concentrations ranged from 12-230 µg/ml. Scans were collected at the maximum allowed rate of data collection. Data were analyzed according to the time derivative method
of Stafford (22-24) using the DCDT software program to yield a
sedimentation coefficient distribution, g(s*),
from which a weight average sedimentation coefficient at a particular
concentration and temperature can be calculated by integration over the
distribution function with respect to s* as described
previously by Stafford (22). The weight average sedimentation
coefficient at zero concentration, s0, was
determined by linear extrapolation of the experimental weight average
values to infinite dilution. The
s20,w0 was calculated from the
s0 value using SEDNTERP.
The time-derivative method of sedimentation velocity analysis can also
be used to determine an apparent sedimentation,
sapp, and an apparent diffusion,
Dapp, coefficient at a particular concentration and temperature by the following relationships (25)
|
(Eq. 4)
|
where rm is the radial position of the
meniscus (cm), t is the equivalent sedimentation time
(seconds), and
s(s*g) is the
standard deviation of the g(s*) versus
s* curve determined by fitting to Equation 5
|
(Eq. 5)
|
where A and
s(s*g) are constants, and
sapp is the apparent sedimentation coefficient
given by the maximum position of the g(s*)
versus s* curve. Values obtained in this manner for
sapp and Dapp were used
to calculate NSF's mass with the Svedberg equation
|
(Eq. 6)
|
where the terms all have their normal meanings.
The NSF frictional coefficient,
f20,w0, was calculated from the
s20,w0 using an alternate form
Svedberg equation (Equation 6) where D = RT/Nf. SEDNTERP was used to estimate a value for NSF
hydration (
= 0.4071) from the amino acid composition. Simple
interpretation of the frictional coefficient to estimate the NSF axial
ratio using an oblate ellipsoid of revolution as a model was also
accomplished using SEDNTERP. More complex interpretation of the NSF
hydrodynamic shape was accomplished by bead modeling approaches where
bead models of the "ATP" and "ADP" forms were created for
subsequent analysis using the algorithm HYDRO (26). The beads were
arranged so that the mass was distributed according to the size and
shape observed for the ADP and ATP forms of NSF by electron microscopy (see Fig. 4B in Hanson et al. (16)). The mass
distribution in the ADP hexagonal ring could be modeled with only 12 beads, whereas the ATP form required 18 beads in order to incorporate
the six globular "feet" surrounding the central hexagonal ring. The
bead radii used for the ATP and ADP bead models were 20.25 and 23.25 Å, respectively, so that both bead models would have the calculated masses corresponding to an NSF hexamer.
Electron Microscopy--
For scanning transmission electron
microscopy (STEM), 2-20 µM His6-NSF-Myc was
either stabilized with 0.05% (v/v) glutaraldehyde for 30 min on ice
and then diluted to 100-500 nM in buffer A lacking glycerol or directly diluted without the glutaraldehyde step. 3-5-µl
aliquots were adsorbed to thin carbon films (supported by thicker
perforated carbon layers on gold-coated copper EM grids) which had been
rendered hydrophilic by glow discharge in a plasma cleaner. They were
subsequently washed on 6 drops of quartz bidistilled water and freeze
dried in the STEM HB-5 microscope. Images were recorded at a nominal
magnification of 200,000 using doses of 200-400
electrons/nm2 and an accelerating voltage of 80 kV.
Individual particles were manually selected from electron micrographs
for mass analysis and processed as described (28). The beam induced
mass loss was determined experimentally by repeatedly scanning the same grid region and monitoring the change in sample mass. The main mass
analysis data sets were corrected accordingly (28).
For transmission electron microscopy (TEM) and image processing,
100-250 nM His6-NSF-Myc (diluted in buffer A
lacking glycerol) was absorbed to carbon coated grids which had been
rendered hydrophilic as above. The grids were washed once with water
and stained with 2% (w/v) uranyl acetate (pH 4.5). Electron
micrographs were recorded with a Phillips EM420 at a nominal
magnification of 36,000 and an accelerating voltage of 100 kV. The TEM
images were digitized in an Eikonix model 1412 camera with a pixel size
of 0.43 nm × 0.43 nm at the specimen level. NSF particles were
selected from several EM images and subjected to single particle
analysis and principle component analysis contained in the SEMPER and
EM system program packages (29, 30). The particles were laterally
aligned by correlation methods using arbitrary reference molecules and subjected to principal component analysis afterward (31). The first two eigenvectors indicated a significant staining inhomogeneity which resulted in a density gradient across the molecule. Thus, the
gradient was substracted prior to a further principal component analysis and classification with respect to eigenvectors
representing the symmetry properties of the molecules. A class of
415 particles (out of 1746) was averaged and 6-fold symmetrized.
Size-exclusion Chromatography and Multiangle Laser Light
Scattering--
Analytical size-exclusion chromatography with
multiangle light scattering was performed at 25 °C using a 30 cm × 7.8-mm TSK-Gel G3000SWxl gel filtration column (TosoHaas) at
a flow rate of 0.5 ml/min. The column was equilibrated with 20 mM HEPES at pH 7.0, 150 mM NaCl, 4 mM MgCl2, 5 mM DTT, 1% glycerol,
and 1 mM ATP. The gel filtration column was followed
in-line by a Mini-DAWN light scattering detector (Wyatt Technologies)
and an interferometric refractometer (Wyatt Technologies) for protein
concentration determination. Molecular weight calculations were carried
out using ASTRA software (Wyatt Technologies). The change in refractive
index as a function of protein concentration is approximately constant
for proteins and a value of 0.193 mol
1 was used (27).
These experiments were carried out using NSF expressed from a pET15b
expression vector (Novagen) from which the NH2-terminal
His6 tag was removed by thrombin treatment followed by
repurification using size exclusion chromatography.
 |
RESULTS AND DISCUSSION |
A Hexamer Is the Predominant NSF Species in
Solution--
Sedimentation equilibrium analytical ultracentrifugation
is a thermodynamically rigorous method for characterizing
macromolecular mass and oligomeric interactions in solution (32).
Analysis of NSF by this method was used to evaluate NSF's oligomeric
state in solution. Typical data are shown in Fig.
1. These data, as well as additional data
collected at different initial loading concentrations and speeds, were
poorly described by a model for a single homogeneous species (Equation 1), and yielded average molecular weights of 483,000-492,000,
considerably higher than the 255,000 expected for the previously
reported trimeric NSF oligomer (12). The data were then analyzed using
models of increasing complexity containing multiple oligomeric species
(e.g. 1-6, 3-6, 6-12, 4-8, 5-10, 3-6-9, 1-6-12) until
global analysis of data collected at different initial concentrations
and speeds indicated a best fit model as evaluated by randomness of the
residuals and minimization of the variance. As can be seen in Fig. 1,
the data collected in 5 mM Mg2+ATP are well
described by a distribution containing trimeric, hexameric and
dodecameric species (Equation 2), where the hexamer is the predominant
species (>90%). Similar results were obtained in buffers containing 1 mM Mg2+ATP, where the best fit model contained
monomeric, hexameric and dodecameric species. In all data sets, global
fits to sedimentation equilibrium data indicated the predominant
species (>90%) to be a hexameric oligomer of NSF. Similar results
were independently obtained by sedimentation equilibrium analytical
ultracentrifugation with the NSF preparations used for the TEM and STEM
analyses shown below.2

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Fig. 1.
Species distribution of oligomeric NSF at
sedimentation equilibrium. The fringe displacement
versus radius profile for 9 µM NSF in 5 mM Mg2+ATP at 7000 rpm at 4 °C is shown in
the lower panel. The open circles are the data
points, and the solid line is the model fit described by
Equation 2. The three dotted lines whose sum gives rise to
the fit represent the distributions of trimeric, hexameric, and
dodecameric species as indicated. The residuals of the fit to Equation 2 are shown in the upper panel.
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Attempts to estimate equilibrium constants describing the relationship
between the trimeric, hexameric, and dodecameric species from data
collected in buffer containing 5 mM Mg2+ATP
were unsuccessful, since fits to models containing equilibrium constants showed increased variances as well as systematic deviations in the residuals. This suggests that the hexamer is not in a
reversible equilibrium with either the trimeric or dodecameric species
on the time scale of the sedimentation equilibrium experiment.
Consistent with this conclusion, dilution of NSF samples from the ~10
µM concentration used in these equilibrium centrifugation
experiments to ~200 nM in sedimentation velocity
experiments (see below) did not change the sedimentation coefficient.
Moreover, dilution to ~100-500 nM for electron
microscopy (16) (see below) did not change the abundance or appearance
of hexameric cylinders. Thus, sedimentation equilibrium shows NSF to be
a stable hexamer, not trimer, in solution.
The D2 Fragment of NSF Is an Oligomerization Domain--
Each
85-kDa NSF subunit consists of three primary domains termed N, D1, and
D2 (14, 15). Electron microscopy of the D1-D2 and the D2 domains showed
ring-shaped structures similar to those formed by full-length NSF (16).
Since the D2 domain appears to be essential for NSF's oligomerization
(12, 39) we examined its oligomeric state by sedimentation equilibrium
which yielded the data shown in Fig. 2.
In buffer containing 0.2 mM Mg2+ATP, the
predominant species in solution is a D2 hexamer. The presence of ATP
appears to be essential for hexamer formation and/or stabilization,
since these hexamers were destabilized in samples from which ATP had
been removed by gel filtration (data not shown). The present findings
are thus consistent with earlier studies demonstrating oligomerization
of the D2 domain (12, 16, 39) and confirm a direct role of both ATP
binding to the D2 domain and of this domain's oligomerization in
organizing NSF's hexameric structure.

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Fig. 2.
Species distribution of oligomeric D2
fragment of NSF at sedimentation equilibrium. The absorbance
versus radius profile for 10 µM D2 fragment in
0.2 mM Mg2+ATP at 13,300 rpm at 4 °C is
shown in the lower panel. The open circles are
the data points, and the solid line is the model fit
described by Equation 3. The two dotted lines whose sum
gives rise to the fit represent the distributions of hexameric and
dodecameric species as indicated. The residuals of the fit to Equation 3 are shown in the upper panel.
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Sedimentation Velocity Experiments Reveal an Unusual Hydrodynamic
Property of NSF--
The observation that hexameric NSF is the
predominant species in solution does not agree with previous
sedimentation equilibrium studies in which NSF was shown to be trimeric
(12). In both studies, however, the same protein construct
(His6-NSF-Myc) and similar purification steps were used. In
an effort to compare the NSF samples evaluated in this study with the
material analyzed in previous studies, the sedimentation coefficient of
NSF was measured by velocity sedimentation analytical
ultracentrifugation. All experiments yielded data described by a
s20,w0 of 13.4 (±0.1) S,
regardless of whether Mg2+ADP, Mg2+ATP, or
Mg2+ATP
S was included in the buffer (see Fig.
3A). This sedimentation coefficient is consistent with the previously reported value of 12.9 S
as well as with the analysis of NSF's sedimentation relative to
standards through glycerol gradients (12, 19).

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Fig. 3.
Sedimentation velocity analysis of NSF.
Panel A shows the apparent weight average sedimentation
coefficients in the presence of 5 mM MgCl2 with
either 0.5 mM ATP (open squares), 5 mM ATP S (open triangles), or 5 mM
ADP (open circles) as a function of total protein
concentration. Extrapolated to infinite dilution, the sedimentation
coefficients for NSF in these three conditions are all consistent with
an s0 value of 8.52 at 4 °C in buffer. This
corrects to an s20,w0 of 13.4 at
20 °C in water. Panel B shows the apparent sedimentation
coefficient distribution function, g(s*)
versus s*, for 0.23 mg/ml NSF in buffer containing 5 mM Mg2+ADP collected at 24,000 rpm at 4 °C.
The error bars represent the standard error of the mean of
the 10 data sets used in this analysis. For clarity, only every fifth
error bar is shown. The solid line is the fit to Equation 5.
Apparent s, D, and Ms/D values
were calculated as described in the text.
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|
A sedimentation coefficient of 13.4 S is low for a 510-kDa protein (the
hexameric molecular mass of His6-NSF-Myc) considering that
the predicted smax values for a spherical
protein of equivalent mass and partial specific volume would be 20.1 S
(anhydrous) and 18.5 S (hydrated, where
= 0.4071, calculated from
NSF's amino acid composition). Such a low sedimentation coefficient
suggests that NSF's frictional coefficient may be unusually high.
Electron microscopy indicated that NSF consists of a ~13 × 10-nm hollow cylinder with a larger surface area and increased
subunit flexibility relative to that expected for a sphere of the same
mass and density (16) (Fig. 4). This
difference in size and shape would indeed be expected to increase the
frictional coefficient and decrease the sedimentation coefficient.

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Fig. 4.
Electron microscopy and average of NSF.
Survey view of negatively stained NSF particles prepared in the
presence of Mg2+ATP. The inset shows the 6-fold
symmetrized average of 415 top view projections. The diameter of the
ATPase is 13 nm. Six protein regions surrounding a central
electron-dense region, indicative of a stain-filled channel or cavity
(diameter 2-3 nm), are clearly recognizable. Scale bar = 100 nm.
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To determine whether the hexagonal ring shape could explain the low
sedimentation coefficient, we constructed several simple models for
NSF's shape and defined their hydrodynamic properties using HYDRO
(26). The models were built using the minimum number of beads required
to form the hexagonal structures observed in EM images of NSF oligomers
either in Mg2+ATP
S or in Mg2+ADP (16) and
are described in detail under "Experimental Procedures." Although
the S value predicted for the ATP bead model (13.8 S, where
= 0.4071) agrees fairly well with the experimental sedimentation coefficient (13.4 S), the value predicted for the ADP bead model (15.9 S, where
= 0.4071) is too high and can only approximate the
experimental S value if
= 0.9-1.2, an unusually high value for
hydration. Thus, although the sedimentation coefficient is significantly reduced by NSF's hexagonal ring shape, the shape alone cannot explain the abnormally low S value as long as
standard estimates of hydration are assumed. Additional
experimentation, such as small angle x-ray scattering, will be required
to further evaluate NSF's structure in solution.
Sedimentation velocity analysis can be extended using a time-derivative
method which provides a means to simultaneously determine the apparent
sedimentation, sapp, and diffusion,
Dapp, coefficients, from which the molecular
weight can be calculated (25) (see "Experimental Procedures"). Even
though both coefficients are hydrodynamic measures of a macromolecule,
the ratio s/D is proportional to the molecular weight by the
Svedberg equation where the hydrodynamic shape and hydration factors
inherent in each coefficient cancel. Fig. 3B shows such an
analysis of NSF sedimentation velocity data collected in 5 mM Mg2+ADP. Using Equations 4 and 5 we
calculated an sapp of 8.43 S and a
Dapp of 1.50 Ficks. When combined in the
Svedberg equation, these yielded a solution molecular mass of 504 kDa.
This value is within 2% of NSF's calculated hexameric molecular
mass.
TEM and Image Processing Demonstrate that NSF Is a Hexagonal
Cylinder with 6-fold Symmetry--
Recent quick-freeze/deep-etch EM
showed that NSF and its domain mutants have dimensions and a 6-fold
symmetry consistent with a hexagonal, and therefore possibly hexameric,
structure (16). Analysis of negatively stained NSF oligomers by TEM and
image processing (Fig. 4) was used to confirm this 6-fold symmetry. Top
view projections of NSF prepared in 1 mM
Mg2+ATP were aligned and subjected to principle component
analysis (see "Experimental Procedures" for details). The molecules
were classified using the most significant eigenvectors which
represented the symmetry properties of the particles. The averages
obtained from these particles demonstrated an apparent 6-fold symmetry which was enhanced by rotational symmetrization (Fig. 4,
inset). According to this average, negatively stained NSF
oligomers had a diameter of ~13 nm, consistent with a previously
reported measurement (33) and with measurements of platinum-shadowed
NSF after substracting the ~2-nm platinum coating (16). The TEM
images also show NSF to contain a central electron-dense region with a
diameter of 2-3 nm representing a stain-filled pore or cavity (Fig.
4).
Quantitative STEM Analysis of NSF Particles Reveals a Mass
Consistent with a Hexameric Structure--
STEM mass analysis of
unstained, freeze-dried NSF particles confirmed a hexameric structure.
This technique enables mass measurements of individual macromolecules
that are independent of their hydrodynamic properties (10, 11). All
experiments with NSF (His6-NSF-Myc preparations 1 and 2)
yielded mass histograms which exhibited a Gaussian distribution with a
single well defined peak at 459-497 kDa (Fig.
5A; histograms not shown),
consistent with the presence of a hexamer. There was no detectable
shoulder or peak in the 200-300 kDa region of the histograms which
would have indicated the presence of significant amounts of a stable
NSF trimer. In addition, a series of experiments allowed us to examine
the effects of chemical cross-linking and freeze-thawing on NSF's
oligomeric state. Comparison of NSF samples prepared and treated in
different ways (Fig. 5A) showed that glutaraldehyde
stabilization, freeze-thawing, and additional purification did not
significantly alter the measured mass. Within experimental variability,
all experiments gave equivalent results showing the presence and
predominance of the NSF hexamer. Pooling the data from all experiments
using one NSF preparation (preparation 2) yielded the histogram shown
in Fig. 5B, with a mass for NSF of 475 ± 91 kDa
(n = 4262).

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Fig. 5.
STEM mass determination of NSF. Results
of STEM mass determinations utilizing two preparations and different
fractions of His6-NSF-Myc eluting from a
Ni2+-NTA column (see "Experimental Procedures") are
listed in A with the corresponding molecular masses,
standard deviations (SD), and number of particles in the
histogram peak (n). Preparation 1, stored frozen
at 80 °C; preparation 2, stored on ice. The data have
been corrected for the ~4% beam induced mass loss incurred during
measurement. Pooled mass data sets from the individual experiments made
with NSF preparation 2, after individual correction for beam induced
mass loss, are displayed as a histogram in B. The Gaussian
fit indicates a mass of 475 ± 91 kDa (n = 4262),
consistent with the presence of hexameric NSF oligomers. There is no
shoulder or peak in the 200-300-kDa region of the histogram, which
would be the mass region of NSF trimers.
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Multiangle Light Scattering and Gel Filtration Support the
Hexameric Structure in Solution--
The finding that NSF is
predominantly a hexamer in solution was further supported by multiangle
light-scattering experiments. This technique offers an additional
shape-independent approach for the determination of molecular mass
(38). Most NSF elutes from size-exclusion columns slightly ahead of the
440-kDa ferritin standard, implying a molecular mass consistent with a
hexameric state (16). Fig. 6 shows a
typical gel-filtration elution profile of NSF together with an in-line
multiangle light-scattering molecular mass determination. The
predominant NSF species, averaged across the peak center (width at half
height), is calculated by multiangle light scattering to be 489 (± 5)
kDa. This value corresponds well to the predicted hexameric molecular
mass of 510 kDa. A smaller peak eluting later consists of an 85 (± 3)-kDa species, which corresponds to monomeric NSF. Distinct peaks of
other possible NSF oligomers are not observed in 1 mM
Mg2+ATP. Since the NSF used in these experiments had its
amino-terminal His6 tag removed (see "Experimental
Procedures"), this result also rules out the unlikely possibility
that the His6 tag itself is responsible for NSF's
hexameric oligomeric state.

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Fig. 6.
Multiangle light scattering of NSF. A
typical elution profile is shown in buffer supplemented with 1 mM Mg2+ATP. The dotted line
represents refractive index on an arbitrary scale, which is
proportional to protein concentration, and diamonds indicate
calculated molecular mass. The molecular mass profile across the peak
at 7 ml reflects a 489 (±5)-kDa, hexameric NSF; the peak at 9.1 ml
reflects an 85 (±3)-kDa monomeric NSF.
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Conclusions--
Analytical ultracentrifugation, transmission
electron microscopy with image processing, scanning transmission
electron microscopy, and dynamic light scattering all reveal that NSF
is a hexamer. This result differs from previously published experiments
which suggested that NSF was a trimer (12). It remains unclear why the
prior sedimentation equilibrium experiments are not consistent with the
current data. The sedimentation equilibrium experiments described here
were confirmed in three independent laboratories with equivalent
results, supporting a hexameric structure (Fig. 1).2
Additional evidence for the previously proposed trimeric structure was
provided by the presence of dimeric and trimeric products in
cross-linking experiments (12). However, several factors including
inefficient cross-linking, low sensitivity of Coomassie staining, and
exclusion of high molecular weight complexes from the
SDS-polyacrylamide gels used for analysis could have prevented the
detection of higher order oligomers (tetramers, pentamers, and
hexamers).
The possible limitations of each technique employed in the present
study were addressed by comparing the results from a variety of
approaches that are based on different physical principles and measure
different parameters of the oligomer's behavior. In particular, the
range of methods chosen allowed NSF's oligomeric size to be determined
in manners both dependent on, and independent of, its shape and
hydrodynamic properties, using protein in solution as well as protein
that had been attached to a solid support. The measurements, which were
carried out over a wide concentration range and in different buffers,
always lead to the same conclusion, NSF is genuinely a hexamer.
Interestingly, the sedimentation coefficient of NSF
(s20,w0 of 13.4 (±0.1) S) closely
matches the low value reported previously (12), suggesting that this
oligomer has a large frictional coefficient for its size and shape.
Further studies will be required to establish whether this is a result
of hydration or conformational flexibility of the oligomers in
solution, perhaps corresponding to the conformational changes seen in
the presence of different nucleotides by quick-freeze/deep-etch electron microscopy (16).
The similarity between NSF, p97, CDC48, and other related ATPases
including the Hsp100 proteins ClpA and Hsp104 thus extends to their
basic structural design of six identical subunits per cylindrical
oligomer (16-20, 34-36). This arrangement is likely to play a role in
the mechanism by which these ATPases interact with and affect the
conformation of their target substrates.
The MATCH algorithm and the NONLIN software
were provided by the University of Connecticut Biotechnology Center
(Storrs, CT). SEDNTERP was developed by J. Philo, D. Hayes and T. M. Laue. The DCDT program was provided by W. F. Stafford III. All
four of these software programs are available from the RASMB homepage
at http://www.bbri.harvard.edu/rasmb/rasmb.html. We thank Donald M. Engelman, John Heuser, Reinhard Jahn, James E. Rothman, Walter F. Stafford III, Olwyn Byron, and Wolfgang Baumeister for important
input and helpful conversations. We thank Rainer Jaenicke and Ariel
Lustig for independently confirming the ultracentrifugation results on
the TEM and STEM samples as well as for critical reading of the
manuscript. We thank Susanne Volker-Mürkl for technical
assistance.