From the Department of Pathology and Laboratory
Medicine, University of Texas, Houston Medical School, Houston, Texas
77030, the ¶ Department of Biosciences at Novum, Karolinska
Institute, 14157 Huddinge, Sweden, and the
Department of
Chemistry and Biochemistry, University of Texas at Austin,
Austin, Texas 78712
Received for publication, February 26, 2001, and in revised form, April 2, 2001
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ABSTRACT |
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Structural studies by three-dimensional electron
microscopy of the Saccharomyces cerevisiae truncated
dihydrolipoamide acetyltransferase (tE2) component of the
pyruvate dehydrogenase complex reveal an extraordinary example
of protein dynamics. The tE2 forms a 60-subunit core with
the morphology of a pentagonal dodecahedron and consists of 20 cone-shaped trimers interconnected by 30 bridges. Frozen-hydrated and
stained molecules of tE2 in the same field vary in size
~20%. Analyses of the data show that the size distribution is
bell-shaped, and there is an approximately 40-Å difference in the
diameter of the smallest and largest structures that corresponds to
~14 Å of variation in the length of the bridge between
interconnected trimers. Companion studies of mature E2 show
that the complex of the intact subunit exhibits a similar size
variation. The x-ray structure of Bacillus
stearothermophilus tE2 shows that there is an
~10-Å gap between adjacent trimers and that the trimers are
interconnected by the potentially flexible C-terminal ends of two
adjacent subunits. We propose that this springlike feature is involved
in a thermally driven expansion and contraction of the core and, since
it appears to be a common feature in the phylogeny of pyruvate
dehydrogenase complexes, protein dynamics is an integral component of
the function of these multienzyme complexes.
The pyruvate dehydrogenase complexes
(PDCs)1 are among the largest
(Mr ~106 to 107) and
most complex multienzyme structures known. A central feature of these
complexes is a 24-mer (Escherichia coli) or 60-mer
(eukaryotes and some Gram-positive bacteria) dihydrolipoamide
acetyltransferase (E2) core with the morphologies of a cube
or a pentagonal dodecahedron, respectively (1-4). The cores have both
functional and structural roles in organizing the multienzyme complex;
the E2 activity is associated with the scaffold to which
the other components are attached. These include the pyruvate
dehydrogenase (E1) and dihydrolipoamide dehydrogenase
(E3), which requires a binding protein (BP) to anchor it to
the core of the yeast and mammalian PDCs, although, in E. coli and Bacillus stearothermophilus PDCs, BP is not
required (1-4).
The E2 subunits have multidomain structures consisting of
one, two, or three amino-terminal lipoyl domains, followed by an E1 and/or E3 binding domain, and a
carboxyl-terminal catalytic domain (1-4). X-ray crystallography (5-9)
and three-dimensional electron microscopy (10, 11) show that the
E2 catalytic domains are arranged in cone-shaped trimers at
each of the 8 or 20 vertices of the cubic or dodecahedral structures,
respectively (7, 8, 10, 11). The trimers are interconnected by bridges
to form an empty cage-like complex with the tip of the trimer directed toward the center of the structure.
Examination of the 4-Å resolution crystal structures of dodecahedral
truncated E2 (tE2) cores from
Enterococcus faecalis and B. stearothermophilus
and the 2-Å resolution crystal structure of a cubic tE2
core from Azotobacter vinelandii show that an anchor residue
in the C terminus of the 2-fold related trimers resides in a
hydrophobic pocket formed by the adjacent subunits (8). Two such
"ball-and-socket" joints on the 2-fold axis maintain the
connections between adjacent trimers. It was proposed that the
different spatial arrangements of the trimers in the cubic and
pentagonal dodecahedron cores are the result of small differences in
the nature and position of these interface residues (8).
Our three-dimensional electron microscopy studies of the
Saccharomyces cerevisiae tE2 and mature
E2 (mE2) reveal flexibility in the arrangement
of the E2 cores that is extraordinary for a macromolecular
protein complex. Cryoelectron microscopy affords a snapshot of the
molecular composition of the preparation at room temperature in the
absence of constraints imposed by a crystal lattice with a shutter
speed of ~10 Enzyme Preparations--
The S. cerevisiae
tE2 subunit, comprising either residues 206-454 or
181-454, was overexpressed in E. coli, and the assembled cores were purified to near homogeneity as described (13, 14). The
expression vector for mE2 was pYE2 m-32TX. The protein has 44 extra amino acid residues at the N terminus, including a
His6 tag and an enterokinase cleavage
site.2 Both tE2
and mE2 exhibited catalytic activity (acetyl transfer from
[1-14C]acetyl-CoA to dihydrolipoamide) similar to
wild-type E2.
Electron Microscopy--
The tE2 preparation was
diluted to 25 µg/ml in 0.25% methylamine tungstate stain containing
10 µg/ml bacitracin and immediately sprayed onto Butvar 76 film with
a carbon backing to minimize shrinkage and to obtain randomly oriented
molecules (15, 16). Application of the specimen to the Butvar film
alone resulted in molecules that were smaller than their
frozen-hydrated counterpart (10). Their application to a carbon film
gave molecules primarily oriented with their pentagonal face bound to
the carbon film and, consequently, were not useful for image
processing. The images of the stained molecules were recorded at
0.2-0.4 µm under focus at a nominal magnification of × 50,000 under conventional irradiation procedures using a JEOL JEM 1200 electron microscope operated at 100 kV. For cryoelectron microscopy, 3 µl of the tE2 and mE2 preparations at 0.1 and
0.4 mg/ml, respectively, were deposited, blotted, and quick-frozen in
liquid ethane on a glow-discharged carbon-coated holey grid. The
vitrified specimens were recorded at ~2-4 µm under focus with a
dose of ~9 e Data Processing--
All data processing steps were carried out
on SGI Octane dual processor workstations (Silicon Graphics, Inc.).
First, individual tE2 and mE2 particles were
boxed out manually using an x-window-based program Emtool (17).
Subsequently, data processing steps included particle size variation
determination and three-dimensional reconstruction of particles in
various size groups, using our programs based on the principles of
Fourier common lines (18, 19). The orientation determination and
three-dimensional reconstruction were carried out using parallel
programs for refinement (20) and reconstruction (21) (see below), which
are based on Fourier common lines (18, 19) and Fourier-Bessel synthesis
(22), respectively.
Orientation Determination and Three-dimensional
Reconstruction--
A list of 28 initial orientation estimates was
generated for each particle image using a program based on self common
lines. A preliminary three-dimensional model was then computed at a
nominal resolution of 35 Å from 10 particles that showed the best self common line phase residuals and had been refined by cross common line
phase-residual minimization among all 10 particles (20). The particles
with incorrect orientations were eliminated from the initial list of
orientation estimates by evaluating the cross common line phase
residual between the particle and projection images of the preliminary
model. The selected orientations were then refined, first by a global
refinement that minimized the cross common line phase residuals across
all selected particles (20) and then by a projection-based refinement
that optimized the match between the image and the projections computed
from a preliminary three-dimensional model. The new model reconstructed from these refinements was used as a template for the next round of
particle selection and refinement, resulting in a further improved model. This process was iterated for two cycles utilizing the entire
data set until no more particles with correct orientation parameters
could be obtained and no improvement was evident in the cross common
line phase residual between particle images and the computed projections.
The final reconstruction was calculated by merging data to 25-Å
resolution (for tE2) or 30-Å resolution (for
mE2) from particle images with defocus values around 2-3
µm, which were determined from the incoherently averaged Fourier
transforms of particle images in each micrograph (23). The final
reconstructions were corrected for the contrast transfer function of
the microscope (24).
Assessment of the Particle Size Variation--
We have developed
procedures to analyze the size variability of the molecules. The
relative size of the tE2 and mE2 molecules were
determined using the program sizeDiff (David Zuckerman and Z. H. Zhou), which is a Unix and Windows NT/2000 program written in C++ based
on Fourier common lines. SizeDiff uses an iterative method that
minimizes the Fourier cross common line phase residuals between the
particle images and a model template derived from the data set as
described below. After the orientation and center parameters of each
particle were determined and the structure was refined to ~30-Å
resolution (see above), a preliminary three-dimensional reconstruction
was calculated by combining all of the refined particles with a phase
residual of less than 40°. Approximately 10-15% of the particles
were eliminated in this step, and these were visually investigated in
order to avoid discarding those particles that may have greater phase
residual values because of a large size variation. However, the subset
of discarded particles was found to be of poor quality. This
"average" reconstruction was then used as the template for the
first round of size determination. Initially, 20 projection images were
generated at regularly spaced orientation intervals, and these were
used as the template set. Each particle image was then isotropically
scaled to best match the template projection by minimizing the averaged
cross common line phase residual between the particle and the
corresponding projection. The isotropic scaling does not accurately
reflect the size variation of the single particle images, because the size variability of the molecule is not isotropic (see "Results") and, consequently, may introduce errors in their alignment, especially when the size difference between the model and the image is large. For
example, after obtaining the structure representative of the 1.0 size
groups, it was used as a model after scaling for the 0.95 and 1.05 size
groups, and these structures were finally used as models after scaling
to align the 0.9 and 1.10 size groups, respectively. This bootstrap
approach was required for the appropriate alignment of smallest and
largest images. Subsequently, the models obtained from the alignment
were refined as described below.
Further classification of the particles and the refinement of the
reconstruction were accomplished by utilizing the particles ± 1% of
the designated size. After repeating a second cycle of size variation
analysis using sizeDiff, ~80% of the particles remained in this 2%
size bin. After eliminating outliers, the remaining particles were
merged to generate a reconstruction to be used as the template in a
third cycle of analysis. Over 96% of the particles were in this 2%
size bin, thus demonstrating the convergence of the data set and that
the final reconstruction had optimal resolution. The convergence of the
data set and the optimization of the resolution support the validity
and utility of our method of size variation analysis. In contrast to
these results, the application of sizeDiff to the rice dwarf virus
images (25) showed that over 99% of the particles were within a 1% size group, indicating that the size variability of the E2
core is not an artifact of the methods described above.
The size variation of the frozen hydrated tE2 molecules was
also analyzed by the polar Fourier transform method (26, 27), and the
size distribution was similar to that shown in Fig. 1 (data not shown).
Three-dimensional Visualization and Comparison with Atomic
Structure--
The three-dimensional visualization was carried out
using the Iris Explorer (NAG, Inc., Downers Grove, IL) with custom
designed modules (28). The maps were displayed at the same threshold using a contour level of ~1
The atomic coordinates of the crystal structure of the dodecahedral
form of B. stearothermophilus tE2 (PDB identification number
1B5S) were initially provided by Izard et al. (8). The areas of interest of the structure were either rendered and exported to Open Inventor format using Ribbons (29) or directly converted to electron density map using a Gaussian filter to a resolution similar to the EM structures (25 Å) (30) for further three-dimensional comparisons using Iris Explorer (28).
Size Variation of the tE2 Molecules--
This study
reveals an ~20% size variation of the tE2 images (Fig.
1). This extraordinary size variation of
a macromolecular complex raised considerable skepticism concerning the
significance of these data and their interpretation. Indeed, our
original observation of a size variation of tE2 and its
subcomplexes was ascribed to magnification variations of the electron
microscope, and consequently, only those particles with sizes that were
within 3% of the average were used in the reconstructions (11). As a
result, the structures of tE2, tE2.tBP,
tE2.BP, and tE2.BP.E3 were
representative of 10-15% of the data sets (11). Below, we examine and
discount three trivial possibilities for the size variation, which, if not discounted, could muddle the interpretation of the findings of this
study.
Magnification Variation of the Electron Microscope--
Electron
microscopy studies of icosahedral virus capsids reveal a maximum
variation of ~1% in their sizes in a given field. This variation in
size was attributed to electron microscope magnification variation,
although the possibility that this size variability was related to the
molecules was not ruled out (31). In any event, a 20% size
distribution of the images in the same field recorded in
stain (10) and vitreous ice (10) (Fig. 2)
in the present study is well outside the range of variation normally attributed to an electron microscope. Moreover, during the recording period of this study, numerous other macromolecular images were recorded without any noticeable size disparity. Therefore, we conclude
that the large size variability of these images is not related to the
electron microscope.
Distortion of the Molecules--
A more plausible explanation for
the size difference is that these flexible ("soft") particles may
be easily squashed to a variable degree between the two air-water
interfaces in vitreous ice or that the particles may collapse in the
stain. We have ruled out both possibilities. A tilt experiment of the
stained molecules shows that they interconvert upon tilting the stage
32° (10). For example, a round particle with a larger profile that
was not imbedded in stain was found to have an oval shape upon tilting the stage, demonstrating that the tilt experiment is a sensitive method
of detecting molecules that are not spherical (10). Similarly, a tilt
experiment employing vitreous ice did not reveal any particles that
were flattened (Fig. 2). Furthermore, analysis of the size distribution
of the particles in the same field showed that the small and larger
particles are co-mingled and are not segregated (10). Finally, since a
size increase due to flattening of the particle would severely disrupt
the icosahedral symmetry of the molecule, the reconstruction
representative of the largest particles should have the poorest
resolution or not be amenable to the icosahedral reconstruction
methods. In this regard, a sensitive gauge of the accuracy of the
alignment of the particle images to their template is obtained by a
comparison of the phase residual values. The average phase residuals
for the 0.90, 0.95, 1.0, 1.05, and 1.1 size groups of the stain data
(Fig. 1) are 19.8°, 17.1°, 18.0°, 18.7°, and 19.7°,
respectively. This exceptionally narrow range and their low numerical
values are strong evidence that the icosahedral symmetry of the
molecules selected for the reconstructions has been well preserved over
the 0.9-1.1 size variation. Moreover, the resolution values ~20-25
Å are comparable for all reconstructions, and most significantly, the
larger reconstruction agrees very well with the comparable x-ray
structure of B. stearothermophilus tE2 filtered
to the resolution of the electron microscope structure (see below).
Incomplete Structures--
The electron microscope fields show
that there are a significant number of particles that lack the full
complement of 20 trimers in stain and ice (see Ref. 10 and Fig.
2C). The E2 components of the
A priori, these incomplete cages may be expected to have the
same radius of curvature and thus the same size of the complete structure, since the nature of the interaction between adjacent trimers
determines the dodecahedral shape of the molecule (8). Shown in Fig.
2C are some images that are representative of incomplete structures based on visual inspection. Interestingly, the missing portion of the molecule does not appear to affect the radius of curvature of the remaining complex, since the size of the images is
comparable with those in Fig. 2, A and B.
Accordingly, any structures lacking a small and undetectable number of
trimers that got included would not be expected to affect the size of the reconstructions.
In any event, we have determined the relationship between the size and
mass of the structures by comparing the relative masses of
reconstructions (which were normalized with respect to the number of
particles in each set) from images in the 0.95 and 1.05 size groups
recorded from one micrograph of frozen-hydrated molecules. Although
this procedure restricts the number of particles in the reconstructions, it offers a more meaningful comparison of the relative
masses, since such variables as defocus, exposure, ice thickness, and
contrast are minimized or eliminated. It should be noted that the 10%
difference in the diameter of the particle corresponds to over a 30%
increase in the volume of the particle, which may be related to a 30%
variation in its mass. A threshold of 2.40 S.D. of the reconstruction
from the 1.05 size group gives a molecular volume that corresponds to
the 1.6 × 106 Mr of the
60-subunit tE2. The same threshold applied to the
reconstruction from the 0.95 size group (34% smaller structure) also
gave a molecular volume that corresponds to 1.6 × 106
Mr. This argument is further supported by a
comparison of the structures representative of the 0.9 and 1.1 size
groups, which vary in diameter ~17% (see below). Again, if this size
variation is related to a difference in their trimer composition, the
smallest structure should have about eight fewer trimers than the 1.1 size group structure. We believe that images representative of such a
disrupted structure could be detected by visual inspection (Fig. 2C). Moreover, since the icosahedral symmetry of these
complexes would be significantly perturbed, the resolution of the
smaller structures should be poorer than that of the larger structures. It is reassuring that the resolution of the reconstructions are comparable and independent of their sizes (see below). Accordingly, we
conclude that the particles in these data sets have similar subunit
composition, and therefore the size variation of the particle images
seen in Fig. 1 is independent of any small subunit variation in the particles.
Comparisons of the Three-dimensional Structures of Truncated
E2--
We have previously proposed that cryoelectron
microscopy affords a reliable means of determining the size variability
and the molecular architecture of macromolecules at room temperature, since the specimen is cooled rapidly to a temperature that freezes their flexibility. The good agreement between the frozen-hydrated and
stain (recorded at room temperature) data sets of the size variation
and their single particle images (Fig. 1) and their reconstructions
(Figs. 2 and 3) strongly support this
proposal.
The images of frozen-hydrated and stained molecules (Fig.
1B) and their reconstructions (Figs. 3 and
4) clearly show the continuous size
variability across the distribution curve, and the intact and cut-away
structures demonstrate that they maintain a very similar dodecahedral
architecture with its 20 triangle-based trimers at its corners
interconnected by 30 bridges (Figs. 3 and 4).
The architecture of the reconstructions from the ice and stained
molecules of corresponding size are very similar, as are their size
variations (Figs. 3 and 4). The ice and stain reconstructions exhibit a
diameter variation of 17% (232-272 Å) and 15% (237-272 Å),
respectively. The good agreement between the estimated size variation
of the data sets supports the versatility and the reliability of the
algorithms used in the sizeDiff program and the protocols developed for
this study.
The overlay comparison of the smallest and largest structures computed
from ice (Fig. 3) and stain (Fig. 4) data provides clues as to the
manner by which their sizes are related. The outer surface of the
largest structure encompasses all of the surface of the smaller one,
whereas both of the cut-away inside views exhibit only the inner
surface of the smallest structure. In other words, the largest
structure is on the outside of the overlays, whereas the smallest
structure is on its inside. Consequently, the largest and the smallest
structures are related by a change in the orientation and separation of
the trimers (see below) in contrast to a global (isotropic) swelling of
the trimers, which would result in the largest structure totally
encompassing the smallest one on both its inside and outside surfaces.
Recall that the smallest and the largest structures have the same mass,
further supporting the proposed relationship between the sizes of these structures.
The size variability of the core is related to conformational changes
in the trimers and their interconnecting bridges (Fig. 5). The views of 1-pixel-thick slices
along the 2-fold axis from the center of the core show small variations
in the protein density distribution and a significant increase in the
indentation between adjacent trimers that is part of the outside
surface of the bridge structure. The views of slices down the 3-fold
axis also indicate that the protein density shifts somewhat from the
corners of the triangular shaped image toward its center. In both
cases, the protein density distribution in the 1.1 structure appears to
best match that seen in the x-ray structure.
The 20-25-Å resolution of the reconstructions would appear to
prohibit a visual detection of the change in the separation of the
trimers, since the 0.90 and 1.10 structures differ in diameter by ~40
Å, corresponding to a 14-Å change in the trimer separation. This is
calculated based on the geometric relationship of the diameter of a
dodecahedron and its bridges. Nonetheless, the visualization of
features smaller than the resolution of the reconstruction has been
documented for numerous three-dimensional EM structures (33, 34). A
comparison of the solid-shaded structures (Figs. 3B and 4B) and their 2-fold slices (Fig. 5) shows
a significant change in the separation of adjacent trimers.
Recombinant mE2 Structures--
We have chosen to
study extensively the structural variation of the tE2
scaffold because the flexible N-terminal half of mE2 may
result in three-dimensional reconstructions that are more difficult to
interpret. Also, the x-ray structure of B. stearothermophilus tE2 more closely corresponds to the
structure of S. cerevisiae tE2. Even so, the
relevance of the size variation of the tE2 to the wild-type
E2 may be questioned, since nearly half of the protein of
the E2 subunit is lacking. Accordingly, we have
investigated size variation of intact, recombinant mE2,
utilizing frozen-hydrated molecules.
A plot of the number of mE2 particles versus
relative size gives the similar bell-shaped curve associated with the
tE2 data sets, except the plot may reflect a skewed
distribution of particle sizes, possibly as a consequence of the
internalization of the N-terminal domains of mE2 (see
below) (cf. Fig. 6A
with Fig. 1A). The 0.95, 1.0, and 1.05 solid-shaded structures of mE2 are similar to
the corresponding tE2 structures (cf. Fig. 6
with Fig. 3). This finding affords structural validation of numerous
biochemical studies (1-3, 35) that showed that the N-terminal half of
intact E2 is composed of flexible domains and, as a
consequence, is not revealed in the images of the mE2
reconstructions.
The overlay structures of the 0.95 and 1.05 mE2
reconstructions are entirely consistent with those corresponding to
tE2 (cf. Fig. 6D with Figs. 3 and
4B) and show that the size variations are similar. The good
agreement between these overlay structures is further validation that
the size variation of the core is related to the variable distance
between adjacent trimers. We were unable to compare structures
representative of the 0.90 and 1.1 data sets because of the more
limited number of images in the data set. However, we have analyzed
about 3000 particle images of recombinant human E2.
SizeDiff gave a size distribution profile very similar to that of
tE2 (Fig. 1),3
further supporting the proposal that the size variability is a
fundamental property of the C-terminal domain of the E2 subunit.
However, there is a difference between the 1.05 size group
tE2 and mE2 structures in that the latter
displays significant density inside the scaffold not seen in the former
(cf. Figs. 3A and 6C). A comparison of
the E2 0.95, 1.0, and 1.05 structures indicates that the
larger scaffold accommodates more internal density. This result is
confirmed by a comparison of the radial density plots (data not show).
The observation that tE2 lacks this internal density (Figs.
3A and 4A) (11) shows that some of the 60 N-terminal lypoyl domains of E2 reside inside the scaffold and the larger scaffold may accommodate more of this portion of the
subunit. In contrast, the outside of the tE2 and
mE2 cores are similar (Figs. 3, 4, and 6), indicating that
the N-terminal domains that reside outside of the core are not
restricted and, hence, do not appear in the reconstruction. The
internalization of the N-terminal flexible domain also occurs in the
cubic core of the branched-chain Visualization of Protein Dynamics by Electron Microscopy--
That
proteins exhibit conformational flexibility has been appreciated for
many years. More recently, this subject has gained in interest because
of its significance to the function of macromolecular complexes (37,
38). For example, recent reports show that the dynamics of enzymes in
regions that are removed from the catalytic site may be crucial to the
enzyme's activity (37). Moreover, proteolysis studies of the
Three-dimensional electron microscopy is well suited to the study of
the details of protein dynamics. The pH-sensitive size changes of
cowpea chlorotic mottle virus (41) and HK97 virus (42) were documented
by cryoelectron microscopy. Particle classification protocols made
possible the identification of structural intermediates in the
conversion pathway and, therefore, further insight into dynamics of the
process (41, 42). In the present study, our sizeDiff program sorts
particles based on their relative size (Figs. 1 and 6A) and
subsequently documents structural (Figs. 3 and 4) and conformational
(Fig. 5) changes that are related to the size variation of the structures.
Mechanisms for the Size Variability of the E2
Cores--
Recall that the overlay of the smallest and largest
structures shows that the surface of the larger structures completely resides on the outside, whereas the surface of the smaller structures is seen to predominate on the inside of the core (Figs. 3B,
4B, and 6D). Moreover, the morphology of the
trimer building blocks and their interconnecting bridges appear
independent of the size of the structure. Thus, we conclude that the
size variability of the dodecahedral scaffold appears to be primarily
related to a change in the variable distance of the trimers on their
3-fold axes to the center of the molecule. Consequently, the distance between adjacent trimers is variable.
The structure representative of the 1.0 size group in the bell-shaped
distribution curve should be energetically most favorable (Fig. 1). We
propose that the size change of the molecule involves a significant
contribution from a synchronous change in the length of the bridges
with their springlike connections. A completely asynchronous or random
change in bridge length would produce molecules of the same size, and
consequently, the 20% size variation observed in this study would not
be detected. The harmonious change in the length of the bridges is
energetically more favorable than the asynchronous alternative, because
the latter disrupts the symmetry of the molecule at an energy cost.
Comparisons of comparable slices of the reconstruction representative
of the different size groups show that the scaffold expansion and
contraction is coupled to changes in the protein density distribution
in the trimers and their bridges (Fig. 5). The different extent of the
demarcation between adjacent trimers appears to be the most significant
conformational change associated with the size variation.
The x-ray structure of B. stearothermophilus
tE2 offers a plausible explanation for the flexible
springlike connectivity between adjacent trimers (Fig.
7). Although the subunits of the trimers exhibit extensive connections between them (Fig. 7B),
adjacent trimers are only held together by interactions of the
C-terminal methionine of one subunit with a hydrophobic pocket afforded
by the adjacent subunit (Fig. 7A) (8). This
"ball-and-socket" connection would be expected to afford
trimer-trimer interaction with rotational flexibility. The x-ray
structure also suggests a springlike connection between adjacent
trimers consisting of a C-terminal loop region (residues 397-402) that
is anchored to a Generality of the Putative Flexible Bridge--
We believe that
E2 cores are exceptional examples of what has been
designated "soft protein" (38). The x-ray structure of B. stererothermophilus tE2 shows that adjacent trimers
are joined through two hydrophobic contacts between adjacent subunits
(Figs. 7 and 8). The association energy due to hydrophobic interactions is similar to thermal energy at room temperature (38).
A common structural feature of the E2s is the trimer
building blocks that reside at the 8 or 20 vertices of a cubic or
dodecahedral structure, respectively. The A. vinelandii
truncated E2 cubic core (7) also shares the ball-and-socket
connection between adjacent trimers found in the E. faecalis
and B. stearothermophilus dodecahedral E2s (8)
and, presumably, the S. cerevisiae and human
E2s. Although the cubic tE2 from A. vinelandii does not have the proline residue near the C-terminal
connection found in the subunit of the dodecahedral cores, it readily
dissociates into trimers upon binding E1 or E3
components. This indicates that the interaction between trimers is weak
(43), which is confirmed by the x-ray structure (7). In contrast,
E. coli truncated dihydrolipoamide succinyltransferase,
which shares the cubic core arrangement with the A. vinelandii truncated acetyltransferase and its ball-and-socket
connection, exhibits additional interactions between adjacent trimers
that add significant stability to this complex (9). Interestingly, the
C-terminal region of the succinyltransferase has all of the features of
the springlike connection of the acetyltransferase (i.e.
loop/cantilever with its associated proline) (9). It is not known if
the cubic 24-mer E2s exhibit the breathing core flexibility
to the extent of the dodecahedral E2s. However, they both
have a similar gap size between adjacent trimers that appears to
accommodate the size variability of the yeast and human E2 cores. It is noteworthy that the B. stearothermophilus,
E. faecalis, S. cerevisiae, and human
dodecahedral E2s have a proline moiety 5-8 residues from
their C termini, and this residue is often associated with a flexible
region in polypeptide chains (44). Furthermore, sequence analyses using
ClustalW (45) show a 49% sequence homology between these truncated
E2s but a 55% homology between the last 29 residues of
their C termini, indicating that they all share the loop region,
cantilever, and ball-and-socket feature seen in the x-ray structure of
tE2 from B. stearothermophilus and E. faecalis and thus similar flexibility.
Functional Consequences of the Protein Dynamics of the
E2 Cores--
The wild-type S. cerevisiae PDC comprises the 60-subunit E2 core, with
BP, E3, and E1 attached. Our initial analyses
of the S. cerevisiae PDC, its subcomplexes, and the
recombinant tE2 subcomplexes indicate that the disposition
and extent of binding of the constituents is related to the size of the
core (data not shown). It is apparent then from these observations and
the present study that the structural organization of PDC cannot be
completely represented by a single reconstruction and that multiple
reconstructions are required to gain an appreciation of the influence
of protein dynamics on its architecture, organization, and function. In
this regard, the E2 core of the wild-type PDC (Fig.
9B) also exhibits size variability. Thus, the complete complex exhibits a similar flexibility to that of the tE2 and mE2. Most interesting,
the E1 on the outside of the core mimics the
expansion/contraction of the underlying core (Fig. 9). The
aforementioned conservation of the sequence homology of the
dodecahedral cores of the S. cerevisiae, human, E. faecalis, and B. stearothermophilus through all
phylogeny indicates that the flexibility associated with the yeast PDC
has an important role in its function. We propose that the breathing
core augments the movements of the lipoamide swinging arms between the
catalytic centers and that the apparent movement of the entire complex
may augment substrate channeling and promote catalysis by methods that
are just beginning to be understood (46, 47). The rapidity of the
expansion and contraction of PDC is unknown but may be investigated by
dynamic light scattering and atomic force microscopy.
Although the PDC is an example without precedence of thermally induced
global protein dynamics, this phenomenon may be more generally
applicable than presently appreciated. For example, the photosynthetic
reaction center of the bacterium Rhodobacter sphaeroides
appears to be augmented by the thermal motion of the protein matrix
(46, 48). Moreover, the studies of the flock house viral capsid
indicate that its complex exhibits global protein dynamics that are
also thermally driven (39). The present study shows that single
particle analysis by three-dimensional electron microscopy is an
appropriate and powerful method of examining protein dynamics, and as
this technology achieves higher resolution structures, the generality
of this phenomenon and its details may emerge. If so, the complexity of
three-dimensional EM analyses will increase significantly.
Nevertheless, these studies should significantly enhance the
understanding of the relationship of protein dynamics to the structure
and function of macromolecular complexes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5 s (12), i.e. the
time required to reduce the temperature of the specimen from room
temperature to approximately
170 °C, at which point molecular
motion is frozen. The tE2 and mE2 molecules exhibit ~20% size variability with a bell-shaped distribution that
is related to conformational changes in the trimers and the bridges
that connect adjacent trimers. Remarkably, the flexibility of
tE2 core is also documented by stain electron microscopy.
We propose that the expansion/contraction phenomenon is thermally driven and is related to a rotationally flexible ball and socket that
forms the bridgelike contacts between adjacent trimers. A cantilever-like feature containing a proline residue may serve to
augment and transmit the flexibility of an upstream loop to the
ball-and-socket connection of the bridge. Sixty of these springlike features, two at each of the 30 bridges, are a common motif of the
dodecahedral cores of B. stearothermophilus, E. faecalis, and presumably S. cerevisiae and the human
pyruvate dehydrogenase complexes. The "breathing" of the core
structures affects the disposition of the bound constituents and may be
an important component of the function of these multienzyme complexes
by promoting the shuttling of the intermediates of catalysis associated
with the lipoamide arm to the numerous reaction centers and by
augmenting the catalysis through the movement of amino acid residues at
the catalytic sites.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/Å2 at × 50,000 magnification. Micrographs were digitized with a Zeiss SCAI
microdensitometer (Carl Zeiss, Inc., Englewood, CO) using a step size
of 2.8 Å/pixel at the specimen scale.
(S.D.) above the mean density of the
map unless otherwise indicated. The radial density distribution profile
was obtained by spherically averaging the three-dimensional density map.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (97K):
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Fig. 1.
Size distribution of the tE2 core
(A) and a comparison of selected images from the
different size groups (B). A, the bar
graph shows the approximate distribution of about half of the
3940 and 3302 particle images of the frozen-hydrated and stained
molecules, respectively, utilized in the sizeDiff analyses. The
distribution profiles from ice and stain data are very similar and
their bell shapes indicate that there is a continual variation in the
size of the images that is supported by the display of selected
particle images from the indicated size groups (B).
B, the circle around the images corresponds to the diameter
of the images from the 1.1 size group and serves as an aid in
determining their relative size. Scale bar, 100 Å in all figures.
View larger version (129K):
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Fig. 2.
Galleries of tilt pairs of tE2
frozen-hydrated molecules from the same field. The
second row consists of corresponding images to
the first row that were recorded with the stage
tilted 35° (tilt axis is approximately parallel to the
bottom of the figure). A and
B consist of images that were visually selected based on
their relative larger (A) and smaller (B) size.
The tilted images corresponding to the larger particles are round,
indicating that their larger size is not related to a flattening of the
molecule. C, tilt pairs of molecules that were judged to be
incomplete. Some of the images 1-5 in the top
row do not appear significantly smaller than those images in
A and B, although half of the structure may be
lacking (e.g. tilt pair in C, column
1, upper row, in which only half of
the molecule is seen). This conclusion results from the spider-like
appearance of the nontilted image and the distinctive cartwheel
appearance of its tilted counterpart in the 5-fold orientation of the
molecule. An image of the complete structure in this orientation does
not exhibit the large openings around the center of the image
(10).
-keto acid
dehydrogenase family of multienzyme complexes are known to form tightly
associated trimers that are held together by weaker connections to form
the cagelike scaffold (7-9). The connections between trimers involve
C-terminal residue(s) of adjacent subunits. In this regard, the
truncated E2 of the dihydrolipoamide succinyltransferase
from E. coli is normally a 24-mer, but it formed stable
trimers when expressed with a C-terminal His6 tag and
failed to associate further (32). These broken and/or incompletely formed cages are probably related to the tenuous connections between trimers, and the incomplete structures are lacking, to a variable extent, some of the trimer building blocks. Although we can readily visualize fragments of the core (Fig. 2C) and avoid these
images in the reconstruction, structures that lack a small number of trimers may not be possible to discern. Hence, it is possible that the
size variation of the images is related to incomplete molecules with
variable number of trimers. In this scenario, only the images near the
1.1 size classification are representative of complete structures, and
consequently, over 90% of the data set consists of molecules with
variable trimer content (incomplete structures, Fig. 1).
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Fig. 3.
A, 2-fold views of the shaded
surface representations of the intact and cut-away reconstructions from
the images of the frozen-hydrated molecules of the selected size
groups; B, the overlay structures representative of the 0.90 (yellow) and 1.10 (light blue) size
groups. The structures were computed from the images
corresponding to the size groups shown in Fig. 1. A,
the circle surrounding the structures has a diameter
corresponding to the 1.10 structure and is shown as an aid in
determining the relative size of the structures. The reconstructions
are rendered at the same threshold (contour level). The structures in
A show that there is a continuous size change of the
molecule across the bell-shaped distribution curve (Fig. 1). The
superimposed semitransparent and wire frame structures (B)
show that the size change is related to a variation of the distance of
the trimers along their 3-fold axes from the center of the
molecule.
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Fig. 4.
A, 2-fold views of the shaded
surface representations of the intact and cut-away reconstructions from
the images of the stained molecules of selected size groups;
B, overlay structures representative of the 0.90 (yellow) and 1.10 (light blue) size
groups. The structures were computed from the size distribution of the
stained molecules (Fig. 1) at the indicated size group and rendered at
the same threshold (contour level). The close agreement between the ice
and stain reconstructions from independently obtained preparations of
the tE2 supports the reliability of the methods used to
analyze the size variability of the particle images and shows that the
frozen-hydrated reconstructions preserve the size variability of the
molecules at room temperature (see "Comparisons of the
Three-dimensional Structures of Truncated E2"). A
reconstruction with 15-Å resolution was achieved, representative of
the most populated 1.0 size group using the close-to-focus images
(0.2-0.4-µm underfocus, structure not shown).
View larger version (35K):
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Fig. 5.
Galleries of the protein density distribution
in 1-pixel-thick slices from the frozen-hydrated reconstructions
representative of the indicated size groups. The slices of the
2-fold view were cut normal to the 2-fold view of the molecule at its
center. The 3-fold view images were cut normal to the 3-fold axis at a
position that was judged to be approximately equivalent in the various
size groups based on similarity of the density surrounding the trimers.
The symmetry axes for the 2- and 3-fold views are parallel and normal
to the long edge of the page,
respectively. The same temperature factor was applied to the
reconstructions before excising the slice. The 2-fold view of the
slices show that the demarcation between adjacent trimers is more
pronounced in the largest structure, and the density at the corners of
the trimers in the 3-fold view appears to become less pronounced in the
largest structure, indicating that the size variability is related to
conformational changes in the trimers and their bridges.
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Fig. 6.
Analyses of frozen-hydrated recombinant
mE2 molecules. A, the size variation of the
data (331 images); B, radial density plot of the
reconstruction representative of the 1.0 size group; C,
shaded surface view of the reconstructions representative of
the size groups indicated; D, superimposed reconstructions
of the largest (green) and smallest (red,
semitransparent) reconstruction and its cut-away inside view. The
resolution of these reconstructions between 30 and 34 Å is ~10 Å different from those from the tE2 data set because of the
fewer particles used in the E2 data set. The E2
images exhibit a similar bell-shaped size distribution of the
tE2 (Fig. 1), and their reconstructions also show a
continual variation in size of the structure (B) across the
size distribution. The radial density plot of the 1.0 structure shows a
significant peak of density inside the core that is attributed to the
insertion of the flexible N-terminal lipoyl domains, presumably through
the pentagonal opening of the core, since the tE2
reconstruction lacks this peak (11). The shaded surface
views show that the largest (1.05) structure accommodates more of the
N-terminal domain presumably because of the increased size of its
internal cavity. Its radial density plot shows that the area under the
internal peak is more than double that corresponding to the 0.95 structure (data not shown). The superimposed structures (D)
support the previous proposal that the expansion of the core is related
to an increase in the distance of the trimers on their 3-fold axes to
the center of the molecule.
-keto acid dehydrogenase complex as
determined by dark field electron microscopy studies (36). Thus, the
large openings in these E2 scaffolds and their empty
interiors readily accommodate a portion of their highly flexible
N-terminal domains.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-peptides inside flock house viral capsid show that they are in
equilibrium with the capsid's surface, and their dynamic domains may
contribute to the initiation of RNA release and translocation (39).
Small angle x-ray scattering studies of the electron-transferring
flavoprotein from Methylophilus methylotrophus indicate
that the protein samples a range of conformations in solutions and,
consequently, renders its structure complementary to its putative
binding site on the trimethylamine dehydrogenase (40).
sheet followed by an
-helix (residues 403-419)
(Fig. 8). Proline 420 at the C-terminal
end of the rod-shaped
-helix introduces an elbow bend that directs
the C-terminal methionine (residue 425) into the hydrophobic pocket of
the subunit of the adjacent trimer. The C-terminal residues (421)
following the proline are part of the bridge-like feature of the
complex. The
-helix/proline/C-terminal residues produce a
cantilever-like structure that may serve to transmit and contribute to
the movement of the flexible loop region. This study shows that the
bridge length varies ~14 Å, which corresponds to an approximate
deviation of ±7 Å in the length of the bridge from its position in
the energetically most stable structure represented by the 1.0 size
group.
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Fig. 7.
Two- and 3-fold views of the
three-dimensional structures of B. stearothermophilus
tE2. The shaded surface
representations were obtained by converting the atomic x-ray
coordinates to electron densities at 25 Å using a Gaussian filter
(30). A, the diameter of the molecule measured between the
outside edge of opposing trimers in the 2-fold orientation is ~256
Å. The ribbon representation of the bridge on its 2-fold axis shows
that there is an ~10-Å gap between adjacent trimers and that the
trimers are interconnected by two C-terminal extensions of the
polypeptide chain from the opposing subunits (red and
blue) that form the bridge. The companion
image shows the orthogonal view of the bridge as denoted by
the arrow. B, the 3-fold view of the trimers in
the ribbon presentation shows the extensive interactions of the three
identical subunits, denoted in different colors,
which comprise the building blocks of the complex. Its orthogonal view
denoted by the arrow further delineates the subunit-subunit
interactions in the trimer. The similarity between the slices from the
largest S. cerevisiae structure (Fig. 5) and the x-ray
structure suggests that the x-ray structure is more representative of
the largest EM structure that also has a pronounced gap between
adjacent trimers.
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Fig. 8.
Ribbon diagram of the residues from the
C-terminal region of the x-ray structure of B. stearothermophilus tE2 that comprise the
putative spring that interconnects adjacent trimers. A loop region
comprising residues 397-403 is anchored to a -sheet. This is
followed by a four-turn
-helix beginning with residue 403 and
disrupted by a proline (residue 420) that directs residues 421-425 to
the subunit of the adjacent trimer. The C-terminal Met (residue 425)
resides in a hydrophobic pocket (HP) of the adjacent subunit
to form the ball-and-socket connection. Residues 403-425 form a
cantilever-like structure, which is attached to the loop at its
N-terminal end. We propose that the assembly of residues 397-425 forms
the springlike connection between adjacent trimers that can adjust to
account for the size variability of the core.
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Fig. 9.
Particle images and reconstructions of the
S. cerevisiae wild-type PDC. The single particle
images (A) are approximately twice the size of
tE2 (cf. Figs. 1 and 2), and the E1
component on the outside completely obscures the underlying core. The
2-fold view of the cut-away structures (B) from images that
vary ~10% in size rendered at the some threshold show that the
disposition of E1 is influenced by the size of the
underlying core. Since the E1 component appears to closely
follow the size change of the scaffold, the rigidity of the connection
may be greater than indicated by its absence in the reconstruction.
These reconstructions and our previous reconstructions of
E1 bound to E2 (49) show that the disposition
of the E1 tetramer is restricted, thus further supporting
the notion that the tether is not highly flexible. The negative
contrast surrounding the E2 scaffold resulting from the
Fresnel fringe effect (50) may significantly mask this connection. The
protein dynamics of the core are an integral part of the structural
organization of the fully assembled PDC.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Marvin L. Hackert and Timothy S. Baker for helpful discussions, Lena Hammer for processing some of the data, and Karon Marek and Jean Williams for secretarial support.
![]() |
FOOTNOTES |
---|
* This work was supported by the American Heart Association, Texas Affiliate, Grant 98BG288; the Pew Charitable Trusts (to Z. H. Z.); United States Public Service Grants HL42886 (to J. K. S.), AI46420 (to Z. H. Z.), and GM06590 (to L. J. R.); a grant from the Foundation for Research (to L. J. R.); Swedish Medical Research Council Grant MFR12175 (to R. H. C.); and National Research Council Grant NFR11691 (to R. H. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains a QuickTime movie.
§ These authors contributed equally to this work.
** Present address: Scirex Corp., 3200 Red River St., Austin, TX 78705.
Present address: Ciphergen Biosystems, Inc., Palo Alto, CA
94306
§§ To whom correspondence should be addressed. Tel.: 713-500-5385; Fax: 713-500-0730; E-mail: James.K.Stoops@uth.tmc.edu.
Published, JBC Papers in Press, April 2, 2001, DOI 10.1074/jbc.M101765200
2 J. E. Lawson, D. B. McCarthy, and L. J. Reed, unpublished results.
3 X. Yu, J. K. Stoops, T. E. Roche, and Z. H. Zhou, unpublished result.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PDC, pyruvate dehydrogenase complex; E1, pyruvate dehydrogenase; E2, dihydrolipoamide acetyltransferase; E3, dihydrolipoamide dehydrogenase; tE2, truncated dihydrolipoamide acetyltransferase; mE2, mature E2; EM, electron microscopy; BP, binding protein.
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