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INTRODUCTION |
-Macroglobulins (
Ms) are
nonspecific, irreversible inhibitors of endoproteinases found in the
circulation of all vertebrates and some invertebrates (for a review,
see Ref. 1). Human
2-macroglobulin (
2M),1 the largest known proteinase
inhibitor (Mr = 720,000), is a homotetramer formed by two protomeric units, each of which contains two 180-kDa subunits linked by two disulfide bonds. It has a vital role in the
clearance of proteinases from the circulation and in regulating their
activity in fibrinolysis, coagulation, and complement activation (2,
3). A single
2M molecule can entrap two proteinase molecules such as chymotrypsin and trypsin and can therefore be considered to contain two functional domains (1). Each subunit of
2M has a bait region with cleavage sites for nearly all
known endoproteinases and an internal thiol ester bond. A proteinase cleaves the two bait regions within both functional units, leading to
an activation and cleavage of the thiol ester bonds. Consequently,
2M undergoes a major structural change resulting in
entrapment of the proteinase and its covalent linkage to the molecule
(1, 4). The bound proteinase, although inaccessible to proteins, may
react with small substrates and inhibitors (2), which is in contrast to
the mode of inhibition of all other natural proteinase inhibitors that
bind at the proteinase's active site. Treating
2M with
a small nucleophile such as methylamine also causes cleavage of
the thiol ester bonds, leading to a structural transformation of the
molecule (1). Electron microscopy reconstructions have shown that the
methylamine-transformed
2M (
2M-MA) has a
similar structure to the
2M-proteinase complex (5).
The remarkable structural rearrangement of
2M has been
the subject of numerous electron microscopy studies (6).
Three-dimensional reconstructions of native (7, 8), and fully
transformed
2M (5, 9) reveal molecules of very different
shapes. A low resolution x-ray map of
2M-MA (10) is in
general agreement with the electron microscopy structures. These
results, as well as other physicochemical studies (2), have shown that
the native and transformed molecules have significantly different
structures. The native structure is more globular, with dense end
regions that are connected by twisted strands (7, 8). The transformed structure has a more compact central region of protein with four arms
extending from its sides, similar to the letter "H" (5, 9, 10). It
also migrates more rapidly on nondenaturing gels than native
2M (1, 2). The trapped proteinases are located in an
internal cavity, symmetrically above and below the minor axis of the
transformed molecule (5).
With structural details of the native and transformed molecules
emerging from electron microscopy reconstructions (5, 7-9) as well as
a low resolution x-ray map (10), conflicting proposals have been
advanced for the transformation of
2M. It was proposed that a lateral compression and unfolding mechanism links
2M and
2M-MA (8). On the other hand, the
observation that the native and transformed structures are constituted
of two twisted, side-by-side strands of opposite handedness has led to
the proposal that an unwinding and rewinding of these constituent
strands leads to proteinase entrapment (7).
2M-half transformed (
2M-HT) provides a
plausible structural link between the native and transformed
structures. This intermediate structure was obtained by reacting the
native molecule with chymotrypsin covalently bound to Sepharose (11).
Two bait regions and two thiol ester linkages are cleaved within this
homotetrameric structure, and the proteinase is not bound.
2M-HT reacts with and traps a proteinase by cleavage of
the remaining bait regions at a slower rate than the native structure.
Its rate of migration on nondenaturing gels is intermediate between
2M and
2M-MA (11).
In this study, the three-dimensional structures of
2M-HT, obtained from negative stain and frozen-hydrated
specimens, are compared and show excellent concordance indicating that
the molecule is well preserved by both methods and that the
reconstructions are reliable. Comparisons of the structure of
2M-HT with the native and transformed structures were
investigated to further understand the mechanism of its structural
rearrangement and identify the functional division in native
2M.
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EXPERIMENTAL PROCEDURES |
Protein Preparations--
Half-transformed
2M was
prepared by reaction of human plasma
2M with
chymotrypsin-Sepharose. Chymotrypsin-Sepharose was prepared by
extensive washing with 50 mM Hepes, 100 mM
NaCl, and 2 mM EDTA (pH 7.4). Approximately 5 g (wet
weight) of the chymotrypsin-Sepharose was incubated with 40 mg of
plasma
2M in a total volume of 12 ml. The suspension was
gently rocked at room temperature, and the progress of the reaction was
monitored by assaying for remaining intact thiol esters. After 5 h, the chymotrypsin-Sepharose was removed by centrifugation and the
supernatant filtered through a 0.45-µm filter. Iodoacetamide (10 mM) was added to react with the free SH groups generated,
and the protein was then dialyzed against 50 mM Hepes, 100 mM NaCl, and 2 mM EDTA (pH 7.4). After gel
filtration on Sephadex G150, the
2M-HT was shown to
contain two cleaved and two intact bait regions as well as two intact and two cleaved thiol esters as described previously (11). However, no
chymotrypsin activity is associated with the
2M, and the
molecule is active, since it traps one molecule of chymotrypsin
(11).
Electron Microscopy--
2M-HT (5 µg/ml) in
0.05 mM sodium phosphate with 10 µg/ml bacitracin and
0.25% methylamine tungstate (pH 7.2) was applied by the spray method
to carbon films (12). Stain images were acquired with a JEOL JEM 1200 electron microscope operating at 100 kV using conventional irradiation
procedures. The 50°-tilted images were recorded with an underfocus of
~1.0 µm at the center of the stage, and the corresponding untilted
images were obtained at 0.5 µm underfocus. For cryo-electron
microscopy, a 3-µl sample of
2M-HT (0.1 mg/ml, in 10 mM citrate (pH 6.0), was deposited on a glow-discharged,
carbon-coated holey grid, and the excess removed by blotting with
filter paper. The grid was then rapidly cooled in liquid ethane.
Specimens were kept below
170 °C in a Gatan cold holder, and
images were recorded at 1.7 µm underfocus at an exposure of
~9e/Å2 using Kodak SO 163 film.
Digitization and Particle Extraction--
Diffraction patterns
from the micrographs were examined on an optical bench (5), and
micrographs showing astigmatism, motion, and drift were rejected. The
remaining micrographs were digitized with an Eikonix 1142 scanner with
12-bit dynamic range using a pixel size of 5.8 Å on the specimen
scale. The digitized micrographs were again subjected to power spectrum
analysis and re-examined for astigmatism and drift. The micrographs of
50°-tilted specimens were utilized in a useful range of underfocus
values so that the first zero in the contrast transfer function of the
electron microscope occurs between 12 and 25 Å.
The SUPRIM image processing software (13) was used for initial particle
picking and processing on Silicon Graphics Iris and Indigo2
workstations. The untilted micrographs were used to select the
characteristic "pseudo-lip" views of
2M-HT (see Fig. 1, a-c and k-m) in 64 × 64-pixel boxes.
The corresponding tilted views were obtained by matching the tilted and
untilted micrographs with 30-50 corresponding fiducial points. The
tilted particles were hand-centered, where needed, and then
extracted.
Image Alignment, Classification, and Random Conical Tilt
Reconstruction--
The entire set of 0° pseudo-lip views was
subject to a reference-free alignment (14) using the SPIDER software
(15) and correspondence analysis, followed by hierarchical ascendant
classification as described previously (16). Clusters best
corresponding to the pseudo-lip shape were selected. A final data set
of 50°-tilted particles (1640 images) was used in a random conical
tilt reconstruction (17). The resolution of this reconstruction was 41 Å as measured by the Fourier Shell Correlation (18) with a Fourier
ring criterion of 0.67 (19). This model was refined using a
three-dimensional projection alignment method (20) employing the tilted
particles (resolution = 38Å).
Stain and Frozen-hydrated Reconstructions from Untilted
Specimens--
The approach developed by Kolodziej et al.
(21) using three-dimensional projection alignment and iterative
reconstruction (20) was employed to compute the structure from stain
and ice images. A set of quasi-uniformly distributed reference
projections (20, 21) using the random conical tilt reconstruction as
the model was generated within
= 0-90° and
= 0-360° using
angular steps of 2°. The stain and frozen-hydrated images consisted
of 4750 and 2233 particles, respectively, from the untilted specimens. Two passes of refinement achieved a stable resolution value. The structures were Fermi-filtered using zero for the temperature (22) to
the resolution value of the reconstruction and the frozen-hydrated reconstruction was corrected for the contrast transfer function of the
electron microscope (23-25).
Display--
For presentation, three-dimensional images were
thresholded to a volume that corresponds to the molecular weight of
2M. Both stain and ice structures were displayed as
solid surface models using SUPRIM and subsequently rendered using the
Explorer software for SGI workstations (version 3.5, NAG Inc.,
Downer's Grove, IL). The images were printed using a Mitsubishi
S3600-3OU dye sublimation printer.
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RESULTS |
Images of
2M-HT in Stain and Ice--
A gallery of
stain and ice images of
2M-HT shows pseudo-lip views
(Fig. 1, a-c and
k-m, respectively), which exhibit similarity to the
"lip" views of native
2M. These oval-shaped images
have small triangular and rounded shapes of higher protein density at
opposing ends connected by two curved strands. These are in contrast to
a similar arrangement of two small triangular shapes of higher protein
density in the lip view of the native molecule (7, 8). Pseudo-lips with
various in-plane rotations in the untilted micrographs interconverted
into a variety of other shapes upon tilting the microscope stage 50°
(data not shown). By observing these conversions, other orientations of
2M-HT were identified in the untilted stain and ice
images and subsequently used for the refinement of the initial random
conical tilt reconstruction using the three-dimensional projection
alignment (20, 21). Some of these orientations, representing side and
end views, are shown in Fig. 1 (d-j and n-t).
It is interesting to note that none of these images resemble the single
particle images of the binary chymotrypsin
2M complex
(26). This difference may result from the secondary reaction of the
trapped proteinase with the remaining uncleaved
2M
subunits in the binary complex preparation (11, 27). In the present
study, the
2M-HT preparation was found to contain two
free thiols and thiol ester moieties per molecule when the electron
microscopy images were recorded.

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Fig. 1.
Stain and frozen-hydrated galleries of
half-transformed human 2M. The unstained images are
presented in reverse contrast to facilitate comparison with the stain
images. The pseudo-lip shape is represented for the stain and ice
images by a-c and k-m, respectively. Other
images represent off-axis orientations of the molecule. The scale
bar in this and subsequent figures corresponds to 100 Å, and the
gray scale bar indicates relative protein density high
(white) and low (dark).
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Images of
2M-HT molecules in methylamine tungstate stain
are very similar to their corresponding views in vitreous ice,
indicating that the molecular architecture is faithfully reproduced by
the stain (Fig. 1). The Euler angle distribution plot of the projection directions for the untilted stain specimens exhibits an isotropic distribution of views showing that the molecules do not assume a
preferred orientation in stain on the carbon film (Fig.
2A). In contrast, the ice
images show a tendency to cluster about the
axis and therefore
under-represent the end views of the molecule (Fig. 2B).

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Fig. 2.
Distribution of the Euler angles obtained
from the untilted stain (A) and ice (B) data
sets. In this polar plot, represents a rotation about an axis
normal to the major axis of the molecule in the front view orientation
(Fig. 5) followed by a rotation about the major axis of the
molecule and plotted as the radius (20). The pseudo-lip view
corresponds to the origin. The stain data set shows a more uniform
distribution than the ice data set and therefore provides a structure
with more uniform resolution.
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Three-dimensional Reconstructions--
Accordingly, the easily
recognizable pseudo lip images were used in the initial stain random
conical tilt reconstruction (41-Å resolution), and this structure
served as a model to align the nontilted stain images in multiple
orientations using the three-dimensional projection alignment method as
described previously (20, 21). The wide distribution of projection
angles (Fig. 2A) ensures a reconstruction with more uniform
resolution than the random conical tilt structure. Using the previous
structure as a model, iterations of refinement were carried out until a
stable resolution value was achieved (32 Å, Fig.
3). The frozen-hydrated images were
aligned using the random conical tilt stain structure as a model, and further refinement was achieved using the resulting ice structure as
the model (34-Å resolution). An ice reconstruction using the refined
stain model was similar, and the resolution was not improved. The
random conical tilt structure is significantly narrower than the
refined stain and ice structures (height-width ratios of 1.5 and 1.6, respectively), and the vertical strand across the front of the random
conical tilt structure is incomplete (Fig. 3). These differences may be
related to the "missing cone" of information associated with the
random conical tilt structure (17).

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Fig. 3.
Comparisons of the initial random conical
tilt (RCT) and the refined stain and ice structures.
The structures were thresholded to a volume that corresponds to the
molecular weight of 2M and low pass-filtered to their
resolution. The random conical tilt structure is displayed at two-third
the size of the refined structures. The pseudo-lip (left:
stain, n = 41; ice, n = 10) and oblique
side (right: stain, n = 151; ice,
n = 10) average images are shown. The average images
were obtained from the candidate images that match the projection of
the structure in the pseudo-lip and oblique orientation over a range of
15° (20). Averaging greatly improves the signal-to-noise ratio of the
particle images. The oblique view was obtained by a 131° clockwise
rotation of the lip view of the structure within the plane of the page
and 75° clockwise rotation parallel to the left edge of
the page. The good agreement between the structures and their
projections and average images indicates that the reconstructions are
reliable.
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The stain and ice structures, their projections and the corresponding
average images show good concordance (Fig. 3). Comparisons of the
protein density distribution between 5.8-Å-thick slices of the stain
and ice structures also show good agreement (Fig. 4). It is interesting that the less
robust features associated with the central slices (8-16) in the stain
and ice structures have similar densities. This contrasts with a
comparison of the stain and ice structures of the Saccharomyces
cerevisae fatty acid synthase where the stain was found to enhance
the less robust features (21). The agreement obtained in the present
comparisons indicates that the architecture of
2M-HT has
been faithfully reproduced by both methods of imaging the molecule and
that the reconstructions are reliable.

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Fig. 4.
Relative protein density distribution in
slices from the stain and ice structures. Slices (5.8 Å thick)
were cut normal to the major axes of the structures and contour lines
were included as an aid in depicting the relative protein density
distribution. The good agreement between the corresponding slices
supports the proposal that the reconstructions are reliable.
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Stereo views of the stain structure show an oval-shaped structure (195 height × 130 width × 137 Å depth) with four large openings ~45 Å in diameter leading into an internal cavity (Fig.
5). The top of the structure is
chisel-shaped, whereas its bottom is broad and bulbous. The two ends of
the structure are joined by four strands (Fig. 5) whose protein
densities are considerably diminished from those associated with its
two ends (Fig. 4). The variability between the size and density of the
strands and different shapes of the two ends results in a structure
that lacks symmetry. The absence of 2-fold symmetry on the major axis
of the structure was unexpected, since it is not apparent in the
particle images (Fig. 1). It is possible that this minor asymmetry is
introduced during sample preparation or is exaggerated by image
processing. The following considerations lead us to believe it is a
valid structural feature of the molecule. As indicated above, the
preparation of
2M-HT has two intact thiol esters
moieties and is functional, since it traps one molecule of proteinase
(11). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed
that chymotrypsin-Sepharose cleavage is specific for the bait region
(11). The oblique side view average images in Fig. 3 also exhibit this
asymmetry and the projections of the structure in 15° angular bins
agree well with the average images from the candidates assigned to
these bins by the three-dimensional projection alignment method (data not shown). Finally, a refinement using a random conical tilt model
with 2-fold symmetry imposed on its major axis gave a structure similar
to that shown in Fig. 5 (data not shown). These results indicate that
the data set is self-consistent and further support our proposal that
the reconstructions are reliable.

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Fig. 5.
Stereo views of the stain structure. The
structure is asymmetric and has four large openings to the cavity which
may facilitate the entrapment of the proteinase. The major shape
difference between the top and bottom of the structure and the top's
similarity to the native structure indicate that the structural change
has primarily occurred in the bottom half of the molecule.
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A threshold level that doubled the volume of the structure resulted in
a global swelling but revealed no additional features.
Structural Comparisons of
2M,
2M-HT,
and
2M-MA--
The protein density distributions in
serial slices 5.8 Å thick cut normal to the major axes of the
structures show the variation in the central portion (body) of the
molecules (Fig. 6). In this figure, the
native and half-transformed structures are aligned so that the tops of
the chisel-shaped bodies are in the same orientation and the strands
associated with the native and
2M-MA structures are
oriented in corresponding positions in slice 12. In the native structure, the strands at the top and bottom (slices 1-5 and 20-25, respectively) are joined; they separate on either side of its middle
(slices 6-9 and 15-18) to form two openings to the cavity approximately 25 Å in diameter. As the strands course through the body
of the structure, they exhibit a 90° clockwise twist. The strands
associated with the chisel-shaped body of the
2M-HT structure are significantly larger than they are in the native molecule
(slices 1-6), and they separate into four lower density, thin strands
in the body of the structure (slices 7-18) and coalesce into a broad
oval-shaped body at the base of the molecule (slices 19-24).
Significantly, the strands exhibit little rotation through the
structure's body, and the four openings are approximately two times
larger than those associated with the native molecule. The
2M-MA structure (fully transformed) consists of four
higher density filaments which are joined by protein of lower density to form a pair of strands. The two strands that traverse the fully transformed structure twist 90° with a handedness opposite to that of
the native structure and form external grooves. The major top-bottom
asymmetry associated with the
2M-HT structure and the
similarity between the chisel-shaped body at its top with that of the
native structure suggest that the lower portion of the molecule has
reacted with the proteinase (see below).

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Fig. 6.
Comparisons of the relative protein density
in slices obtained from native, half-transformed, and fully transformed
2M ( 2M-MA). The top panel
shows the solid shaded structures viewed from the front and
top. The slices were produced by cutting the structures normal to their
major axes. The strands rotate clockwise and counter clockwise 90° in
the native and 2M-MA structures, respectively, and
separate into four strands without a twist in 2M-HT. It
is apparent that the cavity is more open and therefore accessible to
the proteinase in 2M-HT. The 2M-MA is
from Andersen et al. (10) and was filtered to 30-Å
resolution.
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DISCUSSION |
Proposals for the Structural Transformation of
2M--
The unusual structural change that permits
2M to function as a universal proteinase scavenger is
not readily apparent by a comparison of the rather dissimilar
structures of the native and transformed molecules. Their dissimilarity
has resulted in numerous and divergent proposals concerning the
proteinase trapping phenomenon from electron microscopy studies (6, 7,
8, 10, 16, 28, 29). The structure of the intermediate functional state
represented by
2M-HT can be analyzed for possible
similarities linking it to native and transformed
2M.
Hypotheses based on three-dimensional structures to explain the
transformation of
2M (7, 8, 10) can then be further
examined.
Boisset et al. (8) proposed that the transformation of
2M occurs with a compression along its major axis
coupled with a lateral expansion. Thus, the lip view of
2M changes into a shape that corresponds to the end view
of
2M-MA. However, it is difficult to reconcile the
shape of
2M-HT with their proposed rearrangement.
Andersen et al. (10) simulated the structure of native
2M by a vertical displacement of the subunits and some rearrangement of an internal region of protein density of their x-ray
structure. The shapes of the four individual subunits in the structure
were left unchanged. Their proposed transformation is inconsistent with
the reversal of the handedness of the twist in the two strands between
2M and
2M-MA (7) and does not account for
the partial untwisting and separation of the strands in
2M-HT.
Kolodziej et al. (7) showed that the two strands that
traverse the body of the native and
2M-MA structures are
a common feature and are arranged with the opposite handedness (Fig.
6). They proposed that the rearrangement involves a separation of the
strands at the opposite ends of the native molecule, their untwisting
to open the proteinase binding cavity and a retwisting around the
proteinase with the opposite handedness to entrap it (Fig.
7). The untwisting-retwisting hypothesis
of proteinase entrapment is supported by the present studies (see
below). This structural change is based on the consideration that the
two oppositely twisted strands are the protomeric units that are
noncovalently bound by interactions occurring primarily at the ends and
near the middle of the native and transformed molecules, respectively
(Fig. 7, a and c). Each protomeric unit contains
two disulfide-linked 180-kDa subunits in an anti-parallel arrangement
(7, 30), and the four thiol ester bonds maintain the strands in the
twisted state. In this model, the lateral arm-like extensions on
2M-MA (Fig. 7c) result from a separation of
the strands that are joined to form the chisel-like ends of
2M. This hypothesis is supported by immunoelectron
microscopy studies of monoclonal Fab-labeled
2M and
2M-MA. The Fabs are located near the chisel-shaped
bodies of the native structure and near the arms of
2M-MA (7).

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Fig. 7.
Diagram of the structural transformation of
2M. For clarity, the arrows only
indicate changes in the front half of the structures. Equivalent
portions in the back half of the image rotate in the opposite
direction. Initial proteinase (P) attack on the lower half
(a) is followed by a second proteinase attack on the top
half (b) to give the completely transformed
2M (c). RBDs denote receptor
binding domains (see text).
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Structure-Function Relationships--
It is established that the
initial proteinase reaction with
2M involves the
cleavage of two bait regions and the hydrolysis of two thiol esters to
give an intermediate form that reacts more slowly with the second
proteinase (1). We propose that
2M-HT is representative
of the intermediate structure. The
2M-HT structure supports the proposed rearrangement and gives further insight into
proteinase entrapment. The Sepharose-bound chymotrypsin cleaves two
bait regions located on the same side of the minor axis of the
structure resulting in the untwisting of the strands (Fig. 6) and their
partial separation in the bottom half of the structure to form its
bulbous shape (Fig. 7b). The complete separation of the
strands and their retwisting to form the arms associated with the
transformed structure are evidently hindered by the intact thiol esters
in the upper half of the molecule. In this proposal, one-half of each
protomeric unit is cleaved in the bottom portion of the structure and
therefore the functional division is on the minor axis. This assignment
of the functional division is supported by the three-dimensional
structure of the
2M-chymotrypsin ternary complex, which
showed that two molecules are encapsulated on either side of its minor
axis (5). In contrast, the top half of the structure has the
chisel-shaped feature of the native structure, which is probably
maintained by the two intact thiol esters. However, the changes in the
bottom portion of the structure result in a significant enlargement of
the chisel-shaped body and a partial separation of the strands in its
upper half (Figs. 6 and 7). These changes may be related to the reduced
reactivity of the remaining bait domains to subsequent proteolysis (1,
11). It is interesting, though, that the remaining thiol esters exhibit
the same reactivity to methylamine as in the native structure (11).
Their unchanged reactivity may be related to their similar disposition
in the two molecules.
In addition to the untwisting of the two strands after the initial
cleavage event, they separate to form four strands of lower protein
density resulting in a central cavity that is more accessible to the
proteinase (Figs. 6 and 7). The proteinase enters the open cavity of
2M-HT by passive diffusion or is pulled into the cavity as a consequence of its binding to the bait domain. In this regard, the
bait domain is not accessible to proteolysis in
2M-MA, suggesting that it is internalized (31).
We have ascribed the major top-bottom asymmetry of
2M-HT
to the preferential cleavage of the bait and thiol ester domains on one
side of the structure's minor axis. The minor left-right asymmetry
associated with the variable size and densities of the four strands may
be related to the multiplicity of cleavage sites for chymotrypsin in
the bait region (32).
Disposition of Sites of Biological Interest--
The cleavage of
the thiol esters by methylamine or as a result of reacting
2M with a proteinase exposes receptor binding domains.
As a consequence, the transformed
2M binds to receptors on hepatocytes and is internalized and degraded (1, 2). Immunoelectron
microscopy showed that these domains are associated with the arms of
the transformed
2M (33). We propose that these domains
are sequestered in the native structure where the two strands meet to
form the chisel-shaped bodies at both ends of the structure. The
separation and untwisting of the strands upon thiol ester cleavage
exposes these domains near the tips of the arms of the transformed
molecule (Fig. 7). It is not known if these domains are exposed in
2M-HT.
The four thiol groups (Cys-949) resulting from cleavage of the
functional thiol esters by methylamine have been located on the
interior walls near the center of the x-ray structure (10). The
internal distance between the thiols varies from 31 to 44 Å, which is
in agreement with values reported from fluorescence spectroscopy (34).
The location of the thiol esters in the native structure is not
known.
It was proposed that the four bait domains are located inside the
cavity of
2M-MA 11-17 Å from the Cys-949 (10, 35) and that this domain is also inside the native structure (10). Since new
inter and intramolecular disulfide bonds were formed in a variant that
contained a single cysteine residue within the bait region, it was
proposed that the four bait domains are located in close proximity in
the center of the molecule (36). However, the location of the new
disulfide bonds in the amino acid sequence was not determined so that
it is equally possible that they involve residues outside the bait
regions and consequently their location and putative proximity remains
ambiguous. Our structural studies suggest that the small ~25-Å
diameter openings of the native structure (7) appear too small to allow
access to an interior bait region by proteinases which vary in
molecular weight from 25 (chymotrypsin) to 85 kDa (plasmin).
Furthermore, the
2M-HT structure suggests that the
remaining bait domains reside near the chisel-shaped body where they
may function to maintain the contact between the two strands (37). An
alternative proposal is that the bait domain lies externally near the
bridge structures that serve as a gateway to the internal cavity (7).
(The bridge structures are two strand-like features that form the
external wall of the cavity near its middle). The
2M-HT
appears to lack these bridges that may be related to the diminished
reactivity of the bait regions to proteolysis.
We thank Dr. Jens Nyborg for the x-ray density
map of
2M-MA. We also thank Norman Nolasco, Steven
Kolodziej, Dr. Z. Hong Zhou, and Dr. John Schroeter for useful
discussions. We recognize the invaluable aid of Abigail Jimenez in the
production of the manuscript.