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INTRODUCTION |
Human
2-macroglobulin
(
2M1, Mr = 720,000)
is an essential protein present at a high
concentration in the serum (~2 mg/ml) that has the unusual
physiological role of a nonspecific proteinase scavenger (1-3). In a
presently poorly understood mechanism, native
2M
irreversibly traps almost all known endoproteinases by undergoing a
structural change that involves a large alteration in its shape.
Evolutionarily related proteins performing a similar physiological
function, termed
-macroglobulins, are present in all vertebrates and
several invertebrates (1). Recently, an impairment in the
2M gene has been implicated in the etiology of
Alzheimer's disease (4).
2M is a glycoprotein assembled from four identical
180-kDa subunits that are disulfide-linked in pairs to form two
protomers, which, in turn, are noncovalently associated (1). Each
subunit contains an approximately 40-residue-long sequence termed the "bait" region, which displays target sequences for a variety of proteinases (5). Bait region cleavage by a proteinase in turn causes
the activation of a functionally important internal thiol ester bond
between Cys949 and Glx952 of the subunit, which
rapidly undergoes a nucleophilic attack (1). Cleavage of the thiol
ester moiety triggers a major shape change, aptly termed the
"mousetrap mechanism," that causes
2M to internally
sequester the proteinase, which typically retains its catalytic
activity but is inaccessible to its target proteins (6).
An attacking proteinase cleaves two of the four bait regions of
2M in rapid succession (1).
2M can
therefore entrap up to two proteinases the size of chymotrypsin
(Mr = 25,000). Significantly, a direct
nucleophilic attack by methylamine on the thiol esters of native
2M results in a structural change similar to cleavage by
a proteinase (1). Thus, thiol ester cleavage has a pivotal role in the
shape change that accompanies the entrapment of the proteinase.
Transformed
2M obtained by either mechanism exposes receptor binding domains (RBDs) that allow its rapid endocytosis by
cell-membrane receptors principally displayed by hepatocytes but also
by a variety of other cells (1, 7).
Native and transformed
2Ms display significantly
different physico-chemical properties, including migration speeds on
nondenaturing gels (8) and Stokes radii (9). Three-dimensional electron microscopy reconstructions have shown that the two forms also exhibit
markedly different shapes (10-12). The ambiguity in relating structural features between the native and transformed molecules has
therefore led to conflicting models for the structural rearrangement involved in the transformation of
2M (10, 12). The
recently published structure of half-transformed
2M
(
2M-HT), which has only two cleaved bait regions and two
hydrolyzed thiol esters in its bottom half, has provided an important
link in understanding the process leading from native to transformed
2M (13). In the present study, we have employed
cryoelectron microscopy to obtain structures of native
2M (
2M-N),
2M-HT, and
2M-methylamine (
2M-MA) labeled with four
monoclonal Fab fragments that bind to a common epitope on all three
structural forms of the molecule and permit an assignment of related features.
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EXPERIMENTAL PROCEDURES |
Protein Preparations--
Purification protocols for native,
half-transformed, and methylamine-transformed
2Ms have
been previously described in Kolodziej et al. (12), Qazi
et al. (13), and Schroeter et al. (11), respectively. Monoclonal antibody 6E8 binds to a 55-kDa fragment obtained from a papain digest of transformed
2M that
lies principally within the N-terminal half of the 180-kDa
2M monomer (12, 14). Fab fragments from 6E8 were
previously shown to recognize both native and fully transformed
2M with a stoichiometry of approximately 4 mol of Fab
bound/mol of
2M (12). This electron microscopy study has
further confirmed the binding of four Fabs to the native, half-transformed, and fully transformed
2Ms.
Electron Microscopy--
A 6-8 M excess of 6E8
monoclonal Fab was added to the
2M-N,
2M-HT, or
2M-MA (transformed
2M) preparations so that the resulting
2M
concentration was 0.1 mg/ml. A 3-µl sample of each Fab-labeled
2M was added to a glow-discharged carbon-coated holey grid for cryoelectron microscopy. After removing the excess sample by
blotting with filter paper, the grid was rapidly cooled by immersion in
liquid ethane. A Gatan cold-holder was used to maintain the specimens
below -170 °C. Images were then acquired with a JEOL JEM 1200 electron microscope operating at 100 kV with an underfocus of ~1.7
µm and an exposure of ~9 e/Å2 on Kodak SO 163 film
(15).
Digitization and Particle Extraction--
Micrographs were
digitized using an Eikonix 1412 scanner with a 12-bit dynamic range and
a pixel size of 5.7 Å on the specimen scale. Power spectra from the
untilted micrographs were analyzed for astigmatism and drift.
Micrographs showing frost, significant astigmatism, or drift were
rejected. Representative particles were selected in 64×64-pixel boxes
using the SUPRIM software package (16). The
2M-N,
2M-HT, and transformed
2M data sets
contained 2567, 3498, and 2900 particle images, respectively.
Three-dimensional Alignment and Classification--
Previously
obtained unlabeled reconstructions (15) from single-particle images
using the methylamine tungstate stain and carbon support film (17) were
used as initial models for the alignment and refinement,
i.e. three-dimensional projection alignment and iterative
reconstruction, using the SPIDER software (18, 19).
In the case of
2M-N and
2M-HT, the
particles were initially aligned to isotropic projections from the
unlabeled stain models spaced 20° apart (19). Correspondence
analysis, followed by hierarchical ascendent classification (20) using
the SUPRIM software, was then used to identify and remove misaligned
particles in each projection direction. The edited particle data sets
(1564 particles,
2M-N; 3026 particles,
2M-HT) were then aligned to projections of these
reconstructions at 2° intervals and used to obtain reconstructions
with resolutions of 41 Å (
2M-N) and 38 Å (
2M-HT) using a Fourier shell correlation criterion of
0.67 (21).
For
2M-HT, a further pass of refinement was carried out
with 2-fold symmetry imposed on its major axis. This was done to give
equal prominence to all Fabs. The final reconstruction, which was not
further symmetrized, had a resolution of 38 Å.
The
2M-MA data set was initially examined using a
reference-free, K-means clustering algorithm in SPIDER with 50 clusters and 100 iterations (22). Of these, 41 average cluster images were
retained and aligned to a recently obtained, refined
2M-MA stain
model.2 A reconstruction from
these averages, which prominently displayed the Fabs, was used to
obtain a final refined reconstruction (39-Å resolution) from the
entire 2900 particle data set.
Display--
The reconstructions were corrected for the contrast
transfer function of the electron microscope as described previously
(23-25). Bandpass Fermi filtering (26) was applied to retain
information between Fourier space radii of 12 pixels (1/30.4
Å
1; close to the contrast-transfer function cutoff and
providing the best match with the unlabeled structures) and 1 pixel
(1/364.8 Å
1), with a temperature parameter of 0.01.
2M-N and
2M-MA reconstructions revealed
2-fold symmetry along their major axes, and consequently they were
222-symmetrized for display. The distal ends of the Fabs appeared
wedge-shaped in the average images (see Fig. 2) and mushroom-shaped in
the surface-rendered structures (data not shown), presumably because
their site of attachment is not rigid. In the solid-shaded structures,
their external ends were trimmed to give them a rod-like appearance so
that their contact with the surface can be more readily discerned.
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RESULTS |
Electron Microscopy--
In the galleries of frozen-hydrated
images (Fig. 1), four Fab fragments from
the monoclonal antibody 6E8 clearly bind to each of the three
structural variants of
2M. On most particles, the Fabs
can be discerned as rod-shaped protrusions with knob-like extremities
(Fig. 1). Typical shapes such as "lip" views of
2M-N (particles a, c, and g) (12), "pseudo-lip"
views of
2M-HT (particles j and m)
(13), as well as "H" views of
2M-MA (particles
t and u) (11) can be identified and are similar
to their unlabeled counterparts (11-13). Thus, the Fabs do not appear
to perturb the
2M molecules (11-13).

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Fig. 1.
Galleries of frozen-hydrated specimens of
Fab-labeled variants of human
2M. The four Fabs appear as
rod-like extensions with bead-like ends that bind to the external
surface of the molecules. The particle data sets, represented in
reverse contrast, contain a number of off-axis views. The
scale bar in this and subsequent figures corresponds to 100 Å, and the gray scale bar denotes relative protein density
from white (high) to dark (low).
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Cryoelectron microscopy was utilized as the imaging technique in the
present study because it permits the use of higher protein concentrations in the sample preparation than stain electron
microscopy. This assured that the four Fab binding sites are occupied
(Fig. 1) (dissociation constants of the
2M-Fab complexes
are in the nM range (12)). As often observed in the imaging
of nonviral proteins in vitreous ice, the
2Ms tended to
assume a preferred orientation in relation to the air-water interface
(27-29). However, we obtained a sufficient number of off-axis,
"rocking" views in all data sets, which permitted the computation
of three-dimensional structures with significant spatial information in
all directions (27).
Three-Dimensional Alignment and Reconstruction--
Our previous
reconstructions of the unlabeled
2Ms (11-13) (with
resolutions near 30 Å) were obtained from single particles imaged in
an amorphous layer of methylamine tungstate stain containing 10 µg/ml
bacitracin over carbon support film (17). Specimen application by the
spray method (17) and the inclusion of bacitracin minimized the
interaction of the particles with the support film and provides
multiple orientations of the molecules (28). Our technique provides
high contrast images of well preserved molecules, and the multiple
orientations of the particles result in reconstructions with uniform
spatial resolution (11-13). The stain structures (11-13) were used to
align (19) data sets of untilted, frozen-hydrated, single particles of
Fab-labeled
2M-N and
2M-HT or the
averages representing the data set for
2M-MA, obtained
using the reference-free technique of K-means clustering (see
"Experimental Procedures" (22)). The model-based three-dimensional
projection alignment method is a powerful procedure that bypasses
technical difficulties encountered in three-dimensional reconstructions
from tilted frozen-hydrated specimens. The method of using methylamine
tungstate stain reconstructions to align and reconstruct from untilted
frozen-hydrated data sets has been previously validated (13, 27, 28),
and the present study further demonstrates its utility in determining
structures of the corresponding Fab-labeled molecules.
A comparison of the projections of the Fab-labeled three-dimensional
structures and the corresponding two-dimensional average images (Fig.
2) clearly indicate that the processes of
alignment and reconstruction have correctly preserved the location of
the Fab labels. The Fabs are somewhat less prominent on the
2M-N structure, probably because of the predominance of
lip views in the data set.

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Fig. 2.
Solid-shaded views of Fab-labeled
native, half-transformed, and transformed
2Ms. Surface renderings,
projections, and matching average images for the three structural
variants prominently display the Fabs as rod-like extensions. The
arrangement of the four Fabs confirms the anti-parallel orientation of
the individual 180-kDa subunits in the tetramer. The three-dimensional
structures were filtered and thresholded to best match their unlabeled
stain analogs shown at the top of the figure at a volume that
corresponds to the molecular weight. The close agreement between the
projections of the structures and their corresponding average images
indicates that the reconstructions are reliable. The height, width,
depth of the structures are: 2M-N, 200 × 150 × 135 Å; 2M-HT, 195 × 135 × 130 Å; and
2M-MA, 200 × 155 × 140 Å.
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Three-dimensional Structures and Location of Fab Labels--
All
three Fab-labeled reconstructions of
2M closely resemble
their unlabeled stain analogs that were used as the initial models for
three-dimensional alignment, further supporting the reliability of the
reconstructions (Fig. 2). The 222-symmetric
2M-N
structure presents an overall twisted appearance. In its characteristic
lip view, the structure appears as two Z-shaped protein strands that
merge at the top and bottom to form regions of high protein density.
The strands exhibit a 90° clockwise rotation about their major axis
and form the walls of the cavity (Fig. 4a). The two dense
ends of the molecule clearly display a chisel-like appearance as
observed in the unlabeled stain structure (Figs. 2 and
3). Furthermore, the elbow-shaped bends
of the two Z-shaped strands are superficially connected by low density,
bridge-like features. Four small (~20-Å diameter) openings to the
interior of the structure are located above and below each of the
surface bridges on the front and back of the molecule.

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Fig. 3.
Views of solid surface renderings of the
three-dimensional structures of native, half-transformed and
methylamine-transformed 2Ms.
The structures are displayed at a reduced threshold to enhance the
presentation of the Fabs. The four Fabs are shown in
black.
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The end view of
2M-N shows that the Fabs bind on the
elbow-like bends of the Z-shaped protein strands 120 Å apart and
protrude diagonally outward from either side of the base of the chisel (Fig. 3). A closer examination of the "figure-8" end views shows that the Fabs are arranged in a staggered configuration,
i.e. located slightly away from the symmetry planes
bisecting the structure along its major axis. On each Z-shaped
protomer, the two Fabs are located near the ends of antiparallel-linked
subunits and consequently form an angle greater than the 90° rotation
exhibited by the strands (Figs. 3 and 4).

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Fig. 4.
Protein density distribution in slices of
the 2M structures
(a) and the proposed arrangement of the protomers
(b). a, slices 5.7 Å thick were cut
perpendicular to the major axis of the 2M-N and
2M-HT stain structures and the labeled
2M-MA ice structure (with the Fabs removed) as shown at
the top of the figure. An extensive comparison of the slices has been
previously presented (13). Two major strands that appear to split in
2M-HT are the putative dimeric pro tomers that constitute all three structures. The
proteinase-entrapping mechanism appears to involve rotation and
separation of the two strands at the top and bottom of
2M. The two Z-shaped protomers of 2M-N
rotate 90° clockwise between slices 2 and 6 and merge at each end of
the structure to form chisel-like features. In 2M-MA,
the two protomers rotate by 45° in the anticlockwise sense between
slices 1 and 7, forming significant connections with each other near
the ends of the structure, giving it a cage-like appearance. In
2M-HT, the chisel-shaped top splits into two relatively
untwisted strands that split further (slice 4), remerge, and undergo a
45° rotation accompanied by a broadening near the bottom of the
structure without the separation seen in 2M-MA
(b). The arrangement of the protomers in 2M
and 2M-MA is apparent upon displaying the structures at
a high threshold level. Each protomer has been shaded at a different
gray level for ease of viewing. The arrows indicate the
approximate locations of the Fab epitopes.
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2M-HT is a functional intermediate between the native
and transformed molecules in which two of the four bait domains and thiol ester moieties have been cleaved by chymotrypsin bound to Sepharose (30). Its top has a chisel-like shape analogous to
2M-N and is similarly flanked by two Fab labels 120 Å apart on either side, analogous to the native structure. However, the
center of the molecule shows an approximately 2-fold widening of the openings to its internal cavity, whereas the superficial, bridge-like features of
2M-N are absent (Fig. 3). The bottom of
2M-HT presents a bulbous, rounded appearance. The two
strands that comprise the native molecule appear to have split into
four that exhibit little twist (Fig. 4a). The two Fabs that
bind at the bottom appear to be rotated ~50° with respect to the
Fabs at the top of the structure and are located laterally 120 Å from
each other as in
2M-N (Fig. 3).
2M-MA forms a more compact, cage-like structure, with
four arm-like features that extend, two from each end of the molecule (Fig. 3). As seen in the H and X views, the
molecule is formed by two relatively straight strands of protein
density that form major connections near the two ends of the structure.
The H view exhibits a groove, which appears as a pronounced
gap at an increased threshold and separates the body of the structure
into two strands of protein density that twist 45° counterclockwise
(Fig. 4, a and b). The side X view
shows that each strand is broad at the center and tapers at the two
ends to form the arm-like extensions (Fig. 3). This structure is
similar to the
2M-MA reconstruction reported previously
without the Fab labels (11). Two Fabs are located at each end near the
tapered base of its arm-like extremities 160 Å apart, in a noticeably
staggered fashion. The Fabs show the same ~45° rotation exhibited
by the two strands (Figs. 3 and 4a). In the three
structures, the epitopes are located near the upper and lower ends of
their internal cavities with a vertical separation of ~145 Å.
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DISCUSSION |
Structural Organization of the
2M Variants--
A
variety of hypotheses have been proposed for the structural
transformation that links the quite dissimilar structures of the native
and transformed
2Ms (10, 12). The bases for these proposals have ranged from a correlation of surface features (10) to a
comparison of the protein density distributions in the two structures
(12, 13). As discussed below, previous immunoelectron microscopy
studies of individual stained particles have provided significant
structural information for the location of domains in transformed
2M (31, 32). However, a structural correspondence was
not established with antibody-labeled
2M-N, because the
images of stained specimens exhibited variable shapes, making their
interpretation questionable. The acidic uranyl salts (pH ~ 4)
used in these studies seemingly perturbed the more labile native
molecule. We have shown that a reconstruction obtained from a specimen
stained with the neutral pH methylamine tungstate (27) gives excellent
correspondence with that obtained from frozen-hydrated specimens (Fig.
2).
Our Fab-labeled three-dimensional structures have provided the first
definitive structural comparisons of antibody-labeled
2Ms, making it possible to relate the morphological
changes upon transformation by methylamine or chymotrypsin. An initial
consideration of the locations of the four Fab binding epitopes, near
the ends of all three structural forms of
2M, clearly
shows that individual 180-kDa subunits are present in an antiparallel
or head-to-tail orientation within the two disulfide-linked
protomers that noncovalently associate to form the tetrameric
2M. This is a structural validation of the proposal from
previous sequencing studies that the two 180-kDa subunits in each
protomer are linked antiparallel by two disulfide bonds near their N
termini (1, 33) and a previously reported comparison of two-dimensional
average images of Fab-labeled
2Ms (12).
A comparison of the three structures of
2M (Fig. 3)
indicates that in each case, the top and bottom pairs of Fab epitopes are separated along the major (long) axis by a constant distance of
approximately 145 Å. However, the lateral distance between the
epitopes in each of these pairs is variable, ranging from 120 Å in the
native and half-transformed structures to 160 Å in the transformed
molecule (Fig. 5). This indicates that
the transformation of
2M, which allows the physical
entrapment of a proteinase after the bait domains and thiol ester
moieties of
2M have been cleaved, involves a
rearrangement of protein density about its major axis to this regard,
the "accordion folding" model in which a lateral compression and
vertical stretching of the molecular was proposed for this
transformation of
2M is inconsistent with the disposition of the Fab
labels on the native and transformed structures (10). Our observation
agrees with the proposed localization of the two disulfide-linked
dimers in the three variants of
2M reported here (12,
13), one on either side of the major axis of the structure, and is
supported by studies that showed dimeric variants are incapable of
trapping proteinases (34). Slices of
2M and
2M-MA obtained perpendicular to their major axes (Fig.
4a) reveal two twisted strands of high protein density that
form major connections near the ends of the molecules thereby leading
to the structural division proposed (Fig. 4b).

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Fig. 5.
Location of receptor binding domains in
2M-N and
2M-MA. The chisel-shaped features
at the two ends of the native molecule sequester the RBDs
(hatched oval). After thiol ester cleavage, the chisels
split and rotate, exposing the RBDs near the tops of the arm-like
features of 2M-MA. For clarity, the rotation and
translation are depicted with arrows only at the top of the
molecule. The Fabs at the bottom rotate in the opposite
direction.
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Relatedness of Structural Features in the
2M
Variants and Locations of the RBDs--
The Fabs used in the present
study bind transformed
2M near the base of the arm-like
extensions on the top and bottom of the molecule. Previous
immunoelectron microscopy studies with other monoclonal antibodies
showed that the RBDs are located near the tips of these arms and allow
for the rapid endocytosis of
2M-proteinase complexes
(32, 35, 36). It was further shown that an antibody to the RBD
prevented the binding of
2M to its receptor (32, 35).
However, the antibody did not bind
2M-N, indicating that
the RBDs are internally sequestered (35). As the antibody 6E8 used in
the present study binds native, half-transformed, and transformed
2Ms, its binding site appears to be distinct from the
RBD.
The disposition of the epitopes of 6E8 on
2M-N and
2M-MA (Fig. 5) reveals a structural relationship of
major significance in the biology of
2M. A comparison of
the end views of the native and transformed structures (Fig. 5) shows
that the two protomers that merge to form the chisels in
2M-N undergo a separation of 40 Å, along with an
opposite 90°-rotation at each end. This results in the emergence of
arms and exposure of the RBDs at the two ends of the arm-like features
of the transformed structure. It appears that the chisels of
2M-N enclose the RBDs (Fig. 5), and this finding nicely
correlates with the inability of the RBD binding antibody (7H11D6) to
bind to the native molecule (35).
Structural Basis of Proteinase
Entrapment--
2M-HT is a functional intermediate that
was obtained by reacting an excess of
2M-N with
immobilized chymotrypsin so that bait domain cleavage and thiol ester
hydrolysis occurred in only two of its four subunits (30). In the top
half of
2M-HT, the chisel-shaped feature and the
arrangement of the two Fabs closely resemble
2M-N,
whereas the bottom half is broad and bulbous, and the two Fabs have
rotated ~45° (Fig. 6). Therefore, we
conclude that the bait domain and thiol ester on one subunit in each
dimer that may be in close proximity have reacted with chymotrypsin in
the bottom portion of the structure. Therefore, the minor axis of
2M-N represents its functional division. The intact
subunit in each protomer maintains the shape of the chisel-like feature of the native molecule. This proposal agrees with the antiparallel arrangement of the subunits described above.

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Fig. 6.
Structural relationship between
2M-N and
2M-HT. The Fabs indicate that the
strands rotate 45° in the bottom portion of the molecule after
cleavage of two bait and thiol ester domains by chymotrypsin.
Consequently, the openings to the cavity are enlarged, permitting the
entrance of the proteinase (Figs. 3 and 4).
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The untwisting of the strands in
2M-HT (Fig. 6) appears
to lead to an approximately 2-fold widening of the openings to the cavity (Fig. 4, cf.
2M-N and HT), making the
cavity accessible to proteinase entry. However, the Fabs show that
there is no separation of the strands at the ends of the molecule, and
the arm-like features of
2M-MA have not formed.
Consequently, the RBDs are not exposed (Fig. 3). In an apparent
contradiction to this proposal, a binary chymotrypsin
2M
complex that may share the cleavage arrangement of
2M-HT
(two bait domains and thiol esters cleaved) appears to interact with an
anti-RBD antibody (35, 37). However, it was noted (30, 37) that thiol
ester cleavage is more extensive in the binary complex preparation, and
this may have resulted in the IgG binding to some of the
2M molecules seen in immunoelectron microscopy (35). We
propose that transformed
2M, which is targeted for
endocytosis, requires cleavage of intact thiol esters in both ends of the molecule. Such an arrangement would ensure the ability of
2M-HT to encapsulate a second molecule of proteinase
before the RBDs are exposed and the complex is removed from the
circulation. Thus, the presence of two intact bait domains and thiol
ester moieties in the native half of the intermediate structure imposes constraints on the complete transformation of
2M (13).
In this regard, a recent study (38) suggested that the four bait
regions are in contact with one another near the center of the
structure. It was reported that disulfide cross-links between two
dimeric protomers blocked the structural change induced by thiol ester cleavage that, however, occurred upon cleavage of bait domains. The
need to cleave bait regions in both halves of the
2M
tetramer to enable complete transformation of the molecule shown in
this study may, therefore, arise from the need to allow for complete reorganization of the dimer-dimer interface.
The entrapment of two proteinases by
2M is known to
occur in two steps with a fast reaction occurring between
2M-N and the first targeted proteinase (1). The
proteinase may enter the cavity through the enlarged openings where the
-amino group of its lysine moiety is typically cross-linked to the
Glx952 of
2M (39). The thiol esters are
therefore analogous to harpoons that tether the proteinase to the
interior of the cavity of the
2M and, thus, may have a
role in maintaining its irreversible attachment. The intermediate or
half-transformed
2M can subsequently undergo further
cleavage by a second proteinase at a reduced rate (30) in the native
portion of the structure, resulting in a similar entrapment, followed
by a 45° counterclockwise rotation of the strands to encapsulate the
proteinases and expose the receptor binding domains (Fig. 5).