(Received for publication, July 10, 1995)
From the
The structure of methylamine-treated human
-macroglobulin (
M-MA), a 720-kDa
tetrameric inactivated proteinase inhibitor from plasma, has been
determined to a resolution of 10 Å. Data were collected with
synchrotron radiation at 120 K, and phases were calculated by multiple
isomorphous replacement and solvent flattening. A novel feature of the
structure of
M is present in its proteinase-binding
cavity, dividing it into two compartments. The potential sites for
proteinase entrapment in these compartments are sterically restricted.
The positions of the thiol groups appearing from the functional
important thiol esters upon their cleavage have been determined. They
are found at the walls of the compartments at the center of the
structure. The overall structure of
M-MA is much more
sphere-like than previously inferred from electron microscopy studies.
However, several aspects of the structure are well described by recent
three-dimensional reconstructions. Possible models for the monomer, the
disulfide bridged dimer, and native
M are discussed.
Human -macroglobulin (
M) (
)is the best studied member of the class of
proteinase-binding
-macroglobulins (for reviews, see (1, 2, 3) ). One subunit of
M contains 1451 residues of which eight are
glycosylated. This subunit has a mass of 180 kDa(4) . Two
subunits form disulfide bridged dimers(5) . Two such dimers
make noncovalent contacts to form the 720-kDa functional tetramer.
The native form of M can form complexes with
various proteinases. This complex formation is initiated by specific
limited proteolysis of the bait region (6) found at residues
667-705(7) . The cleavage of the bait region initiates a
series of conformational changes in the
M subunits
resulting in entrapment of the attacking proteinase inside the
tetramer. The final result of these changes is the transformed form of
M. The native and transformed forms of
M appear in electrophoresis as the slow and fast forms
of
M(6, 8) . The native form of
M contains internal
-Cys-
-Glu thiol esters,
formed from Cys-949 and Glu-952 in each subunit. During complex
formation, these thiolesters become activated, and this activation
results in covalent binding of the proteinase primarily through
-Lys(proteinase)-
-Glu(
M) cross-links (9, 10, 11) . The bound proteinase is still
active, but it is only accessible to small substrates and inhibitors.
Two small proteinase molecules the size of chymotrypsin, but only one
large proteinase like plasmin, can be bound to
M(12) . A final result of the conformational
change is that sites in the C-terminal domains (residues
1314-1451) (13, 14, 15) become exposed
for interaction with the cellular receptor for
M-proteinase complexes. This receptor has been found
to be identical to the low density lipoprotein receptor-related
protein(16, 17, 18) . Incubation of
M with methylamine also leads to thiol ester cleavage
and covalent binding of methylamine(9, 19) . The
conformation of the resulting molecule,
M-MA,
resembles that of the fast form
M-proteinase complex (20, 21) . In
M-MA the bait regions
are intact but poorly accessible. Therefore,
M-MA is
inactive in proteinase complex formation(9, 19) , but
the receptor recognition sites are exposed in a manner similar to that
of transformed
M(22, 23) .
Transformed M resembles the Cyrillic letter
&cjs3997; (later referred to as the H-view) when studied by
electron microscopy (EM)(24) . Its dimensions are 180-200
Å, 120-140 Å, and 80-90 Å as estimated
from projections of different orientations and from three-dimensional
reconstructions(25, 26, 27, 28, 29, 30, 31, 32, 33) .
Receptor recognition sites are located at the tip of each of the arms
of the H(27, 34) . When studied by EM, native
M has various shapes, which among others resemble a twisted cross(3, 26) , a doughnut(25) , and a padlock(26, 35) .
It has recently been shown that all of these shapes correspond to one
single structure(36) . In a three-dimensional reconstruction of
native
M, the dimensions of the molecule were
estimated to be 200 and 140 Å (35) with an internal
cavity of cross-section 40 and 60 Å. In addition, molecules
probably representing intermediates in the transition from native to
transformed
M have been observed (3, 37, 38) . The proteinase(s) in the
M-proteinase complex appear to partially fill a large
elongated cavity(27, 28, 31, 33) ,
which seems to be empty in
M-MA(28, 29, 30, 33) .
The flexible bait regions (39, 40) are relatively
close to the thiol esters(41, 42) . The latter are
probably located in the center of the molecule at the inner surface of
the cavity(29, 30, 43) . Thus, the two key
functional sites of each
M subunit have an internal
location in the
M tetramer as also indicated by the
location of cross-links in
M-proteinase
complexes(11, 12) .
To provide a model of
M at higher resolution than currently obtained by EM,
we have initiated x-ray crystallographic studies of several crystal
forms. Tetragonal bipyramidal crystals of
M-MA and of
several
M-proteinase complexes diffracting to a
maximum of 9-11 Å resolution were reported
earlier(44) . Hexagonal crystals of
M-MA were
recently found to be suitable for structural
investigations(45) , although the limited diffraction power
would not allow a detailed structural analysis. Crystals of
M-MA from other species (46) and of the
homologous complement component C3 (47) show similar resolution
limits even with the use of high intensity synchrotron radiation and
data collection at cryogenic temperature. None of the crystal forms
found so far has shown diffraction to better than 8 Å resolution.
In this paper, we describe the three-dimensional crystal structure
at 10 Å resolution of human M-MA. The electron
density at this resolution does confirm several of the results already
obtained by EM. Because of a higher resolution than normally obtained
by EM, it reveals additional features of the molecule. The positions of
the thiol esters are now firmly located at the inner surface of the
central cavity. The tetramer has strict crystallographic 222 symmetry.
The molecule is shown to be much more spherical than earlier suggested
by the EM structures. The electron density reveals a large structure
within the central cavity. This cavity body has not previously been
seen in any of the EM studies. The low resolution structure presented
here does allow some speculations about a possible model for the
structural transition from the native to the transformed form of
M.
Native M at least 95% active in terms of
titratable SH-groups after reaction with excess trypsin was prepared as
described previously(9) . Three different heavy atom
derivatives of hexagonal crystals of
M-MA were
prepared for data collection. The complex between
M-MA
and the mercury cluster compound TAMM(48) , which reacts with
the free thiol groups induced by the methylamination, was prepared by
the following procedure. 50 mg of
M in 0.1 M
Na
HPO
, pH = 8.0, A
= 3.04, was made 0.2 M in methylamine, and pH was
adjusted to 8.0 with 0.5 M Na
PO
. After
4 h of incubation at 20 °C, the protein was desalted at 4 °C on
a Sephadex G-25 column equilibrated in 20 mM
Tris-SO
, pH = 7.7. The pool from this column was
made 10 mM in glycyl-glycine by the addition of 100 mM glycyl-glycine. The concentration of
M-MA was 3.0
µM after this treatment. 1 mM TAMM solubilized in
100 mM glycyl-glycine was added to 1.1 times the concentration
of free thiol groups assuming 100% activity. The pH resulting from
these additions was 7.9 at 20 °C and was not further adjusted. The
reaction mixture was incubated for 1.5 h at 20 °C and then made 10
mM in iodoacetamide and immediately gel filtered as above and
concentrated in Centricon 100 cells (Amicon Corp.). The amount of
mercury bound to the protein (0.35% by weight) was determined by cold
vapor atomic absorption spectrometry. The theoretical content assuming
100% thiol ester activity of the protein and binding of mercury only at
the thiol groups is 0.446%. Assuming that the thiol groups are far more
reactive than other nucleophilic groups, the occupancy for substitution
at the thiol groups is 78.5%. Crystals of the TAMM derivative of
M-MA were grown as described previously(45) .
Because the crystals of
M-MA are not stable for
prolonged time in protein-free solution, the two other derivatives were
prepared by adding solid Ta
Br
(49) or
PIP (48) directly to the drops such that solid material was
still present during the whole soaking period of 3-4 days. The
crystals did not show any sign of deterioration, and the crystals
soaked in Ta
Br
developed a weak green color
indicative of the presence of the compound.
Data from heavy atom
derivative crystals were obtained using synchrotron radiation and low
temperature data collection at station 9.6 at SRS in Daresbury, UK. The
data were indexed, integrated, and scaled as described
previously(45) , except that the program INST_HAMBURGD (50) was used for autoindexing. Completeness was 95% for native
data, 79% for the TAMM derivative, 80% for the TaBr
derivative, and 70% for the PIP derivative. The maximum
resolution was 10 Å for native and TAMM-derivative data, while
the maximum resolution for the Ta
Br
and PIP
derivatives was 11 Å. The programs CAD, SCALEIT, RSPS, FFT,
MLPHARE, and PEAKMAX from the CCP4-package were used for scaling,
patterson search, positional refinement, MIR phasing, and map
calculations(51) . Using the program RSPS, one site was
identified for the TAMM derivative, and two sites were identified for
the Ta
Br
derivative. These sites were refined
with MLPHARE. The resulting phases were used in the calculation of a
difference Fourier map for the PIP derivative revealing two sites. Data
at 35-12 Å was used for the TAMM and
Ta
Br
derivatives, while data at 35-14
Å was used for the PIP derivative in the phasing. The final FOM
from MLPHARE was 0.524 for 1863 reflections phased between 35 and 12
Å. The MIR map clearly showed the shape of the molecule and the
solvent region when contoured at 2
by the graphics program
O(52) . The map contoured at 1
showed a large part of the
solvent region occupied by low density. From this map, it was evident
that the asymmetric unit contained one monomer of
M-MA
or three molecules in the unit cell and not one dimer as previously
stated(45) .
After MIR phasing, 1523 reflections with FOM
> 0.2 were selected for input to a solvent flattening
procedure(53) . The do-all procedure described in the PHASES
documentation (54) was used for 16 cycles of flattening
followed by 12 cycles of combined flattening and phase extension. A
solvent content of 75% was assumed in the solvent flattening
calculations. The resulting electron density map showed the molecule
clearly distinct from the solvent with little density left in the
solvent region when contoured at 1. Difference Fourier maps were
calculated for all three derivatives. Six additional sites were found
for the PIP derivative, one additional site was found for the
Ta
Br
derivative, while none was found for the
TAMM derivative. Phasing statistics for the final round of MIR phasing
are shown in Table 1. All sites for the three derivatives were
inside or very close to the molecular boundary in the electron density
map contoured at 1
. Reflections between 35 and 10 Å were
included for the TAMM and Ta
Br
derivatives,
while reflections in the range 35-12 Å were included for
the PIP derivative. For the final round of solvent flattening 2168 MIR
phased reflections at 35-10 Å with FOM > 0.2 were
selected. Sixteen cycles of flattening followed by 12 cycles of
flattening and phase extensions produced 3201 reflections with mean FOM
= 0.816. The final R-factor was 22.3%. The final
electron density map was contoured at 1
and used in the program O
for the presentation in this paper. In spacegroup P6
22, the
hand of the structure is similar to that presented in the
three-dimensional reconstruction published by Boisset and
co-workers(31) . A more comprehensive description of data
processing and phase calculations is given elsewhere(55) .
The electron density map was skeletonized with BONES(56) . At the low resolution of this work, the skeleton obviously does not represent any recognizable secondary structures. However, it does give a useful representation of the electron density. The resulting skeleton was inspected together with the electron density to erase minor nonconnected parts. From the final skeleton, it was possible to isolate one molecule and to select the individual parts that could give suitable skeleton objects representing tetramers, dimers, and monomers. These skeleton objects and the map_cover option in program O were used to extract the relevant electron densities from the complete unit cell. The isolated densities were used to produce graphical presentations of the complete electron density of one molecule in the crystal. In order to compare the crystallographic structure with two recent EM reconstructions, model A (31) and model B(33) , these models were contoured such that the longest dimension in the H-view was approximately equal to that of the x-ray structure. The models were finally aligned manually with the move_obj option in program O.
Figure 1:
The unit cell. The electron density of
the unit cell is shown as contoured at 1 after MIR phasing and
solvent flattening. a, two indefinite solvent channels runs
along the crystallographic c axis, which is shown toward the
viewer. The tetramer in the center of the unit cell is completely
within the unit cell. b, the c axis is horizontal,
and the view is perpendicular to the one seen in a. The third
major solvent channel is seen. The almost spherical tetramer is seen in
the middle.
Figure 2: The tetramer. This and the following figures are shown in stereo. The middle and the right frame can together be seen in stereo using parallel eye view, i.e. focusing a long distance behind the picture. This is normal stereo, which can also be seen using special stereo-viewers. The middle and the left frame can be seen in stereo using cross-eye view. The figures are shown in three views: the H-view, the X-view, and the End-view. The X-view is seen when the H-view is rotated by 90° around a vertical axis. The End-view is seen when the X-view is rotated by 90° around a horizontal axis. a, the tetramer is shown in the H-view orientation as known from EM studies. The RBD is colored orange, the front part is gray-green, the back part is gray, and the midlayer is orchid. Notice that the RBD is attached to the back part but has the major part of its mass leaning over the front part. The midlayer is obviously separated from the two exterior bodies composed of RBDs, front, and back parts. b, two front parts and half of the midlayer has been removed to show the interior in the H-view. This view clearly shows the cavity body. It also indicates the ellipsoidal compartment in front of the cavity body. The compartment runs from lower left to upper right. The symmetry related ellipsoidal compartment can be glimpsed behind the cavity body. The positions of the TAMM clusters found in the phasing process is shown by red dots labeled Cys949. Two cluster positions with an internal distance of 44 Å in one ellipsoidal compartment are clearly visible. c, in the X-view, the spherical shape of the core of the tetramer is seen. It also shows the protrusion from the sphere of the four RBDs. The RBD is connected to the back part. Two back parts interact at the center of this view. d, two front parts and two back parts have been removed in the X-view, and this reveals the wheel-like shape of the midlayer. The view shows that the cavity body has a small hole in the center. The ring of the wheel containing four symmetry related parts is seen to have weak connections along both vertical and horizontal lines. e, if also the midlayer is removed, a section behind the cavity body shows the inverted S-shape of the cavity. Two cluster positions with an internal distance of 31 Å are marked. f, in the third major orientation, the End-view, the elongated lobes in the midlayer are connected to the back structures and are seen to pack together in an antiparallel mode. A hole through which the cavity body is visible is present in the midlayer. g, two RBDs, two front parts, two back parts, and half of the midlayer have been removed in the End-view to show the cavity body and two cluster sites. The cavity body is seen end-on. The distance between the two cluster positions is 39 Å.
In the H-view (Fig. 2a) and in the End-view (Fig. 2f), the tetramer is seen to be organized in three major laminar bodies separated by low levels of density. The central one, termed the midlayer, is not further subdivided in the figures and thus contains four copies of the same density. That it is a separate entity can easily be seen in the H-view sections (Fig. 2, a and b) and in the End-view sections (Fig. 2, f and g). In the H-view, the dimensions of the midlayer are approximately 20 and 120 Å (Fig. 2a). The midlayer is for the most part separated from the exterior bodies by a space of approximately 10 Å in thickness. The dimensions of the midlayer in the X-view are approximately 120 and 130 Å (Fig. 2d). In the End-view (Fig. 2f), the midlayer is located in a large cleft between the two exterior bodies. In this view, it is easily seen that it contains two elongated lobes packing together in an anti-parallel mode. Its wheel-like shape (Fig. 2d) reveals that the midlayer surrounds a large internal cavity and that it includes a cavity body. From the indentions in the density, one unique quarter belonging to one monomer can be proposed (Fig. 2d).
The two exterior bodies as seen in the H-view (Fig. 2a) have a thickness of about 50 Å and are identical because of the molecular symmetry. Each body contains pairs of identical parts that can be separated into three unique ones by low levels of electron density. These are termed the front, the back, and the RBD (Fig. 2a). The tetramer contains four copies of each due to the crystallographic 222 symmetry. In the X-view (Fig. 2c), the two exterior bodies with their RBDs located at the ends are crossed by an angle of 70°. The dimensions of one exterior body (including the RBDs) in this view are approximately 180 and 110 Å. The front part of an exterior body has a rather compact structure (Fig. 2, a, c, e, and g). The back part is much more loosely organized and contains several large depressions with openings toward the surrounding solvent (Fig. 2, b, c, and g). Two back parts pack together in the central part of one exterior body (Fig. 2, c and e). Two front parts and two back parts together make up the core part of one exterior body. This has a flat disc-like shape (Fig. 2, a, c, and f). The distance between two RBDs located at the periphery at the same end of the molecule is 120 Å (Fig. 2f). The RBD has the appearance of an almost closed ring (Fig. 2, a and c). It is connected to the back part (Fig. 2c), although its major mass is leaning over the front part (Fig. 2, c and f).
The cavity body is part of the midlayer (Fig. 2d) and consists of four identical units related by 222 symmetry. These units cannot easily be separated in the density. The cavity body has the shape of an irregular cylinder of dimensions 28, 18, and 52 Å. This cylinder is, however, squeezed on one side at the middle of the cavity body (Fig. 2b) to the extent that a hole appears (Fig. 2d). The irregular cylinder can thus be seen as composed of two distorted tetrahedrons sharing one side. Four small protrusions on the cavity body (Fig. 2, b and d) make the only connections to the rest of the midlayer.
The central cavity creates a continuous cylinder of solvent around the cavity body (Fig. 2, b, d, and g). The most narrow part of this cylinder is around the equatorial region of the cavity body (Fig. 2, b and d). Two symmetry related compartments are seen on each side of the cavity body (Fig. 2d). Close to the cavity body they have an elongated irregular ellipsoidal shape (Fig. 2b). In Fig. 2b, the long axis of the ellipsoid is from lower left to upper right with a length of about 100 Å. The other axis has a length of around 40 Å. Seen in the H-view, the two symmetry related ellipsoidal compartments together form an irregular X-shaped cavity having a depth of about 80 Å. The angle between the two ellipsoidal compartments is approximately 70°.
In the X-view (Fig. 2d) the two ellipsoidal compartments are seen largely along their longest axis. The distance between the edge of the cavity body and the walls in this view is typically 30 Å (Fig. 2d). The cavity body prevents objects with dimensions larger than 15-20 Å to move between compartments. Behind the cavity body in the X-view (Fig. 2e), the central cavity has the shape of a mirror image of a letter S. This inverted S consists of two halves of the ellipsoidal compartments and thus makes a connection between them.
The complex M-MA reacts slowly with
proteinases and is from a physiological point of view
inactive(9, 19) . However, it is recognized by the
M receptor (22, 23) , and the
structures of
M-MA and
M-proteinase
complexes appear to be very similar in EM studies, where models of
M-MA have been used as the basis for localization of
the trapped proteinase(s) in
M-proteinase
complexes(28, 59) . The present investigation provides
new and more detailed information on (i) the overall structure of the
M tetramer, (ii) the location of the receptor-binding
domains of
M, (iii) the location of the thiol group of
Cys-949 appearing as a result of thiol ester cleavage, and (iv) the
detailed shape of the large cavity within
M, which is
the site where proteinases become trapped. Compared with crystals of
most other proteins, the crystals of
M-MA contain an
unusually high amount of solvent (87%). As seen in Fig. 1, the
unit cell contains three large solvent channels ranging from 190 to 40
Å in diameter. Interactions in the crystal between the individual
tetramers only take place at four small lobes of electron density
probably representing the RBDs. The core, which comprises about 90% of
the total mass of the protein, is weakly connected to the RBDs. Another
source of high flexibility of the core is indicated by the few
connections between various parts of the tetramer that could well give
additional loss of order in the crystal lattice. The high solvent
content and the small area involved in crystal packing provides an
explanation for the limited resolution of the data obtained. Attempts
at crystallizing many different members of the
-macroglobulin
family over the last 5 years have all resulted in crystals diffracting
to about 10 Å
resolution(44, 45, 46, 47, 55) .
It is thus likely that all these crystals have a packing and a solvent
content similar to the one described here.
This study demonstrates that it is possible to determine the structure of a very large macromolecule at 10 Å resolution with currently available methods and software. This finding can be important for other attempts at determining crystal structures of large macromolecules or macromolecular complexes with molecular masses in the million dalton range.
If RBD is flexible
relative to the core, as indicated by the single connection, this may
be important when an M-proteinase complex binds to two
adjacent receptors at the same time as proposed earlier(62) .
The flexibility might relieve steric strain in the complex between one
molecule of
M-proteinase and one to three molecules of
the
M-receptor complex(63) .
For small molecules of dimensions less than 10
Å, the central cavity in M-MA is readily
accessible through the many holes in the structure, while objects
larger than 20 Å seem to have difficult access. This, together
with the shape of the cavity and the presence of the cavity body,
provides an explanation for the sluggishness of proteinase-binding by
M-MA. Not only are the entrances to the cavity too
narrow, but there is also insufficient space within the cavity (see
further below).
The overall dimensions of model A are 144, 193, and 130 Å(31) . Thus the reconstructed molecule is larger than the tetramer in the crystallographic structure, especially at the long axis in the H-view. In the H-view, model A describes the front and back features quite well. In the equatorial region, the model is too slim and missing the delicate structures around the back-back interface. In the X-view, model A suffers from the fact that the dimension in the vertical direction is too long compared with the crystallographic structure. This masks the spherical shape of the core. The rotation of the two exterior bodies relative to each other is 20° in model A(31) . This is very different from the value of 70° found in the crystallographic structure. One major reason for this is that the external position of RBD is not observed in model A. In the End-view, the midlayer is described fairly well. Two small protrusions indicate the positions of the RBDs. Model A describes the overall shape and dimensions of the central cavity very well in both the H-view and the X-view, although the cavity body is missing. Finally, the ellipsoidal compartments with axes of 135 and 35 Å in this model are more elongated than found here (31) .
The overall
dimensions of model B are 118, 150, and 103 Å(33) . The
model does not have internal 222 symmetry. In the H-view model B is
rather smooth on the surface such that the front and back features are
missing, and this model is also too slim in the equatorial region. This
model too does not describe the external position of RBD. The relative
rotation of the two exterior bodies is again significantly smaller in
this model than in the crystallographic structure of
M-MA(33) . In the End-view, the midlayer is
present, but with an orientation different from that in the
crystallographic structure. The internal cavity in model B is
funnel-shaped with a narrow waist in the
center(33, 35) . The crossed ellipsoidal compartments
observed in both model A and the crystallographic structure are not
present in the model. The dimensions of a parallel reconstruction of
the
M-chymotrypsin complex are 138, 175, and 125
Å(33, 35) . Hence, the overall dimensions of the
M-chymotrypsin reconstruction appear to be closer to
those found in the crystallographic structure of
M-MA.
It appears from fluorescence spectroscopy that Glx-952 is
positioned within 10-25 Å from the thiol group of Cys-949
in transformed M(64) . The maximum separation
of two such Glx positions has been estimated to be 50
Å(10) , which is not much larger than the separation
between the TAMM sites (Table 2). This indicates that the Glx
residue is located at the surface of the wall lining the same
ellipsoidal compartment as the thiol group of the corresponding
Cys-949.
The
M-MA has a well-defined central cavity. However,
docking experiments show that this cavity is unable to accommodate
proteinases the size of, for example, chymotrypsin (not shown). The
cavity body is the major obstacle to this docking since it occupies
much space inside the cavity. Hence, it is likely that the
M-proteinase complex and
M-MA have
different structures, especially within the central cavity, although
they probably have the same overall molecular shape as they both have
crystallized in the tetragonal crystal system with the same space group
and with similar cell parameters(44) . During complex
formation, the cavity body might change its central symmetrical
location or perhaps collapse to accommodate the trapped proteinase(s).
An asymmetric location of the cavity body could create extra space for
a trapped proteinase, while leaving the rest of the tetramer almost
unchanged.
Figure 3: Possible subunit organization. a, one possible monomer is shown. The monomer has a cavity made up of the midlayer, the front, and the back parts. b, another possible monomer. c, a possible disulfide bridged dimer composed of two monomers as in a.
Irrespective of the actual shape of the monomer there are two ways of creating a dimer from two monomers. First of all, consider the midlayer (Fig. 2d). A dimer can be created either by a vertical or a horizontal cut. None of these two possible interfaces has large areas of contacts. However, the cavity body is squeezed along the horizontal line (Fig. 2b), and the contact areas in the ring are smaller along the horizontal line (Fig. 2a) than along the vertical line (Fig. 2f). Secondly, consider the front and back parts of the exterior body (Fig. 2c). The interactions found here appear to involve considerably larger areas than those in the midlayer and are therefore likely to contain the major part of the noncovalent interactions between monomers. Here again there are two possible lines of cutting the tetramer into dimers. One of these, from the lower left to the upper right corner, seems to run through the smallest contact areas. At the same time, this line is closest to the probable horizontal cut in the midlayer. By cutting along these two weakest interfaces, a proposal for a disulfide bridged dimer made from two monomers as in Fig. 3a is presented in Fig. 3c.
Although appealing, a proposal for a dimer
based on the argument that it should have a weaker interface to the
other dimer than internally between the monomers is of course not
necessarily correct. However, a location of the disulfide bridges
probably in the midlayer of the End-view (Fig. 2f) has
earlier been suggested by Delain et al.(3) . If dimers
of native M induced by mild acidic treatment react
with proteinase, half-molecules resembling the greek letter
or
the upper half of an H are produced (69) . Both of these
observations support the model of the covalently linked dimer shown
here. On the other hand, the model given here is very different from
the dimer presented in Fig. 8 of (35) . This dimer is created
by the alternative cut through the tetramer. Results from bivalent
cross-linking of proteinases do not clarify this discussion of possible
dimers. It has been suggested that such cross-linking involves one
Glx-952 from each of two disulfide bridged dimers(70) .
However, other experiments suggest that bivalent cross-linking can
occur not only between disulfide bridged dimers but also within a
single dimer(11) . Interpretation of these results are further
complicated by the uncertainty in the exact location of the proteinases
in the
M structure.
An attempt to simulate the structure of native
M based on the crystallographic model of
M-MA is shown in Fig. 4. In that model, the
midlayer has been separated into its dimeric parts, and both have been
translated vertically in the H-view. The RBDs have been rotated toward
the midlayer as rigid bodies around their connections to the core. The
positions of the front and back parts are not changed. This simulation
gives a density distribution similar to that in the
reconstruction(35) . In the simulation, the two halves of the
cavity body have been separated together with the rest of the midlayer.
However, the location of the cavity body could be unchanged in the
center of the cavity. During the conformational change from native
M to transformed
M, the RBDs could
move as rigid bodies to the locations observed in
M-MA, thereby exposing those surface patches
responsible for receptor recognition that are buried in native
M(27) . The midlayer and the RBDs seem to be
the most flexible parts in the
M-MA structure. It is
thus plausible that these parts are involved in the conformational
change. Furthermore, the locations of Cys-949 (see above) make it
likely that a signal to trigger the transformation can be transmitted
to the midlayer after cleavage of the thiol esters. The changes in the
front and the back parts are probably not very large, as indicated by
the similar overall architecture of the reconstructed native
M and the crystallographic structure of
M-MA. If this simulation is correct, the mechanism for
the conformational change from native to transformed
M
would be considerably simpler than those considered
earlier(3, 26, 35, 37) .
Figure 4:
Simulation of native aM. The
simulation is presented in three orientations. The H-view, which most
likely is the lip-view in the EM reconstruction, is the same view as
shown in Fig. 2a. If the H-view is rotated by 45°
around a vertical axis, the Padlock view of the EM construction is
obtained. The X-view, which is the figure eight in EM, is the same as
in Fig. 2c. a, compared with Fig. 2a, the front and back parts are in unchanged
positions. The RBDs are rotated toward the midlayer, and the midlayer
is split into two halves and translated vertically. Notice the large
hole also seen in the three-dimensional EM reconstruction. If the
cavity body is not moved with the rest of the midlayer, the hole would
still be seen as a large depression in the surface. The cleft running
from lower right to upper left is also clearly seen in the EM
reconstruction. b, in the Padlock view, a large depression in
the upper part of the molecule is seen. If the cavity body is left at
the center of the molecule, this depression will be a hole as seen in
the reconstruction. c, the X-view shows a surface similar to
the large S shape of the EM reconstruction.
The bait
regions in native M are rapidly cleaved by many
proteinases(1, 2) . This implies that they are readily
accessible for molecules with overall dimensions of 40-50
Å. If the bait regions are located within the internal cavity
(see above), the large hole in the center of the molecule observed in
both the simulation (Fig. 4) and the reconstruction (35) would allow access to the internal cavity and to the bait
regions for an attacking proteinase. If the bait regions are located
within the cavity body and protrude toward the walls of the cavity, the
bait regions in native
M might provide steric
hindrance for access to the thiol esters, as previously
suggested(71) . This could shield the thiol esters from large
nucleophiles. The increased reactivity of the thiol esters after bait
region cleavage would thus result from the breakdown of such a
shielding.