Department of Biochemistry and Molecular Biology, University of
Illinois at Chicago, Chicago, Illinois 60612-4316
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INTRODUCTION |
Inhibition of proteinases by human
2-macroglobulin
(
2M)1 results
from a series of conformational changes that are initiated by the
proteinase and that result in the physical sequestration of the
proteinase within the closed cage-like structure of the conformationally altered
2M (1, 2). The initiating event is a proteolytic cleavage by the attacking proteinase near the center
of the
2M polypeptide in a region termed the bait region (3, 4). Cleavage anywhere within the bait region also results in
activation of an internal thiol ester toward cleavage by nucleophiles. From studies on the kinetics of cleavage of the thiol ester by nucleophiles and of the subsequent conformational change within the
2M, it has been shown that the conformational change
occurs cooperatively after both thiol esters within one half of the
2M tetramer have been cleaved (5, 6).
Knowledge of the structural relationship between the thiol ester and
the bait region and hence of the details of the activation mechanism is
limited by the absence of a high resolution x-ray structure of either
native or conformationally altered human
2M. From
fluorescence resonance energy transfer measurements, it was shown that
the four cysteines that form the four thiol esters of the human
2M tetramer are centrally located and are about 35 Å apart (7). This was subsequently confirmed by a low resolution (~10
Å) x-ray structure of conformationally altered
2M (8). NMR measurements on
2M showed that the bait region of
each monomer was unusually flexible (9, 10) and that it lies close
(10-17 Å) to the cysteine of the thiol ester in the transformed
protein (11). However, the location of the four bait regions is still not known. Studies from this laboratory on
2M variants
in which large portions of the bait region had been removed showed that bait region truncation prevented the disulfide-linked
2M
dimers from associating noncovalently to give the functional tetramer (12) and that, despite normal reactivity of the thiol esters toward
nucleophiles, there was no cooperativity of conformational change
between subunits. This implicated the bait region in formation of the
2M tetramer and raised the possibility that the bait
regions may lie in the central cavity of
2M in contact
with one another. This is made more plausible by the presence of a
symmetrical body of electron density in the central cavity of
methylamine-treated
2M, termed the "cavity body,"
that is absent from proteolytically cleaved
2M (8).
To test the hypothesis that the bait region is involved in the
noncovalent association of
2M dimers and in the
mediation of cooperative subunit-subunit interactions that are involved in the gross trapping conformational changes of
2M, we
used a scanning mutagenesis approach in which four single residue
substitution variants of human
2M containing a cysteine
at distal, central, or proximal ends of the bait region have been
prepared and characterized (G679C, M690C, V700C, and T705C) (Fig.
1). The variants folded normally, as
judged by several properties, including the formation of tetrameric
2M, the presence of thiol esters with normal reactivity toward nucleophiles, and the ability to undergo a proteinase-induced conformational change. However, the extent and rate of conformational change induced by methylamine cleavage of the thiol esters was compromised for all of the variants. Although all of the variants could
still inhibit proteinase, the stoichiometries were reduced compared
with wild-type recombinant
2M or plasma
2M. These perturbed properties correlated with the
presence of new interdimer disulfide bonds formed between the new
cysteine residues in two otherwise noncovalently associated
disulfide-linked dimers. Such cross-linking resulted in covalent
tetramers with either one or two new disulfides between the dimers. The
extent of formation of such new disulfides was a function of the
position of the cysteine within the bait region. This suggests that the
bait regions are involved both in dimer-dimer association and in
mediation of trans-dimer conformational changes and that there is a
requirement for unrestrained reorientation of the bait region
interface. In addition, the M690C variant showed evidence of a new
intradimer disulfide, suggesting that bait regions within a dimer
are also close to one another. These findings suggest that all four of
the bait regions are at the center of the
2M cavity and
may constitute all or part of the cavity body seen in the low
resolution x-ray structure of transformed
2M.

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Fig. 1.
Bait region primary structure of human
2M showing the sites of introduction of cysteine residue
substitutions and the location of the primary proteinase cleavage sites
(+++). The hypervariable region that defines the bait region
stretches from residue 667 to residue 705 in human 2M.
Beyond these residues, toward the N and C termini, respectively, there
is very high sequence homology between different members of the
-macroglobulin family. Within the bait region, there is very high
variation both in length and in composition (4).
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MATERIALS AND METHODS |
Site-directed Mutagenesis and Expression of Recombinant
2Ms--
For circumstantial reasons, the four mutations
were not all created by the same procedure. For the variants G679C and
V700C, site-directed mutagenesis was performed in a single-stranded
M13mp18 construct containing the 1690-base pair BamHI to
BsiWI fragment from plasmid p1167 (13), which covers the
coding region for the bait region. Mutagenesis was carried out using
the Amersham Corp. in vitro Mutagenesis System 2.1 according
to the manufacturer's protocol. The mutations were confirmed by
sequencing, after which the mutated BamHI to
BsiWI fragment was excised and ligated back into the p1167
expression vector. The M690C variant was created by mutagenesis using
the Altered Sites II Mutagenesis System (Promega) on the 2669-base pair
BamHI to ClaI fragment from p1167 subcloned into
pAlter (Promega). The 28-base oligonucleotide 5
-T GAG TCA GAT GTA
TGT GGA AGA GGC CAT-3
was used for the mutagenesis. After confirmation of the mutation in pAlter, the mutated BamHI to
ClaI fragment was excised and religated into p1167. The
T705C variant was created in two stages. In the first step, an
XmaI site was introduced at base 3572 of p1167 using the
Amersham Corp. in vitro Mutagenesis System 2.1 protocol. The
mutation at residue 705 was then introduced by replacing the
XmaI to BsiWI fragment of p1167 with a synthetic
duplex containing the desired change. This duplex was formed by
annealing oligonucleotides 5
-CC GGG TGT GAG ACC-3
and
5
-C TAC CGT CTC ACA C-3
. The mutation was confirmed by
sequencing in p1167. Stably transfected BHK cell lines expressing each
of the four
2M variants were established by the
co-transfection and drug resistance selection procedures previously
described (14, 15).
Purification of Recombinant and Plasma
2Ms--
Plasma
2M was isolated from
outdated human plasma obtained from Rush University Hospital Blood
Bank. The
2M was precipitated in a 4-12% cut with
PEG8000 and collected as a pellet by centrifugation. The pellet was
resuspended in 0.1 M sodium phosphate buffer, pH 6.5, containing 0.8 M sodium chloride and chromatographed on a zinc chelate matrix, as described previously (16). Recombinant
2Ms were obtained from the growth medium of the stably
transfected BHK cells that were grown to confluence in roller bottles
and cycled between serum-containing and serum-free medium, as described previously (15). Purification was by zinc chelate chromatography, as
for plasma
2M, but without the PEG8000 precipitation
step.
Polyacrylamide Gel Electrophoresis--
Polyacrylamide gels run
under denaturing and reducing conditions (8% acrylamide) or denaturing
and nonreducing conditions (3.5% acrylamide cast on Pharmacia Gel Bond
film) were run according to the procedure of Laemmli (17).
Nondenaturing gels (5% acrylamide) were run as described previously
(18). Urea polyacrylamide gels (5% acrylamide) contained 5 M urea and were run in the same way as nondenaturing
gels.
TNS Fluorescence Measurements--
Both kinetic and wavelength
scan fluorescence measurements were made on an SLM8000
spectrofluorometer. Kinetic measurements used excitation at 316 nm and
observation of emission at 410 nm, with slits of 4 and 16 nm for
excitation and emission, respectively. Wavelength scans used excitation
at 316 nm and emission monitored in 2-nm steps from 360 to 600 nm.
Slits of 2 nm for both excitation and emission were used.
Quantitation of Thiol Esters and Kinetics of
Cleavage--
Determination of both the number and kinetics of
cleavage of thiol esters present in the variants were carried out by
continuous monitoring of the free SH (sulfhydryl) released from
the thiol ester by reaction with methylamine, by spectrophotometric
measurement of the TNB
released by reaction of the free
SH with DTNB. TNB
was quantitated from the change in
absorbance at 412 nm using an extinction coefficient of 13,600 M
1 cm
1 (19). For kinetic
measurements, the
2M (final concentration, 1.0 µM) was preincubated in assay buffer containing 100 µM DTNB. The reaction was initiated by addition of amine
hydrochloride stock solution (5 M, pH 8.0) to give the
desired final concentration. Measurements were made in a dual beam
Shimadzu UV2101PC spectrophotometer; the blank contained all components
except
2M. Data were fitted by nonlinear least squares
analysis to a monoexponential using Scientist (MicroMath, Salt Lake
City, UT).
Trypsin Inhibition Assay--
Stoichiometry of trypsin trapping
was determined by measuring the soybean trypsin inhibitor-resistant
trypsin activity (20, 21) at different trypsin:
2M
ratios.
2M (120 nM) was incubated with
different molar ratios of trypsin for 5 min at 25 °C. The reaction
was diluted 1:1 with a solution of soybean trypsin inhibitor (final
concentration, 1.25 mM). After 1 min of incubation,
residual trypsin activity, representing
2M-trapped
trypsin, was assayed by addition of 10 µl of the reaction mixture to
190 µl of assay buffer containing 200 µM (final
concentration)
N-benzoyl-L-isoleucyl-L-glutamylglycyl-L-arginine-p-nitroanilide (S-2222, Chromogenix) in a microcuvette. Change in absorbance at 410 nm, relative to a reference cell containing only substrate, was
monitored using a dual beam Shimadzu 2101PC spectrophotometer. Trypsin
activities were determined from the slope of the resulting time
courses, fitted by least squares linear fit using the program Origin 1.16 (MicroCal Inc., Amherst, MA). All reactions and assays were
carried out in 20 mM sodium phosphate buffer, pH 7.4, containing 0.1 M sodium chloride, 0.1 mM EDTA,
and 0.1% PEG8000.
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RESULTS |
Electrophoretic Mobility of Variants on Nondenaturing
PAGE--
Following reaction with methylamine or proteinase, human
plasma
2M undergoes a major change in conformation that
results in an increase in electrophoretic mobility on polyacrylamide
gels run under nondenaturing conditions (22). This "slow to fast" conversion is frequently used as a diagnostic for this major
conformational change, with the result that
2M
conformations are often referred to as the slow form or fast form. In
addition, there is an intermediate mobility species in which
conformational change has occurred in only two of the four subunits (6,
23). We tested the ability of the four
2M variants to
undergo this slow to intermediate or slow to fast form interconversion
upon reaction with either methylamine or proteinase. All four variants
had the expected slow mobility prior to reaction (Fig.
2) that is characteristic of tetrameric
2M in the native state. Reaction with a small excess of
proteinase (trypsin) for 10 min was sufficient to completely convert
each of the variants from slow to fast conformations (Fig. 2). However,
methylamine treatment overnight produced only partial conversion to a
mixture of slow, intermediate, and fast forms for all of the variants,
with each variant giving different extents of conversion to
intermediate and fast forms (Fig. 2). The M690C variant was least
converted to fast or intermediate mobility species, whereas the other
three showed the presence of about one-third to one-half fast form and
a smaller amount of intermediate mobility species. Recombinant
wild-type
2M was completely converted to the fast form
by methylamine treatment under the same conditions (Fig. 2), as was
plasma
2M (not shown). The failure of the variants to be
completely converted to the fast form was not due to incomplete cleavage of the thiol esters by methylamine (see below).

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Fig. 2.
Conversion of recombinant wild-type and
variant slow form to faster mobility species on polyacrylamide gel run
under nondenaturing conditions following treatment with methylamine or
trypsin. For each 2M, there are three consecutive
lanes corresponding to native protein (N), protein incubated
with 0.2 M methylamine overnight (M), and
protein reacted with 2.2 equivalents of trypsin (T), based
on active site titration for 10 min. The 2Ms are as indicated above each set of lanes. S, I, and
F indicate the positions of the slow, intermediate, and fast
forms of the tetramer, respectively; Dimer indicates the
position of small amounts of dimeric 2M.
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We also followed the rate of conversion of the variants from slow to
intermediate or fast conformations as a function of time of reaction
with methylamine at pH 8 by nondenaturing PAGE. The appearance of the
intermediate and fast mobility species was complete within 2 h,
after which no further changes were observed up to the longest time
examined of 24 h (data not shown). Therefore, although the
variants initially have uniform slow mobility, they are heterogeneous
with respect to the ability to undergo change from slow to intermediate
or fast mobility species.
Quantitation of Free Cysteine and of Intact Thiol Esters and
Determination of the Kinetics of Thiol Ester Cleavage--
Because of
the limited quantities available for some of the variants, the number
of free thiols present initially in each of the variants was estimated
by reaction with 5-iodoacetamido fluorescein rather than by DTNB assay
because this gave higher sensitivity and allowed fluorescence
quantitation. When allowance was made for a small amount of nonspecific
labeling, which was also present in recombinant wild-type
2M, there was little evidence of a significant fraction
of the new cysteine residues being in unconjugated form (Table
I), suggesting that the new cysteines introduced into the bait region were all involved in disulfide linkages, either to other cysteines within the tetrameric protein or to
small thiols, such as glutathione.
The number of intact thiol esters present was also determined by DTNB
assay, which involved reaction of the variants with methylamine and
quantitation of the free SH generated (Fig.
3). From analysis of the time dependence
of the change in absorbance at 412 nm, both the total number of
methylamine-cleavable thiol esters and the kinetics of their cleavage
were determined (Table I). By both criteria, all four of the variants
were similar to both plasma and recombinant wild-type
2Ms, having between two and four intact thiol esters
that could be cleaved at rates that were 63-79% that of recombinant
wild-type
2M. These results indicated that the mutations
had not greatly altered either the ability of the
2M
subunits to form thiol esters or the subsequent reactivity of the thiol
esters toward small nucleophiles. The lower stoichiometry of thiol
esters for the V700C and T705C variants correlated with the presence of
a small amount of intermediate mobility material prior to reaction
(Fig. 2). This form is likely to have only two intact thiol esters.
However, the major species had slow electrophoretic mobility and
probably contained three or four intact thiol esters.

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Fig. 3.
Near normal rates of appearance of free
thiols in variant 2Ms as a result of thiol ester
cleavage by methylamine, monitored by release of
TNB . Reactions were carried out at pH 8.0 with 0.2 M methylamine. The curves represent the
nonlinear least squares fits to the data to a monoexponential. The rate
constants representing these curves are given in Table I.
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Proteinase- and Methylamine-induced Change in Conformation Detected
by TNS Fluorescence--
The conformational changes that accompany
reaction with proteinase were complete within the 10 min used for the
reaction, so the TNS emission spectrum was stable after this time.
Comparison of the TNS emission spectra for unreacted and
proteinase-reacted variants with those for plasma
2M
showed significant differences for both the starting and the finishing
conformations (Fig. 4). TNS in the
presence of plasma
2M gave an emission spectrum with
max at about 440 nm. Upon reaction with proteinase, the
TNS fluorescence intensity increased more than 2-fold, and the
wavelength maximum blue shifted from 440 to about 410 nm (Fig. 4). The
same behavior was seen for wild-type recombinant
2M
(Fig. 4). In contrast, TNS in the presence of any of the four variants
already showed enhanced intensity, and wavelength maxima between those
of native and trypsin-treated wild-type or plasma
2Ms
(Fig. 4). Reaction of the variants with either chymotrypsin or trypsin
produced very little additional increase for the M690C and T705C
variants, and a reduction in intensity for the G679C and
V700C variants. Almost no change in emission maximum was seen for the
M690C and T705C variants after reaction with either trypsin or
chymotrypsin. Although small blue shifts were observed upon proteinase
treatment of the G679C and V700C variants, the wavelength maxima of the
proteinase-treated variant was still not as much blue shifted as
wild-type or plasma
2M. Methylamine treatment resulted
in almost no change in wavelength maximum for any of the variants and
much smaller percentages of enhancement for any of the variants,
particularly T705C, than for recombinant wild-type or plasma
2Ms.

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Fig. 4.
Changes in TNS fluorescence emission spectra
in the presence of control and variant 2Ms as a result
of reaction with proteinase and methylamine. In each panel, the
thin solid line represents unreacted 2M, the
heavy solid line represents methylamine-treated 2M, and the thin and heavy dashed
lines represent 2M reacted with trypsin and
chymotrypsin, respectively. The same 2M concentration (0.1 µM) was used for each set of spectra.
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Ability of Variants to Inhibit Trypsin by Trapping--
The
ability of each variant to inhibit trypsin by the normal bait region
cleavage-induced conformational change was determined by a trapping
assay in which increasing amounts of trypsin were incubated with each
2M variant, and any nontrapped trypsin was inhibited by
addition of a large excess of soybean trypsin inhibitor. The residual
trypsin activity was then determined by chromogenic assay. The
stoichiometry of trapping was determined from the turnover point of a
plot of residual activity against the trypsin:
2M ratio (not shown). Each variant was able to trap trypsin, but the
stoichiometry was greatly reduced compared with plasma and wild-type
recombinant
2Ms (Table I), despite complete and specific
cleavage of all four of the bait regions (Fig.
5). The activity of trypsin trapped by
each of the variants was also reduced from that trapped by wild-type
2M (Table I). The largest reductions in specific
activity were seen for the M690C and T705C variants.

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Fig. 5.
Presence of normal 180-kDa monomers that are
specifically cleavable in the bait region in all four variants
demonstrated by conversion of 180-kDa bands to 90-kDa bands on SDS-PAGE
under reducing conditions, upon reaction with trypsin. The
behavior is the same as for control recombinant wild-type
2M. indicates unreacted, and + indicates after
reaction with a small excess (2.2 eq) of trypsin.
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Origin of Aberrant Properties of Cysteine Variants--
From the
above characterization of the four variants, it is clear that, whereas
they behave normally in forming tetramers with intact thiol esters that
are cleavable by methylamine at normal rates and bait regions that are
still specifically cleavable by proteinase, their ability to undergo
rapid cooperative conformational changes and to efficiently trap 1-2
mol of proteinase has been compromised by the introduction of cysteine
into the bait region. We therefore determined whether the aberrant
properties arose from the presence of new disulfides within the
tetramer.
Plasma
2-macroglobulin is a tetramer, made up of two
noncovalently associated disulfide-linked dimers (24). Upon treatment with either urea (Fig. 6A) or
SDS (Fig. 6, B and C), the noncovalent interactions can be disrupted, and the protein migrates as an ~360-kDa dimer. We found that all of the cysteine variants showed evidence of the presence of additional disulfide bonds between the
dimers, from the presence of nondissociable 720-kDa tetramers (Fig. 6).
Such behavior could result from either one or two additional disulfides
between the dimers. In each case, a minority of the protein lacked such
additional interdimer disulfides and was still dissociable to dimer.
This varied among the variants and was the greatest for the T705C
variant (Fig. 6).

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Fig. 6.
Presence of new disulfide cross-links between
disulfide-linked dimers from presence of large but variable amounts of
nondissociable tetramer on polyacrylamide gels run under different
denaturing/dissociating conditions in the absence of reducing agent.
A, samples incubated and run in a gel containing 5 M urea. B and C, samples incubated and run in 1% SDS on either 4% total acrylamide (B) or
3.5% total acrylamide (C). Note the different types of
dimer that are resolvable in C (indicated by 1 and 2).
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Another difference between variants was that the M690C variant showed a
different mobility for its dimer compared with the dimers of the other
variants when dissociation was carried out in SDS (Fig. 6C),
but less so when carried out with urea (Fig. 6A). This may
be due to the presence of a new intradimer disulfide in a fraction of
the subunits that may constrain the conformation of the SDS-unfolded
protein so that it has a different mobility from the equivalently
unfolded dimers that do not contain such an additional disulfide.
SDS-PAGE run under reducing conditions confirmed that all of the
variants were composed of the expected 180-kDa monomers and that the
bait regions were all accessible to proteinase and could be cleaved
upon exposure to trypsin (Fig. 5).
Kinetics of Fast Phase Conformational Change--
The time-course
of change in mobility of the variants on nondenaturing PAGE showed that
only a fraction of each variant showed conversion at rates similar to
that of wild-type or plasma
2M when reacted with
methylamine (see above). To test whether there was an equivalent
fraction that also showed normal kinetics of conformational change, we
followed the time-dependence of the TNS fluorescence change upon
reaction with methylamine (Fig. 7) and
determined the rate constant for the change. For all four variants it
appeared that there was a rapid phase of smaller magnitude than for
wild-type
2M, that was equivalent to the normal
conformational change step of recombinant wild-type and plasma
2Ms, because the fluorescence changes were well fitted
to a monoexponential, as expected for the conditions of the reaction,
and gave rate constants (kc) no more than about 3-fold
slower than those for plasma and wild-type recombinant
2Ms (Table I).

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Fig. 7.
Normal kinetics of conformational change for
the fraction of each variant that undergoes rapid conformational
change, monitored by change in TNS fluorescence. The solid
lines are the best fits to a single exponential decay. The
behavior of plasma 2M wild type (p-wt) and
recombinant wild type (r-wt) are shown for comparison. All
2M species were prereacted with methylamine at an amine
concentration of 2.5 M at pH 8.0 and 25 °C for 30 s. This ensured cleavage of all thiol esters prior to monitoring of
fluorescence change. Samples were then diluted into the assay buffer
containing TNS, and data acquisition was started after a delay of
approximately 20 s. The dilutions were between 40- and 15-fold to
give a constant final 2M concentration.
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DISCUSSION |
We have examined four variants of human
2M, each of
which contains a single cysteine at a different location within the
bait region, to test the involvement of the bait region in dimer-dimer association and in the mechanism of conformational change and proteinase inhibition by determining the tendency of these variants to
form new interdimer disulfides and thereby alter the properties of the
2M. We found that a major fraction of each of the four variants formed new interdimer cross-links, suggesting a very close
approach of bait regions in separate dimers to one another in the
tetramer and consequently a location for the bait regions at the
interface between these dimers. These new cross-links blocked the
conformational change that occurs upon cleavage of the thiol esters by
nucleophiles and suggests that the bait regions not only are in contact
with one another across the dimer-dimer interface but also mediate the
cooperative conformational changes that constitute the slow to fast
form interconversion, perhaps through a rearrangement of their contact
surfaces. Formation of a new disulfide cross-link between pairs of bait
regions across the interface may lock the interface so that
conformational changes can only occur very slowly or not at all
compared with the unconstrained bait regions of wild-type recombinant
or plasma
2Ms. Consistent with this is the greatly
lowered ability of the variants to trap trypsin, even though trypsin
completely and specifically cleaved all bait regions in each of the
variants. The finding that cysteine at different positions within the
bait region is able to cross-link to a bait region of the other dimer
with different efficiencies suggests that the interface is more complex
than a parallel alignment of bait regions. Instead, different parts of
the bait region must approach the equivalent residue in the other dimer
to greater or lesser extents. In addition, the possible formation of a
new disulfide between monomers of the same dimer in the M690C variant suggests that the bait regions within a dimer may approach one another
at this point. This suggests an organization of the four bait regions
of human
2M in the central cavity of the tetramer, perhaps as the cavity body of the x-ray structure, with extensive interaction between pairs of bait regions from different dimers and
less extensive contact between pairs from the same dimer (Fig. 8).

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Fig. 8.
Schematic representation of the location and
degree of contact between bait regions in human 2M.
The structure on the left is a schematic representation of
the transformed 2M tetramer, based on the electron
microscopy image reconstruction structure of Stoops and colleagues (1,
29). The enlargement on the right is a stylized depiction of
the four bait regions and how they might interact. A1 and
A2 are bait regions from the same dimer, as are
B1 and B2. Interactions between A1 and
B1 and between A2 and B2 represent
noncovalent contacts at the dimer-dimer interface. It is envisioned
that residues Gly-679, Val-700, and Thr-705 lie in the region of
greatest contact (hatched), whereas Met-690 may lie outside
this region, in a position (marked with an asterisk) at
which it is close both to a bait region from a monomer of the second
dimer and to the bait region from the second monomer of the same
dimer.
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A possible concern in trying to deduce structural properties of plasma
2M from the properties of these variants is that
formation of a new disulfide between dimers might have grossly
distorted the interface that results in the normal dimer-dimer
association and thus either precluded tetramer formation or driven the
tetramer into a distorted structure that is not representative of
native
2M. However, by several criteria, this appears
not to be the case for these variants, because they possess a number of
structurally dependent properties that are very similar to those of
plasma
2M. Thus, all four variants form tetramer as the
nearly exclusive species, with the exception of M690C and V700C, which
also form a small amount of dimer. This contrasts with more drastically modified bait region variants that we have previously examined, which
had large truncations within the bait region and that consequently formed exclusively dimers that could not associate to tetramer (12).
The present variants also form close to the full complement of thiol
esters that can be cleaved by nucleophiles at rates very similar to
plasma or recombinant wild-type
2M (Table I). They are
also capable of undergoing complete slow to fast conformational change
upon reaction with proteinase. The presence of new interdimer disulfides is therefore likely to result from the juxtaposition of the
new cysteines across the dimer-dimer interface as a consequence of
initial dimer-dimer association in the normal manner. This then
requires the bait regions to be directly involved in dimer-dimer interactions in plasma
2M.
Such involvement of the bait regions of different dimers in the
noncovalent interface of
2M is not surprising, given the cooperative nature of the conformational changes that result from bait
region or thiol ester cleavage. Earlier studies by Roche et
al. on chemical cross-linking of subunits of
2M
(25, 26) had already shown that covalent cross-links could lead to a
rigidification of the
2M interface such that it no
longer responded normally to bait region or thiol ester cleavage.
However, unambiguous interpretation of these earlier studies was not
possible because numerous cross-links were always present and the sites
of cross-linking were unknown. In contrast, it was seen in the present
study that the introduction of one or two interdimer cross-links
between residues within the bait region leads to a blockage of the
thiol ester cleavage-induced conformational change, as evidenced both
by incomplete conversion from slow to fast forms after cleavage of the
thiol esters by methylamine and by the small fraction of each variant
that shows near-normal conformational change reported by changes in TNS
fluorescence. Even when conformational change is brought about by
proteinase cleavage of the bait region, there is evidence from the
greatly lowered efficiency of proteinase trapping (Table I) that the structural rearrangements may have been slowed down. Such changes in
efficiency of proteinase trapping as a function of the relative rates
of cleavage and conformational change have been documented for plasma
2M and trypsin (27, 28). This suggests that when the
thiol esters alone are cleaved, the conformational change that occurs
cooperatively between noncovalently interacting subunits across the
dimer-dimer interface involves a reorganization of the bait region-bait
region contact area. By introducing a disulfide between such pairs of
bait regions, as in any of the four variants considered here, this
reorganization is either halted or greatly slowed down. The observation
that this blockage of movement is much less when the bait region is
cleaved by proteinase, such that all of the
2M is in the
fast form in less than 10 min, is quite consistent with and even
supportive of such a model, in that the cleavage by proteinase, which
is specifically restricted to the bait region, could isolate the
disulfide cross-link from the parts of the bait region that need to
move relative to one another, thereby freeing them to reorient and thus
abrogating the blockage of conformational change.
Our proposed location of the four bait regions at the center of the
2M tetramer in the position of the cavity body found in
the crystal structure of methylamine-transformed
2M is
also consistent with the changes in TNS fluorescence caused by
proteinase treatment of the variants and of the reduced activity of
trypsin in complex with these
2M variants. The location
and size of the cavity body in the crystal structure of
methylamine-transformed
2M is such that it would have to
be removed in whole or in part to accommodate one or two proteinase
molecules in the central cavity. If the bait regions constitute all or
part of the cavity body, their cleavage by proteinase and the
consequent reorientation of the interface, as suggested by results
presented here, could constitute such a restructuring. For the four
variants, we found that TNS fluorescence spectra not only were
different from either native or transformed plasma
2M
but also were much less responsive to change upon reaction with
proteinase than was plasma
2M. This was particularly so
for the M690C and T705C variants (Fig. 4). These are the variants that
also show the lowest specific activity for trypsin that is trapped
(Table I), suggesting that the trypsin may be very much more
constrained in these variants than in plasma
2M. The
G679C and V700C variants show somewhat higher specific activity for
trapped trypsin and also give TNS fluorescence spectra for the
proteinase-treated forms that are more like that of proteinase-treated plasma
2M. These findings for the four variants are
consistent with bait region cleavage giving less complete interface
restructuring than for plasma
2M as a result of the
constraints imposed by the interdimer disulfides, with the consequence
that the cavity body is not cleared out of the way as completely as in
plasma
2M. This could also be the explanation for the
lowered trapping ability of the variants, in that there is less volume
available to form the trap.
In conclusion, we have provided evidence that all four of the bait
regions of human
2M are in contact with or are very
close to one another, probably in the central cavity of the protein, and that the major conformational change that is required for proteinase trapping involves a reorientation of the bait region-bait region contacts both to bring about the trapping conformational change
and to make room in the central cavity to accommodate the proteinase.
Introduction of cross-links across this interface, either by disulfide
formation, as described here, or by chemical cross-linking, as
suggested elsewhere (25, 26), slows or prevents this reorientation with
consequent reduction in rate of conformational change and in efficiency
of proteinase trapping.
We thank Dr. Steven Olson (University of
Illinois at Chicago) for generous access to his SLM8000
spectrofluorometer and Dr. Esper Boel (Novo Nordisk) for the gift of
p1167.