From the Department of Medicine, Klinikum Innenstadt, Ludwig-Maximilians-University, D-80336 Munich, Germany
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ABSTRACT |
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An exciting application of protein engineering is
the creation of proteins with novel functions by the retrofitting of
native proteins. Such attempts might be facilitated by the idea of a mosaic architecture of proteins out of structural units. Even though
numerous theoretical concepts deal with the delineation of structural
"modules," their potential in the design of proteins has not yet
been sufficiently exploited. To address this question we used a gain of
function approach by designing modular chimeric molecules out of two
structurally homologous but functionally diverse members of the
superfamily of serine-proteinase inhibitors, In all but the smallest proteins, crystallography has revealed
that polypeptide chains form several more or less compact units. When
loosely connected to the remaining molecule, such units are usually
referred to as domains, which implicates the possibility of an
autonomous existence (1). In many other cases, the mosaic nature of
proteins is less obvious, and numerous concepts have been developed to
facilitate the delineation of "modules" thought to rule the
folding, function, and biological evolution of proteins (2-8). The
increasing frequency with which functionally unrelated proteins are
found to contain recurrent structural motifs suggests that the number
of natural folds is limited (9, 10) and that complex proteins have
evolved by modular assembly (11). Such evolutionarily refined
structural units are attractive candidates as building blocks for the
design of novel proteins. This concept may be exploited for the
in vitro recombination of homologous, i.e.
structurally related, proteins.
Based on sequence similarities, an ever increasing number of homologous
but functionally diverse proteins are recognized as members of the
superfamily of serine-proteinase
inhibitors
(serpins).1 They presumably
evolved from a common ancestor at least 500 million years ago (12).
Most of more than 100 known members of the serpin superfamily are true
inhibitors of serine proteinases, best exemplified by the archetypical
serpin The crystallographic structures of several serpins have been determined
(reviewed in Refs. 17 and 18). Their highly compact single-domain
structure has a scaffold of three crossed Although So far, it has not been tested whether the inhibitory function and the
ligand-binding function are mutually exclusive within the serpin
scaffold. We now present a novel concept of a modular architecture of
the serpin structure and construction of an Materials--
Construction of Hybrid TBG- Generation of Recombinant Baculovirus and Expression in Insect
Cells--
Sf9 cells (5 × 106 log phase)
maintained exclusively in serum-free medium were cotransfected with 1 µg of linearized wild type virus and 4 µg of each of the transfer
plasmids by lipofection (29). Western Blotting--
Samples were run on 10% continuous
tris/glycine gels under denaturing, nonreducing conditions. For PAGE
under native conditions, SDS was omitted from all buffers. Blotted
nitrocellulose membranes were probed with rabbit anti-TBG or rabbit
anti- Reaction with HLE--
Samples were incubated in assay buffer
(0.4 M NaCl, 0.05% Triton X-100, 0.1 M Hepes,
pH 7.4) with HLE in molar concentrations between 1:1 and 20:1
(proteinase:sample) for 20 min at 37 °C.
Inhibitor Assay--
HLE was incubated at 37 °C for 15 min
with increasing amounts of recombinant proteins in assay buffer (see
above). The residual proteolytic activity was calculated from the
increase in absorbance (410 nm) after the addition of 0.5 mM
N-methoxysuccinyl-A-A-P-V-p-nitroanilide (Calbiochem) as chromogenic substrate. Rates of substrate hydrolysis were constant over the 3-min period used to determine residual activities. The intercept on the abscissa of the plot of the fraction of enzyme remaining (E/E0)
versus the ratio of the initial inhibitor to initial enzyme
concentration (I0/E0) yielded the
apparent stoichiometry of the reaction. Control reactions with
supernatants of cells infected with baculovirus expressing TBG excluded
endogenous HLE inhibitory activity, degradation of HLE, and substrate
loss to endogenous proteinases.
T4 Binding Assay--
Parameters of T4
binding to the recombinant proteins were measured by a method
previously described in detail (31). Briefly, samples were diluted with
270 mM barbital buffer (pH 8.6) or phosphate-buffered saline (pH 8.0) and incubated with [125I]T4
(specific activity, 48.8 MBq/µg, NEN Life Science Products) in the
presence of increasing amounts of unlabeled T4. After
equilibration, protein bound was separated from free
[125I]T4 with anion exchange resin beads
(M-400, Mallinckrodt), and the specific 125I binding was
determined. The affinity constants (Ka) and binding
capacities for T4 were calculated by Scatchard analysis (32).
Heat Denaturation--
The functional stability of recombinant
proteins was quantified by thermal denaturation in a water bath at
60 ± 0.1 °C for various periods of time or at various
temperatures for 20 min, respectively. The samples were then cooled on
ice and centrifuged for 15 min at 13,000 × g to remove
precipitated protein. Residual specific T4 binding capacity
or HLE inhibitory activity was expressed relative to controls kept at
4 °C. The half-lives (t1/2) of heat denaturation were calculated by least square analysis of
semi-logarithmic plots of the remaining specific T4 binding
versus time of incubation.
Design and Construction of Chimeras--
Based on the structure of
To introduce the putative ligand-binding site of TBG into the
Expression of Recombinant Proteins and Reaction with
HLE--
Bv-
Chimera P1T2P3-4 and, to a lesser
extent, P1T2P3T4
retained the inhibitory properties of Inhibitor Assay--
The residual proteolytic activity of HLE
preincubated with increasing amounts of the inhibitors showed a linear
dependence characteristic for tight binding inhibition (Fig.
5). The stoichiometries of inhibition
(SI), defined as mole of serpin required to inhibit 1 mole of HLE, were
1.3 for bv- Analysis of T4 Binding--
Scatchard analysis of
T4 binding showed no detectable T4 binding
activity (Ka < 106
M Heat Stability of the Chimeras--
To examine the effect of
module exchange on the stability of the chimeras, heat denaturation
experiments were performed. Incubation of bv-
Consistent with the increased conformational stability on native PAGE,
P1T2P3T4 displayed no
significant decline of T4 binding after incubation at
temperatures as high as 85 °C for 20 min (Fig. 8B). However, its inhibitor
function was lost at a slightly lower temperature than that of
bv-
The inhibitor function of chimera
P1T2P3-4 was also less stable than
that of bv- Genetic engineering has become a mainstay in elucidating the still
inadequately understood structure-function correlation of proteins.
This information is critical for the understanding of the diversity of
proteins and the design of new drugs. In recent years, research has
moved from the substitution of single amino acids to the concept of a
modular design of proteins. In some proteins, structural and functional
units are readily obvious, e.g. the extra- and intracellular
and the transmembrane domains of membrane-bound receptors. The
identification of discrete units has been used for the successful
construction of chimeric receptors (37). However, in chimeras of
globular proteins so far only similar functions have been substituted
(38-41). In this study, we present a strategy to engineer bifunctional
chimeras from integral parts of homologous proteins. Based on a concept
of a modular architecture of the serpins (Fig.
9), we have combined two different functional properties of the serpin superfamily, proteinase inhibition and ligand binding, into one chimeric molecule. The inhibitory and
ligand-binding characteristics of the chimeras are summarized in Table
II.
1-proteinase inhibitor and thyroxine-binding
globulin. Substitution of two of four
1-proteinase
inhibitor modules (Lys222 to Leu288 and
Pro362 to Lys394, respectively), identified by
-backbone distance analysis, with their thyroxine-binding globulin
homologues resulted in a bifunctional chimera with inhibition of human
leukocyte elastase and high affinity thyroxine binding. To our
knowledge, this is the first report on a bifunctional chimera
engineered from modules of homologous globular proteins. Our results
demonstrate how a modular concept can facilitate the design of new
functional proteins by swapping structural units chosen from members of
a protein superfamily.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-proteinase inhibitor (
1PI).
Serpins are fundamentally important in the regulation of major
proteolytic cascades, such as blood coagulation, fibrinolysis,
inflammatory response, and extracellular matrix turnover (reviewed in
Ref. 13). However, some serpins have lost the inhibitory function and
serve as transport proteins for small ligands, such as
thyroxine-binding globulin (TBG) (14) and corticosteroid-binding
globulin (15). TBG has an exceptionally high binding constant
(Ka = 1010 M
1)
for thyroxine (T4) and a binding energy close to a covalent bond (16).
-sheets (A-C). Inhibitory
serpins are characterized by a reactive site loop (RSL) located between
-sheets A and C. Proteinase inhibition involves the incorporation of
the cleaved RSL into the A-sheet. This structural rearrangement is
accompanied by an increase in stability (stressed-to-relaxed transition
(19)) and the generation of SDS-stable serpin-proteinase complexes
(20). Although the individual serpins have become remarkably
diversified by evolution, they share a common molecular pathology (21).
Inhibitory dysfunction is caused by disturbances of the hinges of the
RSL (P14-P12 of the RSL and strand 1C) (22) or by prevention of
insertion (23).
1PI has no known ligand, its sheet C and part
of sheet B form a twisted
-barrel-like structure, characteristic of
ligand-binding proteins. By affinity labeling the homologous regions
have been shown to comprise the hormone-binding sites of TBG (24) and
corticosteroid-binding globulin (25), both of which share 40% sequence
identity with
1PI.
1PI-TBG chimera with both inhibitory activity and high affinity T4 binding.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1PI M-type cDNA (26) was a
kind gift from R. Foreman (Southampton, United Kingdom). A vector
containing the full-length cDNA of TBG had been constructed
previously (27). Vent DNA polymerase and restriction endonucleases were
obtained from New England Biolabs. Spodoptera frugiperda
Sf9 cells (ATCC catalog no. CRL 1711) and wild type baculovirus
DNA were from Invitrogen. Liposomes for transfection and SF900 II
insect cell culture medium were purchased from Life Technologies, Inc.
Purified TBG and rabbit anti-TBG serum were generously donated by R. Gärtner (Munich, Germany). Rabbit anti-
1PI serum,
human leukocyte elastase (HLE, EC 3.4.21.37), and transthyretin were
from Calbiochem. T4 stock solutions and TBG concentrations
were quantified using commercially available radioimmunoassays (Brahms
Diagnostica, Berlin, Germany and CIS Bio Int., Gif-Sur-Yvette, France).
Inhibition assays and active site titrations were measured on a Beckman
DU 640 spectrophotometer.
1PI Transfer
Vectors--
Human TBG cDNA was subcloned via the KpnI
and HindIII sites and human
1PI cDNA via
the PstI site into the transfer vector pBlueBac4
(Invitrogen). The splicing sites of the chimeras mapped to highly
conserved regions (homology region H1 and H2) and to a putative
permissive surface loop (splicing site RS, C-terminal to the RSL). The
chimeric constructs were then generated by repeated cycles of two-step
polymerase chain reaction overlap extension (28) with the linearized
TBG and
1PI plasmids or the gel-purified intermediate
polymerase chain reaction products (P1T2-4, P1T2P3-4)2
as templates, respectively. The cDNAs were fused sequentially at
homology regions H1 and H2 and splicing site RS with the primers listed
in Table I. Primers P-N and T-C provided
PstI and KpnI linkers, respectively, for
subcloning into pBlueBac4. The correct sequence of the final products
was confirmed by automated sequencing with fluorescent dye terminators
(PRISM System 377, Applied Biosystems).
Oligonucleotide primers for splicing by overlap extension polymerase
chain reaction
1PI
sequence. H1, H2, and RS denote the locations of the corresponding
splicing sites (bold letters) at homology regions H1 (
1PI
numbering: Val218-Met221), H2
(Pro289-Thr294), and the RSL (Pro361,362),
respectively. N or C denote N- or C-terminal external primers, specific
for the TBG or
1PI coding sequences
or their reverse complements, respectively (bold letters).
-Galactosidase-positive recombinant
clones were selected by plaque assay and screened for wild type virus
contamination by polymerase chain reaction (30). For protein
expression, log-phase Sf9 cells from a spinner culture were
seeded in tissue culture flasks and infected with recombinant virus at
a multiplicity of infection of five. The medium was changed 12 h
later and supplemented with 10 µM
1-(L-trans-epoxysuccinylleucylamino)-4-guanidinobutane and 10 µM pepstatin A (both from Roche Molecular
Biochemicals). Forty-eight hours post infection, the culture
supernatants were collected by centrifugation at 1500 × g for 15 min, concentrated, and washed (0.1 M
NaCl, 0.1 M Hepes, pH 7.4) by ultrafiltration (Centriplus
30, Millipore Corp.). Protein concentrations were determined by
Scatchard analysis of T4 binding and by densitometry of
Coomassie Blue-stained gels using purified serum TBG as the standard.
1PI antiserum as primary antibody, respectively,
followed by enhanced chemiluminescence immunodetection with horseradish
peroxidase-conjugated donkey anti-rabbit IgG (Amersham Pharmacia
Biotech) as secondary antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1PI and guided by a distance analysis of its carbon
backbone using a diagonal plot (8, 33), four compact structural units
of the serpin fold were identified (Figs.
1 and
2A). Modules 1 and 3 complement each other to form an
-
sandwich, while modules 2 and
4 constitute a discontinuous
-barrel fold. The segregation into
these two subdomains becomes even more apparent in cleaved serpins with
the RSL inserted into sheet A.
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Fig. 1.
Diagonal distance plot of the
-carbon atoms of intact
1PI. Coordinates were taken from
the Protein Data Bank entry 1PSI (34). The
-carbon atoms of
1PI are plotted left to right
(abscissa) and top to bottom
(ordinate). Increasing distances between pairs of residues
are shown by dark gray. Triangles 1-4
at the diagonal line indicate four modules of residues that
are located close to one another in the molecule. These modules are
separated from one another as shown by the dark areas
between them. The lighter shading in the boxed
intercepts of triangles 1 and 3 (and
2 and 4, respectively) in the plot shows the
spatial association of module 1 with 3 and module 2 with 4.
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Fig. 2.
Modular design of
1PI-TBG chimeras. A,
topological schematic (35) of
1PI with the secondary
structure elements represented by circles (
-helices
(h)) and triangles (
-strands (s)).
The splicing sites for the construction of the chimeras are indicated
by H1, H2, and RS. The molecule is organized in two subdomains, an
-
sandwich motif consisting of the first and third module and a
-barrel consisting of modules 2 and 4. B, schematic
representation of the chimeras analogous to the topology depicted in
A. Gray,
1PI modules;
white, TBG modules.
1PI scaffold, its complete
-barrel was substituted by
the TBG homologue to give chimera
P1T2P3T4 (Figs.
2B and 9). As controls, chimeras containing only module 2 of
TBG in the
1PI scaffold (P1T2P3-4) or module 1 of
1PI in the TBG scaffold
(P1T2-4) were constructed. The boundaries of
the modules coincided with regions that are highly conserved throughout
the serpins (homology regions H1 and H2) or matched to a permissive
loop region (RS, C-terminal to the RSL), thereby reducing the
probability of local structural disturbance in the chimeric proteins.
The corresponding hybrid cDNAs, generated by repeated cycles of
splicing by overlap extension polymerase chain reaction, were used to
produce recombinant baculovirus by in vivo recombination in
insect cells.
1PI, bv-TBG, and the three chimeras were
efficiently secreted by the insect cells with similar expression levels
of up to 5 µg/ml after 60 h in serum-free medium. The structural
integrity of the proteins was evident by their reaction with specific
polyclonal anti-
1PI and anti-TBG antibodies, whereas
there was no detectable cross-reactivity between bv-TBG,
bv-
1PI, or wild type baculovirus with these antisera.
1PI and formed
SDS-stable complexes with HLE (Fig. 3).
P1T2P3-4 and
P1T2P3T4 showed significantly more cleaved inhibitor than bv-
1PI. The
reaction of HLE with
P1T2P3T4 was slower
than with
1PI, as indicated by the large amount of
uncleaved inhibitor at a molar ratio of one. In contrast to the stable
reaction products of cleaved bv-
1PI, increasing HLE
concentrations led to a loss of detectable
P1T2P3T4-HLE complex
concomitant with the disappearance of free
P1T2P3T4 (Fig. 4). As expected, bv-TBG and
P1T2-4 harboring the RSL equivalent of TBG
behaved like pure substrates (Fig. 3).
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Fig. 3.
Immunoblot of chimeras before and after
incubation with HLE. Bv- 1PI, bv-TBG, and the
chimeras were incubated either alone (
) or with (+) an equimolar
amount of HLE for 20 min at 37 °C. Nondigested and digested samples
were separated on nonreducing SDS-PAGE, blotted, and probed with
polyclonal anti-
1PI (upper panel) and
anti-TBG antibodies (lower panel).
P1T2P3-4 formed SDS-stable
complexes with HLE similar to bv-
1PI but also showed a
significant substrate reaction. Chimera
P1T2P3T4 also formed an
HLE-inhibitor complex (detected with both antibodies), but most of the
protein was cleaved. P1T2-4 and bv-TBG showed
pure substrate reactions.
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Fig. 4.
Digestion of
bv- 1PI and
P1T2P3T4 at different
HLE-to-serpin ratios. Samples were incubated with increasing
ratios of HLE to inhibitor (E/I). Reactions were
stopped after 20 min by denaturation at 95 °C in 0.1% SDS. The
fraction of complexed
P1T2P3T4 was smaller
and less stable than that of bv-
1PI.
1PI, 2.1 for
P1T2P3-4, and 11 for
P1T2P3T4. These SI
values were in agreement with the reaction products on the immunoblots
(Figs. 3 and 4). Again, bv-TBG and P1T2-4
showed no inhibition of HLE.
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Fig. 5.
Titration of HLE with
1PI-TBG chimeras. HLE was titrated
with each of the chimeras at 37 °C. After incubation for 15 min,
residual HLE activity was determined. For bv-
1PI,
P1T2P3T4, and
P1T2P3-4, linear titration curves
were obtained irrespective of the substrate concentrations tested (0.1 and 1 mM, Km of 0.15 mM).
The intercept with the abscissa yielded an apparent
stoichiometry of 1.3 for bv-
1PI (
), 2.1 for
P1T2P3-4 (
), and 11 for
P1T2P3T4 (
). Bv-TBG
(
) and P1T2-4 (
) showed no inhibition of
HLE even at a high molar excess.
1) for chimera
P1T2P3-4 and the
bv-
1PI control. However, in
P1T2P3T4, transposition
of the complete
-barrel motif into the
1PI frame
created a high affinity T4-binding site
(Ka = 1.7·108
M
1), comparable with the first binding site
of transthyretin (Fig. 6B).
The additional substitution of module 3 in
P1T2-4 increased the T4 binding
affinity to almost half of that of bv-TBG or human serum TBG
(Ka = 0.5·1010
M
1 versus 1.2·010
M
1) (Fig. 6A), although 35% of
its residues differed from the wild type TBG sequence.
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Fig. 6.
T4 binding of
1PI-TBG chimeras. A,
Scatchard analysis of T4 binding of bv-TBG (
) and human
serum TBG revealed no significant differences in binding affinity
(Ka = 1.2 ± 0.11·1010
M
1). The Ka of chimera
P1T2-4 (
) was only slightly reduced
(0.5 ± 0.14·1010 M
1).
B, the binding affinity of
P1T2P3T4 (
) was 70 times less than bv-TBG, but at 1.7 ± 0.3·108
M
1 it was still higher than the second-best
natural T4-binding protein, transthyretin (
)
(Ka = 0.9·108
M
1).
1PI and
P1T2P3-4 had no specific
T4 binding. The plots are representative of four
independent experiments.
1PI and
P1T2P3-4 at 60 °C caused a
shift in electrophoretic mobility on native PAGE (Fig.
7) compatible with dimerization, as has
been previously shown for human serum
1PI (36). Bv-TBG and P1T2-4 were not stable at 60 °C. In
contrast, P1T2P3T4 exhibited neither multimerization nor loss of soluble antigen even at
80 °C.
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Fig. 7.
Native PAGE analysis of heat-denatured
chimeras. Samples were incubated for 30 min at the indicated
temperatures, separated by native PAGE and probed with
anti- 1PI (bv-
1PI,
P1T2P3-4,
P1T2P3T4) or anti-TBG
(P1T2-4, bv-TBG) antiserum, respectively.
P1T2P3-4 exhibited a behavior
similar to bv-
1PI, with polymerization at 60 °C and
almost complete loss of immunoreactive material at 80 °C.
P1T2P3T4 showed neither
signs of polymerization nor loss of detectable antigen at 80 °C,
whereas P1T2-4 showed a large proportion of
pre-existing polymers and loss of detectable antigen at 60 °C. No
bv-TBG polymers were detectable, but antigenicity was lost after
incubation at 60 °C.
1PI, starting at 55 °C (Fig. 8C). SDS-PAGE analysis of
P1T2P3T4 denatured at
65 °C revealed that this material was still a specific substrate for
HLE but did not form a serpin-enzyme complex (data not shown).
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Fig. 8.
Functional stability of chimeras.
A, rate of thermal inactivation for
P1T2P3T4,
P1T2-4, and bv-TBG as determined by residual
T4 binding capacity. Proteins were heated at 60 °C,
aliquots were removed at the indicated time intervals and centrifuged,
and the residual T4-binding activity was determined. Values
are expressed as protein-bound T4 relative to the basal
levels and represent the means ± SD for three independent
experiments. Plots of the log binding capacities versus time
of incubation were linear, indicative of an apparent first-order
process. Bv-TBG ( ) had a slightly reduced functional stability
compared with human serum TBG (
) (t1/2 of 4.5 versus 7 min), whereas P1T2-4 (
)
was rapidly denatured (t1/2 = 2 min). Note that
P1T2P3T4 (
) is
essentially stable at 60 °C with no significant loss of
T4 binding capacity within 30 min. B, heat
denaturation profile illustrating the markedly increased functional
stability of uncleaved
P1T2P3T4 comparable
with bv-TBG cleaved by HLE (×). C, functional
stability measured by means of the residual HLE inhibitory activity.
P1T2P3T4 lost its
inhibitory potency at temperatures slightly lower than
bv-
1PI (
), whereas
P1T2P3-4 (
) was less stable in
this assay.
1PI and was completely inactivated at
55 °C. The t1/2 (60 °C) of T4
binding of P1T2-4 was reduced to about
one-third of the t1/2 of bv-TBG (Fig.
8A).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
The proposed modular architecture of the
serpins. Ribbon drawing of 1PI with the proposed
compact modules depicted in different colors. Module 1 (Phe23-Met221) is shown in pink,
module 2 (Lys222-Leu288) in blue,
module 3 (Pro289-Pro361) in yellow,
and module 4 (Pro362-Lys394) in
green. The reactive center residues within the RSL are
highlighted as space-filling models. Coordinates were taken
from Protein Data Bank entry 1PSI (34).
Inhibitory potency toward HLE and T4 binding affinity of the
chimeras
In chimera P1T2P3T4 the
transfer of the T4-binding site of TBG into the
1PI frame was achieved by substituting the
-barrel-like structure of
1PI with its TBG homologue
(modules 2 and 4). This chimera exhibited inhibition of and complex
formation with HLE, characteristic of inhibitory serpins such as
1PI. In comparison with the archetypical, evolutionarily
refined
1PI, it was a weaker inhibitor with a higher
apparent stoichiometry of inhibition and a shorter half-life of its
complex. In addition to proteinase inhibition, chimera
P1T2P3T4 also exhibited
a specific, high affinity T4 binding. Although its binding
affinity was 70-fold lower than that of TBG, it was still higher than
that of transthyretin, the next best natural T4-binding protein.
In contrast, the substitution of only module 2 and thus only part of
the 1PI
-barrel including the environment of the
affinity-labeled Lys253 (24) did not result in detectable
T4 binding. Similarly, a chimera harboring only module 4 of
TBG produced a dysfunctional, secretion-deficient protein (data not
shown). Only the substitution of the complete
-barrel, comprising
modules 2 and 4, was sufficient to transfer the high affinity
T4-binding site. Consequently, both modules seem to
participate in avid T4 binding, in agreement with the
demonstration that all parts of the T4 molecule, and thus an extensive surface of interaction of T4 with the binding
cavity of TBG, are essential for its high binding affinity (42).
Furthermore, the functional transfer of the T4-binding site
of TBG into the
1PI frame unambiguously locates the
ligand-binding site to the
-barrel motif of the serpins.
Surprisingly, P1T2P3T4
remained in solution and retained its T4 binding activity
even at remarkably high temperatures (Fig. 8). Serpins tend to
polymerize at elevated temperatures (43) and simultaneously lose their
activity and escape immunodetection as a result of precipitation.
Polymerization is thought to involve the insertion of the loop of one
serpin molecule into either sheet A (44) or C (21, 45) of another
molecule. Both models require detachment of strand 1 from the C-sheet
(46). The extended RSL of chimera
P1T2P3T4, which is
engineered to be 3 or 7 amino acids longer than in TBG or
1PI, respectively (Fig.
10), most likely delays the
heat-induced release of strand 1C from the C-sheet, compatible with the
increased thermal resistance of an
1PI variant with a
C-terminal extended RSL (48).
|
The discrepancy in the functional stability of T4 binding
versus HLE inhibition of
P1T2P3T4 could be the
result of a higher intrinsic stability of the -barrel than the
remaining molecule. Significant heat-induced unfolding might occur
without affecting the
-barrel and thus T4 binding.
However, the cooperativity in the unfolding of serpins (19, 49) argues
against this possibility. More conceivably, a local structural
rearrangement of the RSL is responsible for the observed loss of
inhibitory activity at intermediate temperatures. During heat exposure
the A-sheet of the serpins is supposed to open up and accept a portion
of its own RSL. In
P1T2P3T4 this might
distort the RSL near the scissile bond, resulting in a pure substrate
behavior toward HLE, whereas the extension of the RSL prevents
detachment of s1C and hence both polymerization and loss of
T4 binding. This limited structural transition of
P1T2P3T4 might resemble
the spontaneous conversion of plasminogen activator inhibitor-1 from an
active to a latent conformation in vivo (50, 51).
In conclusion, the successful construction of a bifunctional chimera
clearly demonstrates that ligand binding and proteinase inhibition are
not exclusive within the serpin structure and provides evidence for
their proposed modular architecture. Moreover, because our approach
does not rely on specific features of the serpins but rather uses
general design criteria such as compactness of modules and sequence
conservation at fusion points, it appears not to be limited to this
protein superfamily. There are many examples in which unrelated
functions have evolved within a conserved structural scaffold (52-54),
occasionally recruiting different portions of a molecule as reactive
centers (55, 56). Thus the exchange of homologous modules offers vast
possibilities for the design of chimeric proteins with new functional
properties. Furthermore, the integration of two functions in one
globular protein suggests the potential to introduce novel allosteric
effects, e.g. modulation of enzymatic activities upon ligand binding.
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ACKNOWLEDGEMENTS |
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We thank R. Huber, W. Bode, H. Fritz, and S. Refetoff for helpful discussions.
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FOOTNOTES |
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* This work was supported by Grants DFG Ja671/1-2 and SFB 469/B8 from the Deutsche Forschungsgemeinschaft (to O. E. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Medizinische Klinik,
Klinikum Innenstadt, Ludwig-Maximilians-University, Ziemssenstr. 1, D-80336 Munich, Germany. Tel.: 49-89-5160-5394; Fax: 49-89-5160-4566; E-mail: Onno.E.Janssen{at}lrz.uni-muenchen.de.
§ Current address: Thyroid Div., Dept. of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115.
2
The names of the chimeras illustrate the
composition from modules (the subscript numbers) of TBG and
1PI (preceding letter T or
P), e.g. in
P1T2P3-4, modules 1, 3, and 4 are
1PI sequences, whereas the second module is a TBG
sequence. The denotation of serpin secondary structure elements and
their assignments to TBG are as described in Ref. 17.
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ABBREVIATIONS |
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The abbreviations used are:
serpin, serine-proteinase inhibitor;
1PI,
1-proteinase inhibitor;
bv, baculovirus (recombinant);
HLE, human leukocyte elastase;
PAGE, polyacrylamide gel electrophoresis;
TBG, thyroxine-binding globulin;
RSL, reactive site loop;
SI, stoichiometry of inhibition;
T4, thyroxine;
s, strand (i.e. s1C).
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REFERENCES |
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