Modularity of Serpins
A BIFUNCTIONAL CHIMERA POSSESSING alpha 1-PROTEINASE INHIBITOR AND THYROXINE-BINDING GLOBULIN PROPERTIES*

Helmut Grasberger, Christoph Buettner§, and Onno E. JanssenDagger

From the Department of Medicine, Klinikum Innenstadt, Ludwig-Maximilians-University, D-80336 Munich, Germany

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, alpha 1-proteinase inhibitor and thyroxine-binding globulin. Substitution of two of four alpha 1-proteinase inhibitor modules (Lys222 to Leu288 and Pro362 to Lys394, respectively), identified by alpha -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

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 alpha 1-proteinase inhibitor (alpha 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).

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 beta -sheets (A-C). Inhibitory serpins are characterized by a reactive site loop (RSL) located between beta -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).

Although alpha 1PI has no known ligand, its sheet C and part of sheet B form a twisted beta -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 alpha 1PI.

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 alpha 1PI-TBG chimera with both inhibitory activity and high affinity T4 binding.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- alpha 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-alpha 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.

Construction of Hybrid TBG-alpha 1PI Transfer Vectors-- Human TBG cDNA was subcloned via the KpnI and HindIII sites and human alpha 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 alpha 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).

                              
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Table I
Oligonucleotide primers for splicing by overlap extension polymerase chain reaction
For the internal primers, the first letter of the primer names denotes their 5'-origin from the TBG or alpha 1PI sequence. H1, H2, and RS denote the locations of the corresponding splicing sites (bold letters) at homology regions H1 (alpha 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 alpha 1PI coding sequences or their reverse complements, respectively (bold letters).

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). beta -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.

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-alpha 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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Design and Construction of Chimeras-- Based on the structure of alpha 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 alpha -beta sandwich, while modules 2 and 4 constitute a discontinuous beta -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 alpha -carbon atoms of intact alpha 1PI. Coordinates were taken from the Protein Data Bank entry 1PSI (34). The alpha -carbon atoms of alpha 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 alpha 1PI-TBG chimeras. A, topological schematic (35) of alpha 1PI with the secondary structure elements represented by circles (alpha -helices (h)) and triangles (beta -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 alpha -beta sandwich motif consisting of the first and third module and a beta -barrel consisting of modules 2 and 4. B, schematic representation of the chimeras analogous to the topology depicted in A. Gray, alpha 1PI modules; white, TBG modules.

To introduce the putative ligand-binding site of TBG into the alpha 1PI scaffold, its complete beta -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 alpha 1PI scaffold (P1T2P3-4) or module 1 of alpha 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.

Expression of Recombinant Proteins and Reaction with HLE-- Bv-alpha 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-alpha 1PI and anti-TBG antibodies, whereas there was no detectable cross-reactivity between bv-TBG, bv-alpha 1PI, or wild type baculovirus with these antisera.

Chimera P1T2P3-4 and, to a lesser extent, P1T2P3T4 retained the inhibitory properties of alpha 1PI and formed SDS-stable complexes with HLE (Fig. 3). P1T2P3-4 and P1T2P3T4 showed significantly more cleaved inhibitor than bv-alpha 1PI. The reaction of HLE with P1T2P3T4 was slower than with alpha 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-alpha 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-alpha 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-alpha 1PI (upper panel) and anti-TBG antibodies (lower panel). P1T2P3-4 formed SDS-stable complexes with HLE similar to bv-alpha 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-alpha 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-alpha 1PI.

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-alpha 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 alpha 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-alpha 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-alpha 1PI (open circle ), 2.1 for P1T2P3-4 (black-square), and 11 for P1T2P3T4 (). Bv-TBG () and P1T2-4 (black-triangle) showed no inhibition of HLE even at a high molar excess.

Analysis of T4 Binding-- Scatchard analysis of T4 binding showed no detectable T4 binding activity (Ka < 106 M-1) for chimera P1T2P3-4 and the bv-alpha 1PI control. However, in P1T2P3T4, transposition of the complete beta -barrel motif into the alpha 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 alpha 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 (black-triangle) 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 (open circle ) (Ka = 0.9·108 M-1). alpha 1PI and P1T2P3-4 had no specific T4 binding. The plots are representative of four independent experiments.

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-alpha 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 alpha 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-alpha 1PI (bv-alpha 1PI, P1T2P3-4, P1T2P3T4) or anti-TBG (P1T2-4, bv-TBG) antiserum, respectively. P1T2P3-4 exhibited a behavior similar to bv-alpha 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.

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-alpha 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 (diamond ) (t1/2 of 4.5 versus 7 min), whereas P1T2-4 (black-triangle) 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-alpha 1PI (open circle ), whereas P1T2P3-4 (black-square) was less stable in this assay.

The inhibitor function of chimera P1T2P3-4 was also less stable than that of bv-alpha 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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Fig. 9.   The proposed modular architecture of the serpins. Ribbon drawing of alpha 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).

                              
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Table II
Inhibitory potency toward HLE and T4 binding affinity of the chimeras
The stoichiometries of inhibition (SI) for the interaction of the chimeras with HLE and affinity constants (Ka) for T4 binding were determined as described under "Experimental Procedures."

In chimera P1T2P3T4 the transfer of the T4-binding site of TBG into the alpha 1PI frame was achieved by substituting the beta -barrel-like structure of alpha 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 alpha 1PI. In comparison with the archetypical, evolutionarily refined alpha 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 alpha 1PI beta -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 beta -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 alpha 1PI frame unambiguously locates the ligand-binding site to the beta -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 alpha 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 alpha 1PI variant with a C-terminal extended RSL (48).


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Fig. 10.   Sequence alignment of the RSL regions of TBG, alpha 1PI and P1T2P3T4. The active site residues of alpha 1PI and the cleavage site of TBG by HLE (47) are depicted in bold letters. Note that the RSL of chimera P1T2P3T4 harbors a seven-residue C-terminal extension, compared with the alpha 1PI loop, including the HLE cleavage site of TBG. sec. struct., secondary structure elements.

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 beta -barrel than the remaining molecule. Significant heat-induced unfolding might occur without affecting the beta -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.

    ACKNOWLEDGEMENTS

We thank R. Huber, W. Bode, H. Fritz, and S. Refetoff for helpful discussions.

    FOOTNOTES

* 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.

Dagger 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 alpha 1PI (preceding letter T or P), e.g. in P1T2P3-4, modules 1, 3, and 4 are alpha 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.

    ABBREVIATIONS

The abbreviations used are: serpin, serine-proteinase inhibitor; alpha 1PI, alpha 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).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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