1Department of Molecular Biology, Sejong University, 98 Gunja-dong, Kwangjin-gu, Seoul 143-747 and 2Functional Proteomics Center, Korea Institute of Science and Technology, 391 Hawolgok-dong, Sungbuk-gu, Seoul 136-791, Korea
3 To whom correspondence should be addressed. E-mail: mhyu{at}kist.re.kr
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Abstract |
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Keywords: 1-antichymotrypsin/antithrombin III/serpins/stability/strain
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Introduction |
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The serpin family proteins share a common tertiary structure, composed of three ß-sheets and several -helices (Figure 1). An intriguing structural feature of inhibitory serpins is the strain of the native form (Huber and Carrell, 1989
). The native forms of most proteins, having unique tertiary structures carrying biological activities, are in their most stable state (Anfinsen, 1973
). However, the native forms of some proteins are in a metastable strained state and the native strain has been recognized as a mechanism of biological regulation (Wiley and Skehel, 1987
; Huber and Carrell, 1989
; Stein and Carrell, 1995
; Carr et al., 1997
; Im et al., 1999
; Lee et al., 2000
). The reactive site loop of inhibitory serpins is exposed in the strained native conformation, but is inserted into the major ß-sheet, A ß-sheet, upon formation of an inhibitory complex with the target protease (Engh et al., 1995
; Wright and Scarsdale, 1995
; Huntington et al., 2000
). This process accompanies conversion of the metastable native structure into a more stable conformation. However, ovalbumin, a stable but non-inhibitory serpin, does not undergo a drastic conformational change upon cleavage of the reactive site loop by a protease. Therefore, it is conceivable that the native strain of inhibitory serpins is utilized for the facile conformational change upon complex formation.
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In the present study, we searched for common themes generally applicable to inhibitory serpins to increase stability. There are some residues conserved among inhibitory members of serpins such as 1ACT, ATIII and PAI-1 and hormone-binding globulins, but not in non-inhibitory members such as ovalbumin and angiotensinogen (Huber and Carrell, 1989
). Such residues might be critical for either functionality or the native strain of the inhibitory serpins. Substitutions, which increased conformational stability in
1AT, were also introduced at the equivalent positions in
1ACT and ATIII and mutational effects were investigated. We measured thermostability of the serpins. There is a correlation between the conformational stability of
1AT and its thermostability (Kwon et al., 1994
). Thermostable variants of serpins may be of practical use because heat-induced deactivation of serpins was shown to be a concentration-dependent aggregation process (Lomas et al., 1993
; Kwon et al., 1994
). In addition, thermostability of recombinant
1AT was shown to relate to the biological turnover rate of the protein (Travis et al., 1985
). The results obtained in this study provide valuable information for engineering clinically important inhibitory serpins.
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Materials and methods |
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Porcine pancreatic elastase, bovine -chymotrypsin, human plasma thrombin, porcine mucosa heparin, N-succinyl-(Ala)3-p-nitroanilide, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and a rabbit anti-human ATIII antibody were purchased from Sigma (St Louis, MO). Chromozym-TH was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN). Bac-N-Blue transfection kit and High 5 cells were purchased from Invitrogen (Leek, The Netherlands) and Spodoptera frugiperda (Sf9) cells from Clontech Laboratories (Palo Alto, CA). Econo-Pac heparin cartridge was purchased from Bio-Rad Laboratories (Hercules, CA). All other chemicals were of reagent grade.
Recombinant 1AT and recombinant
1ACT proteins
Plasmids for the wild-type human 1AT and human
1ACT expression in Escherichia coli have been described previously (Im and Yu, 2000
). Substitution mutations of
1AT and
1ACT were generated by oligonucleotide-directed mutagenesis (Kunkel et al., 1987
). Expression of recombinant
1AT proteins and recombinant
1ACT proteins was induced in M9ZB media (1% tryptone, 0.5% NaCl, 0.1% NH4Cl, 0.3% KH2PO4, 0.6% Na2HPO4, 0.4% glucose, 1 mM MgSO4) by addition of 0.1 mM IPTG as described previously (Kwon et al., 1994
).
Recombinant ATIII proteins
Human ATIII cDNA was previously obtained by polymerase chain reaction (Im and Yu, 2000). A pair of complementary oligonucleotides coding for baculovirus gp64 signal sequence were introduced in front of ATIII sequences to direct the synthesized protein to extracellular culture media. Substitutions of ATIII sequences were introduced by oligonucleotide-directed mutagenesis. After dideoxynucleotide sequencing, the DNA fragment containing ATIII gene was transferred to pBlueBac plasmid (Invitrogen) to co-transfect insect cells with a linearized wild-type baculovirus DNA. Recombinant blue plaques formed on Sf9 cell lawns were purified and stored at 4°C. Wild-type baculovirus, recombinant baculovirus harboring wild-type ATIII gene and recombinant baculovirus harboring mutant ATIII gene were used in parallel to infect High 5 cells. Serum-free media (Invitrogen) were used in this study to avoid any contaminating antithrombin activity from serum. Conditioned media were harvested 45 days after infection with recombinant baculovirus and clarified by centrifugation at 9000 g for 20 min. Samples were buffer-exchanged to ATIII assay buffer (0.1 M TrisCl, pH 8.0, 0.12 M NaCl, 0.01% sodium azide and 0.1% bovine serum albumin). ATIII was partially purified on a heparin-affinity column by elution with a NaCl gradient. Expression of ATIII was confirmed by inhibitory activity assays and western blot analyses using a rabbit anti-human ATIII antibody.
Measurement of inhibitory activity
The concentration of porcine pancreatic elastase was determined by measuring the initial rates of hydrolysis of 1 mM N-succinyl-(Ala)3-p-nitroanilide (Travis and Johnson, 1981). After recombinant wild-type or mutant
1AT proteins had been incubated in 50 µl of elastase assay buffer (30 mM phosphate, 160 mM NaCl, 0.1% PEG 6000 and 0.1% Triton X-100, pH 7.4) with 20 nM porcine pancreatic elastase at 37°C for 10 min, the reaction mixture was diluted 10-fold with the assay buffer and the residual proteolytic activity was determined using 1 mM N-succinyl-(Ala)3-p-nitroanilide as a substrate for porcine pancreatic elastase.
The concentration of bovine -chymotrypsin was determined by measuring the initial rates of hydrolysis of 0.1 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. After recombinant wild-type or mutant
1ACT proteins had been incubated in 50 µl of chymotrypsin assay buffer (100 mM TrisCl, 10 mM CaCl2 and 0.005% TritonX-100, pH 7.8) with 20 nM bovine
-chymotrypsin at 37°C for 10 min, the reaction mixture was diluted 10-fold with the assay buffer and the residual proteolytic activity was determined using 0.1 mM N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate.
Thrombin activity was measured using 0.1 mM Chromozym-TH as a substrate. Partially purified recombinant ATIII was mixed with 25 ng of heparin and incubated with 20 nM human plasma thrombin at 37°C for 10 min in ATIII assay buffer (0.1 M TrisCl, 0.12 M NaCl, 0.01% sodium azide and 0.1% bovine serum albumin, pH 8.0). The reaction was diluted 10-fold with the assay buffer and the remaining proteolytic activity was measured using 0.1 mM Chromozym-TH as a substrate.
Thermal deactivation of recombinant serpins
1AT and
1ACT proteins were expressed in M9ZB medium at 37°C as a soluble form. Cells corresponding to A600
1 were harvested by centrifugation and resuspended in 500 µl of buffer (50 mM TrisCl, 50 mM NaCl, pH 8.0). Cells were disrupted by sonication using a W-380 sonicator (Heat Systems, New York) and insoluble materials were removed by centrifugation. Soluble fractions containing recombinant
1AT or
1ACT proteins were assayed for the inhibitory activity against porcine pancreatic elastase or bovine
-chymotrypsin, respectively. Amounts of
1AT or
1ACT proteins, which barely inhibit 90% of the target protease used, were included in each assay, so that subtle changes in serpin activity could be easily monitored. It was previously shown that, at least up to 50 µg/ml
1AT, the concentration dependence of polymerization was negligible in urea-dependent equilibrium unfolding and refolding experiments (Kwon et al., 1994
). Since all mutant
1AT proteins retained comparable inhibitory activity and concentration ranges of active inhibitors (
0.8 µg/ml) used in this study were far below 50 µg/ml, it is not likely that some concentration difference between mutant proteins would seriously modify the kinetics of thermal inactivation. The thermal stability of recombinant
1AT and
1ACT proteins was measured by following the kinetics of inactivation at 53 and 52°C, respectively. Samples were taken at various time points during thermal deactivation and the remaining inhibitory activity was determined as described above.
Thermal stability of the partially purified recombinant ATIII proteins, in the absence and presence of added heparin, was measured by following the kinetics of inactivation at 59 and 64.5°C, respectively. Aliquots of samples were taken along the time course of inactivation and the remaining ATIII activity was determined as described above.
Structural examination of native serpins
The crystal structure of 1AT was from the Protein Data Bank with a coordinate number 2PSI (Elliott et al., 1998
) and 1ATU (Ryu et al., 1996
). The native structure of
1ACT was kindly provided by D.Christianson (University of Pennsylvania). The active ATIII structure in heterodimeric form was from the Protein Data Bank with a coordinate number 1ANT (Carrell et al., 1994
). Structures were examined using the InsightII program (MSI).
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Results and discussion |
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To examine whether common themes are applicable to relieve the native strain of inhibitory serpins, single amino acid substitutions that increased the conformational stability of 1AT were introduced at equivalent positions of
1ACT and ATIII. Especially, substitutions that stabilize
1AT by >1 kcal/mol were chosen. Mutant serpins were expressed and their thermostability was measured through heat deactivation. Figure 2 shows typical heat deactivation results for stable serpin variants. All mutant
1AT proteins, which increased conformational stability presumably by compensating various structural defects of
1AT (Seo et al., 2000
), greatly increased the half-life in heat deactivation at 53°C (Tables I and II; Figure 2A). Especially a cavity-filling mutation at Ala142 and a size-decreasing substitution at Lys335 showed profound effects on thermostability, shifting the half-life upon heat deactivation from 17 min (wild-type
1AT) to >400 min at this temperature. These substitutions increased the conformational stability of
1AT by 2.4 and 6.5 kcal/mol, respectively (Im et al., 1999
; Seo et al., 2000
). Other substitutions also increased the thermal stability of
1AT significantly (222-fold). The thermostability of mutant
1ACT, in which the same kinds of substitutions were introduced at the equivalent positions of
1ACT, was measured by heat treatment at 52°C (Figure 2B). Most mutant proteins showed increased thermostability compared with the wild-type
1ACT (Table I; Figure 2B). For example, cavity-filling substitution at Ala140, in addition to the size-decreasing mutation at Lys335, showed a dramatic increase in thermostability, as in the case of
1AT.
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Inducing native strain in ATIII by reverse engineering
A great stabilizing effect was obtained by substitutions at the residue 142 site (residue number of 1AT): size increases by A142V of
1AT and A140V of
1ACT increased the thermostability of these serpins >25-fold (Table I). Ala142 forms a cavity with surrounding residues in
1AT and
1ACT and filling the cavities at these sites appeared to increase the thermostability. ATIII already has a larger residue, Leu170, at the equivalent position. To confirm the stabilizing effect of cavity-filling substitutions at this position, ATIII was reverse engineered by introducing smaller residues. Size reduction from leucine to valine (valine was the stabilizing residue in
1AT and
1ACT) increased the half-life on thermal inactivation by 1.4- and 4.1-fold in the absence and presence of added heparin, respectively, presumably by allowing better packing at this site (Figure 3). However, substitution of a smaller residue, alanine, which is the wild-type residue in
1AT and
1ACT, diminished the thermal stability of ATIII: L170A mutation reduced the thermal half-life to 0.2- and 0.1-fold of the wild-type ATIII in the absence and presence of added heparin, respectively (Figure 3). The results confirmed that cavity-creating substitutions decreased the thermostability of ATIII. The results also suggest that the wild-type residue, Leu, of ATIII at the site appears to be suboptimally suitable for the site because size reduction to valine appears to allow better packing in the region. It appears that the existence of a cavity at this location is a structural design inducing the native strain of inhibitory serpins.
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Table I summarizes substitutions that showed increased thermostability in all serpins when the theme found with the 1AT study was tested. In the crystal structure of the native
1AT, the stabilizing mutation sites are composed of many unusual interactions, which appear to be the basis of the native strain (Im et al., 1999
; Seo et al., 2000
). From the current mutational studies, the following structural schemes were confirmed as strategies to increase the stability of the native serpin molecule.
Relief of side-chain overpacking
Substitutions at positions 51, 381 and 335 (residue numbers of 1AT) with smaller residues increased the thermostability in all three serpins examined (denoted S in Table I), possibly by releasing the native strain caused by overpacked side chains.
Cavity filling
Residues at positions 142 and 117 (residue numbers of 1AT) constitute internal cavities of
1AT and
1ACT (denoted C in Table I) and filling the cavities by substitutions at these sites increased the thermostability. ATIII, which showed inherent higher thermostability, already has larger side chains at the equivalent sites, Leu170 and Arg145. Likewise, Thr114, Gly164 and Thr102 constitute surface pockets with surrounding residues (denoted SP in Table I). Substitutions by larger residues at these sites increased the thermostability of
1AT and
1ACT. Substitution of Ser142 of ATIII (equivalent to Thr114 of
1AT) with isoleucine also increased stability. The other two surface pockets near Gly164 and Thr102, observed in
1AT and
1ACT, are already filled with Lys193 and Arg129 at the equivalent sites of ATIII.
Others
In the case of S381A and K335A, removal of the unpaired polar groups in hydrophobic environments may contribute to the increased thermostability in all serpins tested (denoted Po in Table I). In the case of Thr68 of 1AT, substitution with alanine increased the thermostability.
1ACT and ATIII already have alanine at the equivalent site. Buried polar groups have been recognized as a means of inducing metastable native structures in other proteins such as influenza A virus, arrestin and the spike glycoprotein of influenza C virus (Chen et al., 1998
; Rosenthal et al., 1998
; Hirsch et al., 1999
). Increased backbone freedom is another theme of stabilization mode in inhibitory serpins. Ala70 of
1AT (equivalent to Asn68 of
1ACT and Asn96 of ATIII) is located at the turn between helix B (hB) and helix C (hC) and substitution with a flexible small residue, glycine, greatly increases the thermostability of the serpin. The existence of flexible glycine at the turn seems to allow better packing of nearby residues, including those in the following helices. Introduction of favorable ionic interactions (denoted I in Table I) may be another means of stabilization. Substitution with arginine at the Thr102 site of
1AT and Thr100 of
1ACT might provide positive charges to neighboring Glu376 and Glu98 of
1AT and Asp384 of
1ACT. ATIII already has arginine at the equivalent site.
Substitutions showing distinct effects among inhibitory serpins
Some substitutions that increased the thermostability of 1AT did not show stabilizing effects when introduced at the equivalent positions of
1ACT or ATIII. Table II summarizes such substitutions. Structural examination shows distinct side-chain interactions at these sites among inhibitory serpins.
Common strain in 1AT and
1ACT but not in ATIII
Some structural strains exist common in
1AT and
1ACT but not in ATIII. Thr113 of
1AT (Ser111 of
1ACT) is located in a surface hydrophobic pocket and substitution with isoleucine at this site stabilized
1AT possibly by filling the surface cavity. However, ATIII has valine at the equivalent site, which is well packed without a surface pocket. Introduction of a larger residue at this position of ATIII destabilized the molecule. Phe189 of
1AT (Phe187 of
1ACT), located on s3A (the third strand of A ß-sheet), is unfavorably overpacked underneath hF and substitutions with smaller, aliphatic side chains increased the thermal stability. However, Tyr220 of ATIII is well packed and there is no unfavorable polarnon-polar interaction with surrounding residues. Substitution to a smaller residue, valine, at this site of ATIII causes destabilization, possibly by introducing a cavity. Overall, ATIII is better packed, compared with
1AT or
1ACT, and it has fewer cavities and surface pockets. The relative lack of cavities and surface pockets in ATIII would explain the moderate native metastability of this serpin.
Strains unique in 1AT
Some unfavorable interactions exist only in
1AT. Leu172 of
1AT is pointing towards A ß-sheet and Lys335 is unfavorably overpacked underneath Leu172 (Figure 4). A size decrease at Leu172 greatly increased the thermostability of
1AT (Table II). Unlike the side chain of Leu172 of
1AT, the side chains of Leu170 of
1ACT and Val201 of ATIII are positioned toward helix F (hF) and interact with Lys160 (3.52 Å) and Val190 (3.73 Å) of hF, respectively (Figure 4). Hence substitution with a residue with a smaller side chain at Leu170 of
1ACT and Val201 of ATIII may reduce favorable interactions with hF and not bring hF and the following turn closer to A ß-sheet. Likewise, microenvironments near Leu286, Ala248 and Lys387 of
1AT are distinct from those of
1ACT and of ATIII. For instance, ATIII has a large residue, isoleucine, at the 279 residue site (equivalent to Ala248 of
1AT). It also has Arg425 (at the equivalent site to Lys387 of
1AT), which suitably fits the site and exposes a positive charge to solvent.
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Two-thirds of the substitutions (12 out of 18) increased thermostability in all serpins tested (Figure 1; Tables I and II). Substitutions at two sites (residues sites 113 and 189 of 1AT) among the other six sites increased thermostability in
1AT and
1ACT but not in ATIII (Table II). Only the substitutions at four sites did not show a thermostability increase in other serpins when the theme found in the
1AT study was applied (Table II). These results suggest that inhibitory serpins are suboptimally folded using common structural strategies at many sites, even though some structural details could vary in individual serpins. The overall packing density (0.67) of the metastable
1AT structure is similar to those of common globular proteins (Rashin et al., 1986
), but local structures of
1AT are made of a mosaic of many over- and under-packed regions (Lee et al., 2001
). The existence of cavities and over-packed regions seems to be a unique structural feature in strained serpin molecules including
1AT. The results of this study suggest that rules found for
1AT can be applied to increase the thermostability of inhibitory serpins. Activity-affecting mutations are highly localized in the regions that are mobilized during the complex formation and stabilizing mutations at other sites did not affect activity. Increased thermostability based on enhanced kinetic stability against heat-induced aggregation has practical implications. The formation of inactive oligomers of inhibitory serpins is frequently found in many pathological, denaturing conditions or upon storage (Stein and Carrell, 1995
). Information obtained in this study, including understanding of the packing modes and stability of serpins, will be a valuable guide in designing and modulating conformational characteristics of the molecules. Especially suppression of heat-induced polymerization of clinically important serpins would contribute to the development of therapeutic serpins suited to long-term storage and administration.
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Acknowledgments |
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Received December 31, 2003; revised April 13, 2004; accepted April 13, 2004.
Edited by Andreas Matouschek