©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Thermostable Mutation Located at the Hydrophobic Core of -Antitrypsin Suppresses the Folding Defect of the Z-type Variant (*)

Jeongho Kim (§) , Kee Nyung Lee , Gwan-Su Yi (¶) , Myeong-Hee Yu (**)

From the (1) Protein Engineering Group, Genetic Engineering Research Institute, Korea Institute of Science and Technology, Taejon 305-333, Korea

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A thermostable mutation, F51L, at the hydrophobic core of human -antitrypsin (AT) increased the conformational stability of the molecule by decreasing the unfolding rate significantly without altering the refolding rate. The mutation specifically influenced the transition between the native state and a compact intermediate, which retained 70% of the far-UV CD signal, but which had most of the fluorescence signal already dequenched. The mutant AT protein was more resistant than the wild-type protein to the insertion of the tetradecapeptide mimicking the sequence of the reactive center loop, indicating that the mutation increases the closing of the central -sheet, the A-sheet, in the native state. The F51L mutation enhanced the folding efficiency of the Z-type (E342K) genetic variation, which causes aggregation of the molecule in the liver. It has been shown previously that the aggregation of the Z protein occurs via loop-sheet polymerization, in which the reactive center loop of one molecule is inserted into the opening of the A-sheet of another molecule. Our results strongly suggest that the hydrophobic core of AT regulates the opening-closing of the A-sheet and that certain genetic variations that cause opening of the A-sheet can be corrected by inserting an additional stable mutation into the hydrophobic core.


INTRODUCTION

-Antitrypsin (AT)() is a member of the serine protease inhibitor (serpin) family, which shares a common tertiary structure composed of three -sheets and several -helices (1, 2) . Two salient features of the serpin structure are the stress of the native conformation and the mobility of the reactive center loop, both of which are considered to be critical for biological function. Inhibitory serpins have a stressed conformation in which the reactive center loop is open to proteolytic cleavage. The cleavage accompanies an irreversible transition to a very stable relaxed form in which the newly created N-terminal portion of the cleaved loop is completely inserted as a strand of the major -sheet, the A-sheet (2, 3, 4) . Structural comparison of various forms of serpins and thermostability studies of serpins complexed with synthetic peptides carrying sequences of the reactive center loop (5, 6, 7, 8) suggest that the enhancement in stability appears to be mainly due to insertion of the loop into the A-sheet, leading to an increase in the number of strands in the sheet and the buried surface area. However, studies have indicated that the stress of the native form of AT is not limited to the reactive center loop or the A-sheet, but may be propagated throughout the molecule (9, 10) . The structural mechanism of the stress including the distribution is not yet understood.

The reactive center loop of the serpins is unusually long and is able to adopt various conformations (10, 11, 12, 13, 14, 15) . Several biochemical studies and recent structural determinations show that the reactive center loop of the active serpin is mobile and partially inserted into the central A-sheet (7, 14, 15, 16, 17) . On the contrary, no insertion of the loop residue was observed with the active form of -antichymotrypsin (10) . Although these apparent contradicting data may argue for a distinctive inhibition mechanism (18, 19, 20) , the loop mobility postulated by Carrell et al. (7) was confirmative.

The ability of the reactive center loop to adopt various conformations is associated with opening of the A-sheet, in which a partial or complete insertion takes place. Some factors that may regulate A-sheet opening have been identified by structural comparison between the cleaved form of AT and a noninhibitory serpin, ovalbumin (5) . Others have been identified by studies on some genetic variations of AT that cause aggregation of the molecule in the liver. It was suggested that the Z (E342K) or S(S53F) mutation of AT enhances loop-sheet polymerization by opening the A-sheet more readily and converting the molecule into a better acceptor for the loop insertion from another molecule (8, 21, 22) . Since the loop insertion into the A-sheet always accompanies an increase in stability, it is likely that the native stress of an inhibitory serpin is associated with A-sheet opening and consequently with loop mobility. However, the structural mechanism of the link between native stress and opening of the A-sheet remains unclear.

Previously, we identified single amino acid substitutions in native human AT that confer increased thermal and conformational stability, but that maintain inhibitory activity (23) . The substitution of Phe-51 with hydrophobic aliphatic residues reduced heat-induced aggregation, and it is likely that the mutation diminished loop-sheet polymerization. If this is the case, the defect of some genetic variants of AT that cause loop-sheet polymerization may be suppressed by the thermostable mutation. In the present study, a stable mutation, F51L, was further characterized, and its effect on the Z or Sgenetic variation was examined. Since the Phe-51 mutation is located at the hydrophobic core of the molecule, the present study addresses the question regarding the link between the stability conferred by the hydrophobic core and the A-sheet opening of the AT molecule.


MATERIALS AND METHODS

Recombinant AT Proteins and Plasmids

Recombinant wild-type and F51L mutant AT proteins produced in Escherichia coli were purified from inclusion bodies after refolding and ion-exchange chromatography as described previously (23) . Concentrations of AT were determined in 6 M guanidine hydrochloride using a value of A1 cm1% = 4.3 at 280 nm, calculated from the tyrosine and tryptophan content of the AT protein (24) and based upon M= 44,250. The Z-type (E342K) or S(S53F) mutation or its combination with the F51L substitution was introduced into the AT cDNA by oligonucleotide-directed mutagenesis (25) . The mutagenic oligonucleotide was a 26-mer, and the mismatched codon was located in the middle. For in vitro translation, the AT expression vector pF(BLG)AT was constructed in which the AT cDNA was connected downstream of the T7 promoter and the 5`-untranslated region of the rabbit -globin gene.

Chemicals

Ultrapure urea was purchased from Schwarz/Mann. [S]Methionine was purchased from DuPont NEN. All other chemicals were reagent-grade. The buffer used for folding experiments was 10 m M phosphate, 50 m M NaCl, 1 m M EDTA, and 1 m M -mercaptoethanol (pH 6.5).

Urea-induced Equilibrium Unfolding Transition

Equilibrium unfolding as a function of urea was monitored by fluorescence spectroscopy and CD spectroscopy. The native protein was incubated in 10 m M potassium phosphate, 50 m M NaCl, 1 m M EDTA, 1 m M -mercaptoethanol, and various concentrations of urea (final pH 6.5) at 23 °C. Samples were allowed to equilibrate for 8 h. Tryptophan fluorescence was measured for each sample (Shimadzu RF-5000 fluorescence spectrophotometer) with excitation at 280 nm and emission at 360 nm. The CD signal was recorded (Jasco J-600) at 222 nm at 23 °C in a cell with a 0.1-cm path length and a 1-nm bandwidth. The protein concentration for the unfolding transition was 10 µg/ml for fluorescence spectroscopy and 50 µg/ml for CD spectroscopy. Experimental data of fluorescence measurement were fitted to a two-state unfolding model as described previously (23) .

Kinetics of Folding and Unfolding

AT in the native or unfolded state was subjected to structural change in various concentrations of urea, and the time required to reach a new equilibrium was analyzed by monitoring the fluorescence intensity (= 280 nm and = 360 nm). The final protein concentration was 10 µg/ml. For refolding, the native protein was unfolded in 7 M urea for 5 min, and refolding was initiated by adding the unfolded protein to the designated urea concentrations. The kinetic data were collected after manual mixing with a dead time of 15 s on average. The major kinetic phase was examined, and the data were fitted to a two-state model to obtain the relaxation time: F( t) = F( i) e

Transverse Urea Gradient Gel Electrophoresis

Gels were prepared with a gradient of 0-8 M urea perpendicular to the direction of electrophoresis with an opposing gradient of acrylamide from 15 to 11% (26) . Four slab gels (100 80 mm) were prepared simultaneously in a multigel caster (Hoefer Scientific Instruments) by using a gradient maker and a single-channel peristaltic pump. The electrode buffer was 50 m M Tris acetate, 1 m M EDTA (pH 7.5). The native protein (20 µg in 100 µl), the protein unfolded in 8 M urea (10 min at room temperature), or the in vitro translation product was applied across the top of the gel. The gels were run at a constant current of 6 mA for 3 h at a controlled temperature of 23 °C.

In Vitro Translation of Cloned Genes

In vitro transcription was performed with the SP6/T7 transcription system (Boehringer Mannheim) at 37 °C for 1 h. In vitro translation was performed with a rabbit reticulocyte lysate system (Promega). A typical translation reaction mixture (final volume of 25 µl) contained 20 µCi of [S]methionine, 0.5 µg of mRNA, and 17.5 µl of reticulocyte lysate treated with micrococcal nuclease. The labeling reaction was carried out at 30 °C for 1 h and was chased with unlabeled methionine (2 m M).

Binary Complex Formation

The binary complex of the wild-type and F51L AT proteins with the tetradecapeptide of the reactive center loop sequence (Ac-Thr-Glu-Ala-Ala-Gly-Ala-Met-Phe-Leu-Glu-Ala-Ile-Val-Met-OH) was formed by incubating the native protein (0.24 mg/ml) in 10 m M phosphate, 50 m M NaCl, and 1 m M EDTA (pH 6.5) with a 230-fold molar excess of the peptide at 40 °C for various period up to 6 h. The degree of complex formation was determined by 13.5% polyacrylamide gel electrophoresis in the absence of urea. The buffer system was identical to the urea gradient gel electrophoresis system described above.


RESULTS

Effect of the F51L Mutation on Thermodynamic, Kinetic, and Structural Aspects of the Conformational Transition of AT

A previous study on urea-induced reversible unfolding of AT showed that the F51C mutation increased conformational stability toward the denaturant in a concentration-independent manner (23) . The thermostable substitution at Phe-51 was further characterized using the F51L mutation as a prototype to avoid possible complications involved with the cysteine residue in the F51C mutation. The F51L mutation also increased stability toward urea as demonstrated by the equilibrium unfolding transitions monitored by fluorescence and CD signals (Fig. 1, A and B). Although the transition monitored by the fluorescence signal was fitted well to the two-state unfolding model (Fig. 1 A) to give midpoints of transition at 1.9 ± 0.1 and 2.8 ± 0.1 M urea for the wild-type and mutant proteins, respectively, the transition monitored by the CD signal (Fig. 1 B) indicates that the unfolding of AT is multiphasic. The increase in equilibrium stability by the F51L mutation was also observed in the transverse urea gradient gel electrophoresis. Fig. 2shows that two major transitions of AT were detected during unfolding and refolding of AT in urea. A major effect of the F51L mutation was a shift in the transition point to a higher urea concentration in the initial unfolding step, which was also confirmed in the refolding transition. The midpoints of the transition of the wild-type and F51L mutant proteins were approximately 2 and 3 M urea, respectively. It appears that the mutation specifically influenced the initial stage of unfolding and had no effect on the later unfolding transition at a higher urea concentration.


Figure 1: A, urea-induced unfolding of the wild-type ( WT; ) and F51L mutant () recombinant AT proteins as measured by the increase in fluorescence emission intensity at 360 nm (= 280 nm; excitation and emission slit widths of 5 nm for both). Samples were equilibrated for 8 h in 10 m M potassium phosphate, 50 m M NaCl, 1 m M EDTA, 1 m M -mercaptoethanol (pH 6.5), and various concentrations of urea at 23 °C. The protein concentration was 10 µg/ml. The transition midpoints of the wild-type and F51L mutant proteins were 1.9 ± 0.1 and 2.8 ± 0.1 M, respectively, as determined by fitting the data to a two-state unfolding model. B, urea-induced unfolding of the wild-type () and F51L mutant () recombinant AT proteins as measured by the change in the CD signal. The loss of CD ellipticity at 222 nm was monitored using a 0.1-cm path length cell as a probe of structural change in the AT protein. The protein concentration was 50 µg/ml.




Figure 2: Transverse urea gradient gel electrophoresis of the wild-type ( WT) and F51L AT proteins. The native protein ( Unfolding) or the protein unfolded in 8 M urea ( Refolding) was applied across the top of the gel. Electrophoresis was carried out at 6 mA of constant current for 3 h at 23 °C. Protein bands were visualized by staining with Coomassie Blue.



There are two ways of increasing the free energy of stabilization of a protein: by stabilizing the native state or by destabilizing the unfolded state. Kinetic studies of unfolding and refolding monitored by the fluorescence signal revealed that the F51L mutation retarded the unfolding rate substantially, while the refolding rate was not affected by the mutation (Fig. 3). This result indicates that the mutation increased the free energy of stabilization by decreasing the energy level of the native state rather than by increasing the energy level of the partially unfolded conformation.


Figure 3: Kinetics of unfolding and refolding of the wild-type ( and ) and F51L ( and ) recombinant AT proteins. The relaxation time of urea-dependent refolding ( and ) and unfolding ( and ) monitored by the fluorescence signal was plotted. AT in the native or unfolded state was subjected to structural change in various concentrations of urea, and the time to reach a new equilibrium was analyzed by monitoring the fluorescence intensity (= 280 nm and = 360 nm). The final protein concentration was 10 µg/ml. For refolding, the native protein was unfolded in 7 M urea for 5 min, and refolding was initiated by the addition of the unfolded protein to urea at the designated concentrations.



The transition influenced by the mutation in urea denaturation may correspond to the transition from the native to the open conformation, in which the major sheet, the A-sheet, is able to accept the reactive center loop of another molecule. To test this, the complex formation was examined between the native AT protein and the tetradecapeptide that mimics the reactive center loop sequence. Fig. 4 shows that the kinetics of the complex formation with the peptide was retarded by the F51L mutation, indicating that the mutant protein is more resistant to the peptide insertion.

The F51L Substitution Increases the Folding Efficiency of the Z-type Variant

If the F51L mutation truly enhances closing of the A-sheet, it might compensate for the defect of those genetic mutations that induce A-sheet opening. The effect of the F51L mutation on Z- or S-type AT was examined by monitoring the folding transition on urea gradient gel electrophoresis with in vitro translation products (Fig. 5). Most of the nascent Z or Spolypeptides were accumulated at the intermediate state. However, on combination with the F51L mutation, part of the population of the Z type, but not the Stype, could fold into the native state. The gel patterns of the wild-type and F51L AT proteins synthesized in vitro were very similar to those of the purified proteins (Fig. 2), indicating that most of these translation products are successfully folded. The results show that the folding defect of Z-type AT was partially relieved by the F51L mutation. The defect caused by the Smutation was not suppressed by the F51L mutation.


Figure 5: Effect of the F51L mutation on the defect of Z and S variants. The translation products of AT labeled with [S]methionine for 1 h carrying the indicated mutations were analyzed by urea gradient gel electrophoresis. The protein bands were visualized by autoradiography.




DISCUSSION

In an effort to understand the relationship between the stress of the inhibitory serpins and the tertiary fold of the molecule, we previously identified a stable but active variant (Phe-51 aliphatic hydrophobic residues) of human AT, which mapped on the B- sheet in the hydrophobic core of the molecule (23) . The existence of such a mutation supports the notion that the stress of the native serpin is not limited to the A-sheet, but may be distributed over the molecule (9, 10) .

Stability Increase in AT by Enhancing the A-sheet Closing from the Hyrophobic Core

What is the nature of the transition influenced by the F51L mutation, which was demonstrated in the unfolding transition monitored by fluorescence and CD spectroscopies and urea gradient gel electrophoresis (Figs. 1 and 2)? The equilibrium unfolding of AT was multiphasic, involving at least two slow steps, although the fluorescence signal could detect only the initial unfolding that fits to the two-state model. The F51L mutation shifted the first unfolding transition to a higher urea concentration. Various data indicate that this transition reflects unfolding of native AT to an open conformation, in which the major sheet (A-sheet) is open to be able to accept the loop insertion. It was shown that loop-sheet polymerization could be induced with wild-type AT under mild denaturing conditions (7) . In addition, the thermostable mutation that shifted this transition (Figs. 1 and 2) was shown previously to retard the aggregation rate (23) . The transition midpoints of the wild-type and mutant AT proteins in the urea gradient gel system were approximately 2 and 3 M, respectively, which are close to those obtained by fluorescence spectroscopy (Fig. 1 A). Trp-194, the major contributor of the signal among two tryptophans (27, 28) , is likely to be a sensitive probe for the A-sheet opening because it is located at the top of strand 3 of the A-sheet, being hydrogen-bonded with residues in strand 5 of the A-sheet both in the intact cleaved (2) and model intact (29) AT proteins. Finally, the nascent polypeptides carrying the Z or Smutation, which causes loop-sheet polymerization in vivo (21, 22) , were arrested at this intermediate state (Fig. 5). Although recent structural determination of antithrombin dimer suggested another kind of polymerization with the C-sheet (14, 15) , C-sheet polymerization of AT has not been reported. These data indicate that the folding intermediate detected in the present study is likely to be an open state of the A-sheet, slightly less compact than the native form, with tryptophan fluorescence being fully dequenched, but containing 70% of the native far-UV CD signal.

The effect of the F51L mutation appears to be specific to the native state. Kinetic data in Fig. 3 show clearly that the F51L mutation increased the thermodynamic stability of AT by lowering the energy level of the native state, indicating that the native state of the molecule is specifically changed by the F51L mutation. In addition, the native fluorescence intensity of the F51L mutant protein was lower than that of the wild-type protein for the same amount of protein, while the unfolded values for both were similar (Fig. 1 A), indicating that the native state of the F51L mutant protein is more packed near Trp-194. If the native state of the F51L mutant AT protein is more stably closed in the A-sheet than that of the wild-type protein, it would be more resistant to the insertion by the peptide of the reactive center loop sequence. The complex formation experiment in Fig. 4 shows that this is indeed the case, suggesting strongly that the change in the A-sheet opening is one of the physical changes caused by the F51L mutation. In the crystal structure of cleaved AT (2) , Phe-51 is located in strand 6 of the B-sheet in the hydrophobic core, interacting not only with conserved hydrophobic residues (Phe-372 and Phe-384) in strands 5 and 4 of the B-sheet, but also with some residues (Ala-336 and Leu-338) in strand 5 of the A-sheet. The mutations at Phe-51 may regulate the opening-closing of the A-sheet through interactions with some of these residues. By decreasing the side chain volume (23) , the stable mutations may provide a more favorable packing in the native form of mutant proteins.


Figure 4: Complex formation of the wild-type ( WT) and F51L AT proteins with a peptide of the reactive center loop sequence. The native AT protein was incubated with a 14-mer peptide (Ac-TEAAGAMFLEAIVM-OH) in 10 m M phosphate (pH 6.5), 50 m M NaCl, and 1 m M EDTA (protein concentration of 0.24 mg/ml; protein/peptide ratio of 1:230; total volume of 10 µl) at 40 °C for 0-6 h. The reaction samples were analyzed by native gel electrophoresis. The gel electrophoresis condition was as described for Fig. 2, except that the gel contained 13.5% polyacrylamide without any urea.



Suppression of the Z-type Folding Defect by the F51L Substitution

The proposed mechanism for the F51L mutation is exactly opposite of that postulated for the genetic defect of the Z (E342K) or S(S53F) mutation, which was attributed to the opening of the A-sheet (21, 22) . A major defect of these genetic mutations is the arrest of the final folding of the nascent chains from the intermediate to the native state (Fig. 5). Upon combination with the F51L mutation, however, some of the nascent Z polypeptides, but not the Spolypeptides, could fold into the native state. The folding arrest could be due to a destabilization of the mutant native protein or alternatively to a kinetic retardation of folding. The Z type could fold into the native form, although very slowly, with a folding rate on the order of several hours.() The F51L mutation allowed part of the population of the Z polypeptides to fold properly, while the rest of the Z polypeptides remained at the intermediate state. It appears that competition exists between the productive folding pathway and a kinetic trap: while the Z mutation induces a partitioning into the folding trap, a more favorable partitioning into the productive pathway is induced by the F51L mutation. It is likely that the F51L mutation, by shifting the equilibrium toward the production of the native form, decreased the amount of the intermediate that can be trapped.

The defect of the Smutation could not be suppressed by the F51L mutation. This may be because the conformational stability of the Sprotein is drastically affected. The Smutation occurs at the N terminus of the B-helix that is comprised in the hydrophobic core of the molecule. Replacement of Ser-53, which initiates the helix and the side chain Ohydrogen-bonded to the backbone nitrogen of Ser-56 (2) , with bulky phenylalanine is likely to have a drastic effect on the stability. The results in Fig. 5are consistent with the Smutation causing a more severe aggregation of the molecule than the Z-type mutation (22) . Our results suggest that some, although not all, genetic variations that cause a secretion block by enhancing the A-sheet opening can be corrected by inserting an appropriate stable mutation at the hydrophobic core. Indeed, a preliminary investigation of the yeast secretion system revealed that the secretion block of Z-type AT can be partially relieved by the F51L mutation.()

The F51L mutation site does not directly interact with the Z mutation site (at the tip of stand 5 of the A-sheet), which is likely to be a critical site for partial insertion of the loop into the A-sheet (2) . Our results support that structural features of the hydrophobic core are tightly linked during folding to A-sheet closing and consequently to loop movement. Identification of more sites that are critical for the A-sheet closing of AT will be essential for understanding the mechanism of the native stress of the serpins.


FOOTNOTES

*
This work was supported by Grant G71142 from the Korean Ministry of Science and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Laboratory of Bioorganic Chemistry, NIDDK, NIH, Bethesda, MD 20892.

Present address: Magnetic Resonance Laboratory, Korea Basic Science Center, Taejon 305-333, Korea.

**
To whom correspondence and reprint requests should be addressed. Tel.: 82-42-860-4140; Fax: 82-42-860-4593.

The abbreviations used are: AT, -antitrypsin; serpin, serine protease inhibitor.

M.-H. Yu, K. N. Lee, and J. Kim, submitted for publication.

H. A. Kang and M.-H. Yu, unpublished results.


ACKNOWLEDGEMENTS

We are grateful to Dr. Soon Jae Park for peptide synthesis and to Dr. Jonathan Weissman for the gift of the pF(BLG) vector.


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