Báo cáo khoa học: The loss of tryptophan 194 in antichymotrypsin lowers the kinetic barrier to misfolding

The Loss of Tryptophan 194 in Antichymotrypsin Lowers the Kinetic Barrier to Misfolding

Tên đề tài: The loss of tryptophan 194 in antichymotrypsin lowers the kinetic barrier to misfolding

Tác giả: Mary C. Pearce, Lisa D. Cabrita, Andrew M. Ellisdon and Stephen P. Bottomley

Lĩnh vực: Biochemistry and Molecular Biology

Nội dung tài liệu: Nghiên cứu này khảo sát vai trò của tryptophan tại vị trí 194 (W194) trong cấu trúc của antichymotrypsin (ACT) đối với sự ổn định và quá trình polymer hóa. Các thí nghiệm cho thấy việc thay thế W194 bằng phenylalanine đã làm giảm đáng kể rào cản động học đối với quá trình gấp sai và tăng tốc độ hình thành polymer. Điều này gợi ý rằng W194 đóng vai trò quan trọng trong việc duy trì sự ổn định của vùng “breach” trong ACT, từ đó ngăn ngừa sự polymer hóa.

Mục lục chi tiết:

• Keywords
• Correspondence
• (Received 21 November 2006, revised 17 May 2007, accepted 23 May 2007)
• doi:10.1111/j.1742-4658.2007.05897.x
• Antichymotrypsin, a member of the serpin superfamily, has been shown to form inactive polymers in vivo, leading to chronic obstructive pulmonary disease. At present, however, the molecular determinants underlying the polymerization transition are unclear. Within a serpin, the breach position is implicated in conformational change, as it is the first point of contact for the reactive center loop and the body of the molecule. W194, situated within the breach, represents one of the most highly conserved residues within the serpin architecture. Using a range of equilibrium and kinetic experiments, the contribution of W194 to proteinase inhibition, stability and polymerization was studied for antichymotrypsin. Replacement of W194 with phenylalanine resulted in a fully active inhibitor that was destabilized relative to the wild-type protein. The aggregation kinetics were significantly altered; wild-type antichymotrypsin exhibits a lag phase followed by chain elongation. The loss of W194 almost entirely removed the lag phase and accelerated the elongation phase. On the basis of our data, we propose that one of the main roles of W194 in antichymotrypsin is in preventing polymerization.
• The serpins (serine proteinase inhibitors) are a large family of metastable proteins with a highly conserved tertiary structure. Metastability of the serpin native state facilitates the rapid structural changes required for efficient inhibition of a range of serine and cysteine proteinases [1]. During inhibition, the exposed flexible reactive center loop (RCL), which acts as bait for the proteinase (Fig. 1), becomes incorporated into the A β-sheet following cleavage by a proteinase. The A β-sheet is composed of five strands, which are referred to as s1A, s2A, etc. This structural rearrangement results in translocation of the proteinase from one pole of the molecule to the other, deforming the covalently bound proteinase against the base of the serpin and thus preventing efficient hydrolysis of the ester bond linking the proteinase and inhibitor [2-4].
• Insertion of the RCL into the body of the serpin, during proteinase inhibition, creates a molecule that is considerably more thermodynamically stable than its initial native state [5]. This energetically favorable conformational change can, however, also occur in the absence of a proteinase to form a variety of misfolded states [6]. In some serpins, the RCL becomes completely incorporated into the central β-sheet while remaining intact, with concomitant disruption of the C β-sheet, which results in the stable, inactive latent state [7]. Several serpins have also been shown to form inactive polymers when the RCL of one serpin inserts into a β-sheet of another [8,9]. This can lead to accumulation of the aggregated serpin both within the cells producing the protein and in the circulation. Polymerization of the plasma serpins, antitrypsin (α1-AT), antichymotrypsin (ACT) and antithrombin III, can be promoted by single point mutations and lead to many human diseases [10].
• Abbreviations
• ACT, antichymotrypsin; α1-AT, antitrypsin; bis-ANS, bis-8-anilinonaphthalene-1-sulfonate (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt); muACT, murine homolog of antichymotrypsin; PAI-1, plasminogen activator inhibitor-1; RCL, reactive center loop; SI, stoichiometry of inhibition; TEM, transmission electron microscopy; TUG, transverse urea gradient.
• The serpin architecture can be split into regions that either deform or remain intact during proteinase inhibition and misfolding [11]. One such critical region that undergoes deformation during inhibition is the ‘breach’, an area that encompasses the top of the A β-sheet and the N-terminus of the RCL (Fig. 1). The breach region is the first point of insertion for the RCL during inhibition, and therefore has evolved to readily accommodate this conformational change.
• Located at the top of the breach, on a loop connecting s3A and s3C, is the residue W194, which is present in over 95% of serpin sequences [12]. The structural and functional roles of this residue have been studied in various serpins, and the results indicate that replacing it with a phenylalanine has variable effects on the overall inhibitory or thermodynamic properties of the protein [13-15]. In plasminogen activator inhibitor-1 (PAI-1), the replacement of this highly conserved tryptophan residue reduced the propensity of the protein to adopt the latent state. This phenomenon has not been observed for any other member of the family. Taken together, these studies do not suggest any reason for the high level of conservation of tryptophan at this position within the family. Therefore, we reasoned that it might play a role in serpin polymerization, an area that has not been previously investigated for proteins carrying this mutation. We have mutated W194 to phenylalanine in ACT, and monitored the effect of this mutation upon inhibition, stability and polymerization. Our data indicate that W194 plays a critical role in maintaining the stability of the breach region and preventing polymerization of ACT.
• Results
• The ‘breach’ has been identified as a region possessing high structural flexibility in serpins, and many of the residues occupying this area are strongly conserved [12,16]. In addition, several disease states have been linked to serpins that contain mutations within this region [10]. In this study, we examined the role of the highly conserved breach residue, W194, in maintaining the structural and functional properties of ACT.
• ACTW194F was generated using site-directed mutagenesis to replace the intrinsic tryptophan at this location with phenylalanine. Both wild-type (wt)ACT and ACTW194F were expressed and purified as previously described [17]. Gel filtration chromatography was used to monitor the aggregation status of the protein after storage and before each experiment. In all the experiments described below, only active, monomeric protein was used. We attempted expression of ACT containing different mutations at position 194, such as Gly, Ile, Lys, Asp and Asn; in all cases, we were unable to purify any monomeric protein.
• Characterization of the inhibitory properties of ACTW194F
• The stoichiometry of inhibition (SI) values for inhibition of chymotrypsin by wtACT and ACTW194F were determined by titration of the proteinase with known concentrations of the serpin. The SI for ACTW194F was 1.4, slightly higher than that observed for wtACT (Table 1). The association rate constant (kass) for
• Fig. 1. Schematic representation of murine ACT homolog, Serpina3n. Ribbon diagram of native ACT, Protein Data Bank 1YXA [1], with W194 (black) and surrounding residues identified (gray). The inset is a close-up of the region around W194. A β-sheet is highlighted in gray.
• Table 1. Inhibitory and stability properties of wtACT and ACTW194F. SI, association rate constant (kapp) and apparent association rate constant (kapp) were determined as described in Experimental procedures. The rates of unfolding (KN→1 and k₁u) in 4 m guanidine hydrochloride were determined as described in Experimental procedures. Each value represents the average of five separate experiments; actual value and standard error are listed.
• wtACT
• ACTW194F
• SI
• 1.1 ± 0.02
• 1.4 ± 0.03
• kapp (x 105 M-1-s-1)
• 8.70 ± 0.58
• 6.39 ± 0.35
• kass (x 105 M-1.s-1)
• 9.59
• 9.22
• TUG transition (M urea)
• 2.2
• 0.8
• KN→1 (S-1)
• 14.45 ± 0.2
• ND
• Kiu (s-1)
• 3.6 ± 0.008
• 3.8 ± 0.005
• Fig. 2. SDS-PAGE analysis of complex formation between chymotrypsin and ACT. ACT and ACTW194F were mixed with chymotrypsin in a 2: 1 molar ratio and allowed to incubate for 30 min at 37 °C, following which the complexes were analyzed by 10% (v/v) SDS-PAGE. Arrows indicate the position of native ACT, cleaved ACT released by the proteinase, and ACT still in complex with chymotrypsin.
• inhibition of chymotrypsin by ACTW194F was similar to that of wtACT (9.22 × 105 M⁻¹.s⁻¹ and 9.59 × 105 M⁻¹·s⁻¹, respectively; Table 1). The ability of ACTW194F to form SDS-stable complexes with chymotrypsin was also examined by incubating 4 µm serpin with 2 µм chymotrypsin for 30 min at 37 °C. The resulting mixture was then analyzed by SDS/PAGE under reducing conditions (Fig. 2). Both wtACT and ACTW194F formed SDS-stable complexes with chymotrypsin, as indicated by the presence of a larger species (Fig. 2, labeled complex). In addition, a small amount of cleaved serpin was present (Fig. 2, labeled cleaved), which is consistent with the SI data. Taken together, these data suggest that the replacement of W194 with phenylalanine had no significant effect upon the inhibitory properties of the protein.
• Characterization of the spectral properties of ACTW194F
• Both fluorescence and far-UV CD spectra were obtained for wtACT and ACTW194F, in order to determine what structural differences may exist between them (Fig. 3). Far-UV CD spectra were obtained for both proteins in the folded and unfolded states (achieved by incubation in 6 M guanidine hydrochloride) at 25 °C and pH 7.8. The native spectra were identical, indicating that both proteins possess a similar fold and that W194 does not contribute to the far-UV spectrum of ACT (Fig. 3A).
• The unfolded spectra for both ACT proteins were also similar, implying that the mutation had no effect on the structure of the unfolded state of ACT (Fig. 3A). These data indicate that the replacement of W194 with phenylalanine did not alter the secondary structure of the variant.
• ACT has three intrinsic tryptophan residues, one of which was mutated to phenylalanine in ACTW194F. Fluorescence emission spectra were obtained for both wtACT and ACTW194F in their folded and unfolded states (Fig. 3B). In both cases, the fluorescence spectra represent the average of all the tryptophan residues in the protein. There was no difference in emission maxima (λmax) between wtACT and ACTW194F in the native state (λmax = 335 nm for both). There was, however, a significant difference in the emission intensity of both proteins. The emission intensity at the spectral peak for ACTW194F was 75% that of the wtACT value, indicating that in the native state W194 contributes 25% to the total tryptophan fluorescence signal of the wild-type form (Fig. 3B). Both proteins were unfolded in 5 M guanidine thiocyanate, as this denaturant is strong enough to completely unfold all structure within ACT [17]. There was no significant difference in the λmax for either unfolded protein. The fluorescence emission intensity of unfolded ACTW194F was, however, two-thirds the intensity of wtACT, which could be attributed to the loss of one of the three intrinsic tryptophan residues through the introduction of the mutation. Both unfolded proteins showed a dramatic decrease in fluorescence signal in comparison to the native protein. This is most likely attributable to the nature of the interaction between the protein and guanidine thiocyanate, as this denaturant readily quenches tryptophan fluorescence in solution.
• The fluorescence emission spectrum of bis-8-anilinonaphthalene-1-sulfonate (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt) (bis-ANS) when bound to proteins can provide information regarding the relative exposure of hydrophobic pockets/surfaces [18]. Figure 3C shows the emission spectra of bis-ANS in the presence of wtACT and ACTW194F. The emission intensity of bis-ANS in the presence of ACTW194F was two times greater than that of wtACT. This could be due to either a small conformational change within ACTW194F that altered the binding affinity of bis-ANS for ACT, or a more accessible hydrophobic core, due, for example, to larger conformational fluctuations in ACTW194F.
• Fig. 3. Spectral properties of wtACT and ACTW194F. In each figure, wtACT is represented by squares, and ACTW194F is represented by circles. (A) Far-UV CD spectrum of native (empty symbols) and guanidine hydrochloride-unfolded (filled symbols) ACT proteins. (B) Intrinsic tryptophan fluorescence emission spectra for native (empty symbols) and unfolded (filled symbols) ACT proteins. (C) Bis-ANS fluorescence emission spectra for wtACT and ACTW194F in the native conformation. Protein was present at 500 nm for each experiment, and each spectrum represents the average of five scans.
• The role of W194 in the stability of ACTW194F
• The stability of wtACT and its variant was determined by transverse urea gradient (TUG) PAGE analysis (Fig. 4). Both proteins demonstrated the characteristic unfolding profile seen for other serpins; in addition, some bands representing polymeric ACT were observed in higher denaturant concentrations. The unfolding transition for ACTW194F occurred at a significantly lower urea concentration (0.8 m) than that of wtACT (2.2 м) (Table 1). Owing to the formation of polymers in high urea concentrations, no spectroscopic equilibrium studies were performed.
• The unfolding kinetics of both wtACT and ACTW194F were determined using stopped-flow fluorescence. In order to directly compare the two proteins, which have different intrinsic fluorescence properties, we followed the unfolding reaction using the change in bis-ANS fluorescence. wtACT unfolded with a biphasic transition that corresponded to the presence of a folding intermediate, which is consistent with previous findings on this and other serpins [19]. bis-ANS fluorescence intensity increased during adoption of the intermediate ensemble, and this was followed by a decrease in fluorescence intensity as the rest of the
• Fig. 4. TUG PAGE analysis of ACT proteins. Both wtACT and ACTW194F were applied to a 0-5 м urea gradient gel, increasing from left to right. (A) wtACT and (B) ACTW194F formed polymers (P) in high denaturant concentrations. The final protein concentration was 10 µm, and each figure shows the best representative of three gels run for each protein.
• structure was lost (Fig. 5). The rates of unfolding, in 4 M guanidine hydrochloride, corresponding to the N to I (kN→1) and I to U (k₁ → u) transitions for wtACT, were 14.5 s⁻¹ and 3.6 s⁻¹, respectively (Table 1). When ACTW194F was unfolded under the same conditions, only a single exponential decay was observed (Fig. 5). The rate of decay was 3.8 s⁻¹, similar to that observed for the second phase of wtACT unfolding. Taken together, these data indicate that the loss of W194 destabilizes ACT and increases the rate at which the protein unfolds.
• Fig. 5. Stopped-flow unfolding kinetics of ACT proteins. wtACT (squares) and ACTW194F (circles) were unfolded in 4 м guanidine hydrochloride, at a final protein concentration of 1 µm in the pres- ence of bis-ANS. The change in bis-ANS fluorescence was monitored over time. The data were analyzed using either single or double exponential equations, using the software provided by the manufacturer. Each dataset represents the average of ten separate traces.
• Characterization of the polymerization behavior of ACTW194F
• The mechanism of ACT polymerization has been characterized previously in two studies [20,21]. A key feature is the presence of a lag phase that can be abolished by addition of preformed polymers, a situation that does not occur for other serpins studied so far. ACT polymerization was initiated by incubating the protein at 48 °C and monitoring the changes in