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Identification of residual structure within denatured antichymotrypsin: implications for serpin folding and misfolding
BBRC Biochemical and Biophysical Research Communications 324 (2004) 729–735 www.elsevier.com/locate/ybbrc
Identification of residual structure within denatured antichymotrypsin: implications for serpin folding and misfolding Mary C. Pearcea, Lisa D. Cabritaa, Harvey Rubinb, Michael G. Gorec, Stephen P. Bottomleya,* a
c
Department of Biochemistry and Molecular Biology, Monash University, Vic. 3800, Australia b Department of Medicine, University of Pennsylvania, PA 19104-6073, United States Department of Biochemistry and Molecular Biology, University of Southampton, Hants SO16 7PX, United Kingdom Received 9 September 2004 Available online 2 October 2004
Abstract The native serpin fold is metastable and possesses the inherent ability to convert into more stable, but inactive, conformations. In order to understand why serpins attain the native fold instead of other more thermodynamically favourable folds we have investigated the presence of residual structure within denatured antichymotrypsin (ACT). Through mutagenesis we created a single tryptophan variant of ACT in which a Trp residue (276) is situated on the H-helix, located within a region known as the B/C barrel. The presence of residual structure around Trp 276 in 5 M guanidine hydrochloride (GdnHCl) was shown by fluorescence and circular dichroism spectroscopy and fluorescence lifetime experiments. The residual structure was disrupted in the presence of 5 M guanidine thiocyanate (GdnSCN). Protein refolding studies showed that significant refolding could be achieved from the GdnHCl denatured state but not the GdnSCN denatured form. The implications of these data on the folding and misfolding of the serpin superfamily are discussed. 2004 Elsevier Inc. All rights reserved. Keywords: Serpin; Conformational disease; Protein misfolding; Residual structure; Aggregation; Antichymotrypsin; Antitrypsin; Protein folding
The metastability of the native serpin fold is the key to both their biological function and dysfunction [1]. This metastability manifests itself in the form of ‘‘molecular tension’’ that allows the serpin to trap and inactivate both serine and cysteine proteinases [2–4]. During proteinase inhibition the exposed reactive centre loop (RCL) becomes cleaved, which releases the stored free energy and results in the rapid translocation of the proteinase from one pole of the molecule to the other [5,6]. This distorts the proteinase, rendering it proteolytically incompetent [6–9].
*
Corresponding author. Fax: +61 3 99054699. E-mail address: [email protected] Bottomley).
(S.P.
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.09.105
The energetic cost of being able to undergo such rapid conformational change means the native state is only marginally stable. Small changes in environment such as elevated temperature or pH cause the native state to rapidly convert to one of two inactive conformations [10–12]. Serpin polymers arise when the RCL of one molecule is inserted into the body of another, forming a long chain [13]. Such polymers have been identified in vivo in people who carry certain mutations, with diseases such as liver cirrhosis and emphysema common amongst those affected [14–16]. The second inactive state involves self-insertion of the RCL, known as the latent state, and has also been identified in vivo [17]. The relationship between these inactive forms and serpin folding has been a matter of interest for some time and it has been hypothesised that one of the folding
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intermediates may be the precursor to both states [1, 18–20]. The interplay between the serpin folding and misfolding pathways is complicated and poorly defined. A number of folding studies have reported the formation of either one or two partially folded ensembles during serpin folding [21–26]. In addition, it has been shown for a1-antitrypsin (a1-AT) that one of these ensembles is polymerogenic [23,27]. A crystallographic study of an antichymotrypsin (ACT) variant managed to trap a potential intermediate, termed the d form, and gives clues to the structure of the polymerogenic structure [28]. In this structure the RCL is partially inserted and the F-helix is partially unwound thereby expanding the A b-sheet and priming the molecule for either polymerisation or latency. The combination of biophysical and structural data suggests that the folding intermediates have a significant amount of native-like structure; therefore, the biggest structural change is in forming these species from the unfolded state. Two recent studies of the a1-AT folding pathway showed a region, termed the B/C barrel, is formed early during the folding reaction and the side chain interactions within are essential for folding to the native conformation [29]. This structure is located within the body of the molecule, involving the B and C b-sheets, as well as several surrounding helices [30]. This study demonstrated that this region possesses significant structure in the presence of 8 M urea, indicative of a highly stable region within the protein. To date, no other protein family has revealed the location of a potential folding nucleus through the use of fluorescence spectroscopy, but rather has required more involved techniques like NMR [31–35]. In the work presented here we have investigated whether the residual structure in the B/C barrel is present in another serpin, ACT. Through the use of mutagenesis and spectroscopic analysis, residual structure was identified and shared many similar features with those observed for a1-AT, suggesting that the early events of serpin folding may be conserved.
original volume. ACT was loaded onto a 5 ml HiTrap SP Sepharose column at 4 ml/min and eluted with a gradient of 0–500 mM NaCl in 10 mM Mes, pH 6.8. Fractions containing inhibitory activity were pooled and diluted to 8 times the original volume with 50 mM Tris, pH 8.0. The diluted protein was loaded onto a 10 ml DEAE Toyo-Pearl column at 4 ml/min and eluted with a 0–500 mM NaCl gradient in 50 mM Tris/HCl, 10 mM EDTA, pH 8.0. Monomeric, active protein was identified by size exclusion chromatography and inhibitory activity assays [37]. The protein was then changed over to a buffer containing 50 mM Tris/HCl, 100 mM NaCl, pH 7.4, and concentrated to a workable concentration. Protein characterisation. Inhibitory activity assays including stoichiometry of inhibition and kass were performed as previously described [37]. Thermal melts were performed in a quartz cuvette with a pathlength of 1 mm, at a protein concentration of 6 lM, as previously described [38]. Spectral studies. Fluorescence spectra were obtained on a Perkin– Elmer LS-50B Luminescence Spectrophotometer at 25 C. Proteins were at a protein concentration of 500 nM and incubated in a range of concentrations of either guanidine hydrochloride (GdnHCl), or guanidine thiocyanate (GdnSCN). Spectra were obtained with slit widths of 3 nm, each spectrum representing the average of 5 scans between 300 and 400 nm, exciting with a wavelength of 280 nm at 25 C. CD spectra were obtained on a Jasco 810 spectropolarimeter at 25 C. Each protein was at a concentration of 4 lM in 50 mM Tris/HCl, pH 7.4, except those unfolded samples which also contained 4 M GdnHCl. GdnSCN could not be used for CD measurements, as the signal to noise ratio was too low. CD spectra represent the average of 5 scans between 250 and 195 nm, at a scan speed of 20 nm/min. Fluorescence lifetimes. Fluorescence lifetime measurements were obtained using a time-correlated single photon counting technique, on a Photon Technology International TimeMaster Fluorescence Lifetimes Spectrometer. Samples containing 5 lM protein in buffer containing 0, 6 M GdnHCl, and 5 M GdnSCN were thermoregulated at 20 C, and L -tryptophan was used in the same buffer systems as a control. The excitation source was a nitrogen/dye laser, the output from which was frequency doubled to produce an excitation wavelength of 290 nm. Fluorescence measurements were obtained at 330 nm, with each acquisition comprised of an average of three complete scans. Refolding studies. Each protein was incubated in 6 M GdnHCl or 5 M GdnSCN at room temperature for 30 min. The proteins were then refolded by diluting an unfolded sample in 50 mM Tris/HCl, 50 mM NaCl, pH 7.4, followed by incubation at room temperature for 30 min. The final denaturant concentrations were 60.1 M. To assess the recovery of active material, each protein was incubated with chymotrypsin at a 1:1.5 mol:mol ratio for 15 min at 37 C, before the substrate pNA (SucAAPF-p-nitroanilide) was added and the absorbance change at 405 nm was recorded for over 10 min. The percentage of active material recovered was calculated as a percentage of the inhibitory activity before and after refolding.
Materials and methods Site-directed mutagenesis. Mutagenesis of ACT was performed using the Stratagene site-directed mutagenesis kit and verified using DNA sequencing. Production of the mutant form of ACT studied here, W276 ACT, involved replacing the other two intrinsic tryptophan (trp) residues with phenylalanine residues. Protein purification. a1-AT was expressed as inclusion bodies in BL21 (DE3) Escherichia coli cells and purified as previously described [36]. ACT was expressed in SG130009 E. coli cells. Following sonication (30 s on/30 s off) and centrifugation (18,000 rpm for 45 min), the supernatant was rocked at 4 C with 10 ml Ni–NTA resin for 2 h. ACT was eluted with 250 mM imidazole, 25 mM sodium phosphate, pH 8.0. Eluant was checked for inhibitory activity against chymotrypsin and pooled, and then diluted with 10 mM Mes, pH 6.8, to three times the
Results Properties of W276 ACT In this study we wanted to introduce a site-specific probe for residual structure within ACT. To accomplish this we replaced two (W194 and W215) of the three tryptophan residues within ACT with phenylalanine. Following mutagenesis and purification W276 ACT was characterised to determine whether the mutations introduced had any adverse effects on structure or
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Table 1 Biochemical and biophysical characterization of ACT, W276 ACT, and a1-AT SI kass Tm (C) % refolded (GuHCl) % refolded (GuSCN)
wt ACT
W276 ACT
a1-AT
1.3 3.35 · 105 61 32 ± 0.02 10 ± 0.03
1.3 3.00 · 105 62 — —
1.1 [26] 4.8 · 105 [26] 59.6 [26] 80 ± 1 <5 ± 0.5
function of the protein. The SI for W276 ACT with chymotrypsin was the same as wild type at 1.3 (Table 1). Association rates between both ACT proteins and chymotrypsin were determined to be 3.35 · 105 M 1 s 1 for wild type ACT and 3.00 · 105 M 1 s 1 for W276 ACT, indicative of there being very little difference between the functional properties of both proteins. Thermal melts were performed to determine whether there were any thermostability differences between the two proteins. A 1 C difference in Tm was observed between the two proteins, suggesting little difference in stability of the proteins (Table 1). Cumulatively these data show that the replacement of W194 with phenylalanine has little effect on the stability and function of the protein. Spectral evidence for residual structure within ACT Fluorescence spectroscopy has previously been used to identify the residual structure within denatured a1AT [29]. Here we attempted to determine whether the structurally related protein ACT also possesses such a structure. The tryptophan at position 276 is located within the B/C barrel of native ACT (Fig. 1). Previous studies have shown that 6 M GuHCl is sufficient to unfold ACT such that no further conformational change is detected using both far-UV CD and fluorescence spectroscopy [21]. This GuHCl concentration was therefore chosen initially to represent the unfolded state. Native wild type ACT had a fluorescence emission spectrum with a maximum at 334 nm (Table 2). As the concentration of GdnHCl was increased, the emission maximum gradually shifted to 347 nm, which remained stable between 4 and 6 M GdnHCl, indicative of increased solvent exposure of the tryptophan residues (Fig. 2A). As a comparison we placed L -tryptophan in 6 M GuHCl buffer, which resulted in an emission maximum centred around 356 nm (Fig. 2E and Table 2). These data therefore indicate that there is a significant degree of tryptophan burial within the denatured state of ACT. When incubated in increasing concentrations of GdnSCN, a stronger denaturant, the emission maximum shifted to 355 nm and remained there in denaturant concentrations between 4 and 5 M (Fig. 2C). These data are indicative of complete solvent exposure of the tryptophan residues.
Fig. 1. A schematic representation of ACT. The A b-sheet is present in red, the B b-sheet in green, and the C b-sheet in yellow. The location of W276 is represented by its Van der Waals spheres (orange). This image was produced using MOLSCRIPT [48]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.) Table 2 Emission maxima for ACT, W276 ACT, and L -Trp in various buffer conditions Protein
kmax (nm) Tris
6 M GuHCl
5 M GuSCN
wtACT W276 ACT L-Trp
335a 331 355
347 344 356
355 354 357
a The emission maxima for each protein represent the average of determinations from at least three different protein preparations.
The emission maximum for W276 ACT in buffer was 331 nm, indicative of a buried residue. Increasing the concentration of GdnHCl resulted in a shift in the emission maximum, however this value only ever reached 344 nm (Fig. 2B and Table 2). No further movement was detected
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Fig. 2. Fluorescence emission and far-UV CD spectra of ACT and L -tryptophan. Fluorescence emission spectra were obtained of wtACT (A) and W276 ACT (B) in increasing concentrations of GdnHCl (native spectra (filled squares), 1 M (filled triangles), 2 M (filled diamonds), 3 M (filled circles), 4 M (empty squares), 5 M (empty triangles), and 6 M (empty circles) denaturant). The vertical line at 355 nm represents the emission maxima of L -tryptophan in water. Fluorescence emission spectra were also obtained for wtACT (C) and W276 ACT (D) in GdnSCN, symbols as described above. (E) Spectra of L -tryptophan in Tris/HCl (solid line), GdnHCl (dashed line), and GdnSCN (dotted line) conditions. (E) Far-UV CD spectra of wtACT (squares) and W276 ACT (circles) in their folded and GdnHCl-unfolded states.
in GdnHCl concentrations greater than 4 M (Fig. 2B). These data suggest that at high GdnHCl concentrations, some structure persists around this residue. Incubating W276 ACT in increasing concentrations of GdnSCN resulted in a blue shift of the peak to 356 nm with no further shift at denaturant concentrations higher than 4 M (Fig. 2D). The emission maximum of the tryptophan residue at position 276 under these conditions was comparable to that of L -tryptophan when completely solvated, demonstrating full unfolding of this region (Fig. 2D). Fluorescence quenching experiments were attempted in order to assess the accessibility of the tryptophan residue in each state, however, due to the quenching effect of the GdnSCN it was not possible to perform these experiments. Far-UV CD spectra were also obtained for wild type ACT and W276 ACT in the folded and GdnHCl-un-
folded states (Fig. 2F). Spectra could not be obtained for the proteins in GdnSCN, as the signal-to-noise ratio under these conditions was too low. Both the wild type and mutant proteins appeared to have very similar secondary structure present, in either the folded or GdnHCl-unfolded states. The residual structure apparently reported by Trp fluorescence can be seen in the GdnHCl-unfolded states of both proteins (Fig. 2F), indicated by a CD signal less than zero. Fluorescence lifetimes Both the wild type and mutant ACT proteins possess tryptophan residues, three in the former and only one in the latter. To determine whether there was indeed structure surrounding W276 in ACT under GdnHCl-denaturing conditions, fluorescence lifetimes were assessed for
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the tryptophan fluorophores within the proteins. The lifetimes of the tryptophan residues were assessed for each protein when in Tris/HCl buffer, 6 M GdnHCl, and 5 M GdnSCN. To ensure that any differences in lifetimes were attributed to residual structure and not buffer effects the lifetimes of L -tryptophan in each buffer condition were also determined. Representative traces of the fluorescence decays for both wild type ACT and W276 ACT in their folded states can be seen in Fig. 3. Table 3 shows the lifetimes observed for wild type ACT, W276 ACT, and L -tryptophan in the various buffer conditions. For each protein and condition the v2 value for the fits was close to 1. A small change in fluorescence lifetime could be seen for L -tryptophan between the various buffer conditions assessed; therefore, lifetimes for the proteins were compared with those obtained for L -tryptophan under the same conditions to determine whether there was structure remaining. Wild type ACT data were difficult to interpret, as the presence of three tryptophan residues significantly complicated the lifetimes. Therefore, the focus was on W276 ACT, as this mutant possessed only the one tryptophan residue. In Tris/HCl buffer, W276 ACT
Fig. 3. Fluorescence lifetime analysis of ACT. Fluorescence intensity decays of wtACT (squares) and W276 ACT (circles) in 50 mM Tris/ HCl, pH 7.8. Each trace represents the average of three traces.
Table 3 Fluorescence lifetimes for ACT, W276 ACT, and various buffer conditions
L -tryptophan
in
Protein
Lifetimes (ns) Tris/HCl
6 M GuHCl
5 M GuSCN
WtACT
6.06 ± 0.50 3.55 ± 0.14 0.89 ± 0.14
3.33 ± 0.10 1.01 ± 0.10
3.59 ± 0.14 1.48 ± 0.05
W276 ACT
4.17 ± 0.10
3.67 ± 0.30 1.18 ± 0.30
2.25 ± 0.02 0.19 ± 0.02
L -Tryptophan
3.12 ± 0.10 0.59 ± 0.16
2.89 ± 0.08 0.50 ± 0.24
2.06 ± 0.08 0.62 ± 0.11
Each lifetime represents the average of three readings.
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had one lifetime of 4.17 ns, which was elevated in comparison to the two lifetimes seen for L -tryptophan under the same conditions (3.12 and 0.59 ns). This correlates with a more buried environment for the tryptophan in the protein in comparison to the free amino acid in solution. When in 6 M GdnHCl, L -tryptophan had lifetimes of 2.89 and 0.50 ns, which were still appreciably lower than those seen for W276 ACT at 3.37 and 1.18 ns, again indicative of structure still remaining around W276. When fully unfolded in 5 M GdnSCN, W276 ACT had lifetimes of 2.25 and 0.19 ns, similar to those obtained for L -tryptophan under the same conditions (2.06 and 0.62 ns), indicating that the structure surrounding W276 had been completely abolished. Disruption of the residual structure affects protein refolding Wild type ACT was denatured in both 6 M GdnHCl and 5 M GdnSCN, and then refolded by dilution to determine whether the different starting conformations effected the yield of active protein. For comparison, a1-AT was also denatured and refolded. Inhibitory activity was used as an indicator of the refolded conformation of the protein. Following refolding the denaturant concentrations were less than 0.1 M. Control experiments showed that this had no effect on the interaction between ACT or a1-AT with chymotrypsin (data not shown). When refolded from GdnHCl wild type ACT regained over 30% of its original inhibitory activity (Table 1). This was dramatically reduced to approximately 10% when refolded from GdnSCN. A similar pattern was seen for a1-AT, where material refolded from GdnHCl regained 80% of its original activity, however incubation in GdnSCN led to an almost complete loss of recoverable inhibitory activity (<5%).
Discussion How large proteins fold and misfold is of great biochemical and biomedical importance. A number of mechanisms have been proposed for small single domain/model proteins which account for the fact that many proteins reliably fold to the native state, despite the seemingly endless number of possible conformations [39]. A common theme of these mechanisms revolves around the idea that the polypeptide chain collapses. This reduces the number of possible conformations and interactions an amino acid can adopt, thereby reducing the search through conformational space, and increasing the stability of the species formed. Recent data indicate that nucleation sites form early in the folding process, which guide the molecule to the native state [40,41]. These sites have also been identified in equilib-
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rium studies using techniques such as NMR, H/H2 exchange, and far- and near-UV CD. These sites commonly consist of hydrophobic clusters and often tryptophan residues are found in their centre. In this study we have been able to identify a region of residual structure in the serpin ACT using intrinsic fluorescence spectroscopy [29]. The location of residual structure is similar to that within a1-AT, which we previously identified. This highly conserved region consists of the B and C b-sheets along with the G and H helices [29]. The folding nucleus of ACT involves interactions between W276, located on the G-helix, and other residues. Tryptophan is conserved at this position in over 60% of all serpins [42]. In the native state the indole ring of this residue is buried within the body of the protein, packed against strands 2 and 3 of the large B b-sheet (Fig. 1). Our fluorescence emission spectra and lifetime data support the idea that residual structure persists around W276 in denatured ACT. In combination with our previous data on a1-AT, this strengthens our hypothesis that this region, known as the B/C barrel, is conserved for folding of the serpin molecule. There are a number of lines of additional evidence that also support the idea that the B/C barrel helps direct folding. First, residues within this region of the serpin are highly conserved, especially those that are hydrophobic [42]. A number of computational and biophysical studies have shown that folding nuclei are hydrophobic in character and conserved within protein families [43,44]. Second, we identified a number of serpins within the Caenorhabditis elegans genome, four of which contained truncated sequences that apparently contain the B and C b-sheets, and the G and H helices [45]. Third, a comparison of the native, cleaved, and latent serpin structures reveals that this core is rigid when compared with the rest of the molecule [46]. These facts suggest that the residual structure may possess an enhanced stability compared to the rest of the structure, which would allow this region to act as a platform for the dynamic conformational changes required for proteinase inhibition. An interesting finding from this study was the difference in refolded yield for both ACT and a1-AT from the different strength denaturants. When residual structure was present, as in 6 M GdnHCl, a significantly higher yield of active protein was obtained than when this structure was disrupted. A simplistic interpretation of these data would imply that this region can act as a scaffold for the rest of the protein to productively fold upon, and when it is disrupted there is more competition from off-pathway reactions. In reality, serpins can fold to their native state, even when this structure is disrupted. This can most likely be attributed to the presence of chaperones in vivo which may allow the B/C barrel to form, thus allowing the rest of the molecule to fold around this. Several serpin mutations have been identified within this region that lead to deficiency of the
effected protein, and in each case the problem arises from a folding defect [47]. This information supports the argument that the region is paramount to directing the serpin to the correct fold. In conclusion, serpins are large single domain proteins that are particularly prone to misfolding and aggregation. It is possible that the evolutionary adaptation of this region of residual structure helps prevent misfolding events, and therefore serpin deficiencies.
Acknowledgments This work was supported by grants from the National Health and Medical Research Council and Australian Research Council to S.P.B. S.P.B. is a Monash University Senior Logan Fellow and R.D. Wright Fellow of the NH&MRC. We are indebted to the BBSRC for funding the purchase of the lifetime fluorimeter. References [1] L.D. Cabrita, S.P. Bottomley, How do proteins avoid becoming too stable? Biophysical studies into metastable proteins, Eur. Biophys. J. 33 (2004) 83–88. [2] J.A. Huntington, P.E. Stein, Structure and properties of ovalbumin, J. Chromatogr. B Biomed. Sci. Appl. 756 (2001) 189–198. [3] M. Bruch, V. Weiss, J. Engel, Plasma serine proteinase inhibitors (serpins) exhibit major conformational changes and a large increase in conformational stability upon cleavage at their reactive sites, J. Biol. Chem. 263 (1988) 16626–16630. [4] C. Schick, P.A. Pemberton, G.P. Shi, Y. Kamachi, S. Cataltepe, A.J. Bartuski, E.R. Gornstein, D. Bromme, H.A. Chapman, G.A. Silverman, Cross-class inhibition of the cysteine proteinases cathepsins K, L, and S by the serpin squamous cell carcinoma antigen 1: a kinetic analysis, Biochemistry 37 (1998) 5258–5266. [5] U. Baumann, R. Huber, W. Bode, D. Grosse, M. Lesjak, C.B. Laurell, Crystal structure of cleaved human a1 antichymotrypsin ˚ resolution and its comparison with other serpins, J. Mol. at 2.7 A Biol. 218 (1991) 595–606. [6] J.A. Huntington, R.J. Read, R.W. Carrell, Structure of a serpin– protease complex shows inhibition by deformation, Nature 407 (2000) 923–926. [7] G. Kaslik, A. Patthy, M. Balint, L. Graf, Trypsin complexed with alpha 1-proteinase inhibitor has an increased structural flexibility, FEBS Lett. 370 (1995) 179–183. [8] M.I. Plotnick, L. Mayne, N.M. Schechter, H. Rubin, Distortion of the active site of chymotrypsin complexed with a serpin, Biochemistry 35 (1996) 7586–7590. [9] D.J. Tew, S.P. Bottomley, Intrinsic fluorescence changes and rapid kinetics of proteinase deformation during serpin inhibition, FEBS Lett. 494 (2001) 30–33. [10] M.K. Chow, D.A. Lomas, S.P. Bottomley, Promiscuous betastrand interactions and the conformational diseases, Curr. Med. Chem. 11 (2004) 491–499. [11] L.N. Benning, J.C. Whisstock, J. Sun, P.I. Bird, S.P. Bottomley, The human serpin proteinase inhibitor-9 self-associates at physiological temperatures, Protein Sci. 13 (2004) 1859–1864. [12] G.L. Devlin, M.K. Chow, G.J. Howlett, S.P. Bottomley, Acid denaturation of alpha 1-antitrypsin: characterization of a novel mechanism of serpin polymerization, J. Mol. Biol. 324 (2002) 859– 870.
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Identification of residual structure within denatured antichymotrypsin: implications for serpin folding and misfolding Mary C. Pearcea, Lisa D. Cabritaa, Harvey Rubinb, Michael G. Gorec, Stephen P. Bottomleya,* a
c
Department of Biochemistry and Molecular Biology, Monash University, Vic. 3800, Australia b Department of Medicine, University of Pennsylvania, PA 19104-6073, United States Department of Biochemistry and Molecular Biology, University of Southampton, Hants SO16 7PX, United Kingdom Received 9 September 2004 Available online 2 October 2004
Abstract The native serpin fold is metastable and possesses the inherent ability to convert into more stable, but inactive, conformations. In order to understand why serpins attain the native fold instead of other more thermodynamically favourable folds we have investigated the presence of residual structure within denatured antichymotrypsin (ACT). Through mutagenesis we created a single tryptophan variant of ACT in which a Trp residue (276) is situated on the H-helix, located within a region known as the B/C barrel. The presence of residual structure around Trp 276 in 5 M guanidine hydrochloride (GdnHCl) was shown by fluorescence and circular dichroism spectroscopy and fluorescence lifetime experiments. The residual structure was disrupted in the presence of 5 M guanidine thiocyanate (GdnSCN). Protein refolding studies showed that significant refolding could be achieved from the GdnHCl denatured state but not the GdnSCN denatured form. The implications of these data on the folding and misfolding of the serpin superfamily are discussed. 2004 Elsevier Inc. All rights reserved. Keywords: Serpin; Conformational disease; Protein misfolding; Residual structure; Aggregation; Antichymotrypsin; Antitrypsin; Protein folding
The metastability of the native serpin fold is the key to both their biological function and dysfunction [1]. This metastability manifests itself in the form of ‘‘molecular tension’’ that allows the serpin to trap and inactivate both serine and cysteine proteinases [2–4]. During proteinase inhibition the exposed reactive centre loop (RCL) becomes cleaved, which releases the stored free energy and results in the rapid translocation of the proteinase from one pole of the molecule to the other [5,6]. This distorts the proteinase, rendering it proteolytically incompetent [6–9].
*
Corresponding author. Fax: +61 3 99054699. E-mail address: [email protected] Bottomley).
(S.P.
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.09.105
The energetic cost of being able to undergo such rapid conformational change means the native state is only marginally stable. Small changes in environment such as elevated temperature or pH cause the native state to rapidly convert to one of two inactive conformations [10–12]. Serpin polymers arise when the RCL of one molecule is inserted into the body of another, forming a long chain [13]. Such polymers have been identified in vivo in people who carry certain mutations, with diseases such as liver cirrhosis and emphysema common amongst those affected [14–16]. The second inactive state involves self-insertion of the RCL, known as the latent state, and has also been identified in vivo [17]. The relationship between these inactive forms and serpin folding has been a matter of interest for some time and it has been hypothesised that one of the folding
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intermediates may be the precursor to both states [1, 18–20]. The interplay between the serpin folding and misfolding pathways is complicated and poorly defined. A number of folding studies have reported the formation of either one or two partially folded ensembles during serpin folding [21–26]. In addition, it has been shown for a1-antitrypsin (a1-AT) that one of these ensembles is polymerogenic [23,27]. A crystallographic study of an antichymotrypsin (ACT) variant managed to trap a potential intermediate, termed the d form, and gives clues to the structure of the polymerogenic structure [28]. In this structure the RCL is partially inserted and the F-helix is partially unwound thereby expanding the A b-sheet and priming the molecule for either polymerisation or latency. The combination of biophysical and structural data suggests that the folding intermediates have a significant amount of native-like structure; therefore, the biggest structural change is in forming these species from the unfolded state. Two recent studies of the a1-AT folding pathway showed a region, termed the B/C barrel, is formed early during the folding reaction and the side chain interactions within are essential for folding to the native conformation [29]. This structure is located within the body of the molecule, involving the B and C b-sheets, as well as several surrounding helices [30]. This study demonstrated that this region possesses significant structure in the presence of 8 M urea, indicative of a highly stable region within the protein. To date, no other protein family has revealed the location of a potential folding nucleus through the use of fluorescence spectroscopy, but rather has required more involved techniques like NMR [31–35]. In the work presented here we have investigated whether the residual structure in the B/C barrel is present in another serpin, ACT. Through the use of mutagenesis and spectroscopic analysis, residual structure was identified and shared many similar features with those observed for a1-AT, suggesting that the early events of serpin folding may be conserved.
original volume. ACT was loaded onto a 5 ml HiTrap SP Sepharose column at 4 ml/min and eluted with a gradient of 0–500 mM NaCl in 10 mM Mes, pH 6.8. Fractions containing inhibitory activity were pooled and diluted to 8 times the original volume with 50 mM Tris, pH 8.0. The diluted protein was loaded onto a 10 ml DEAE Toyo-Pearl column at 4 ml/min and eluted with a 0–500 mM NaCl gradient in 50 mM Tris/HCl, 10 mM EDTA, pH 8.0. Monomeric, active protein was identified by size exclusion chromatography and inhibitory activity assays [37]. The protein was then changed over to a buffer containing 50 mM Tris/HCl, 100 mM NaCl, pH 7.4, and concentrated to a workable concentration. Protein characterisation. Inhibitory activity assays including stoichiometry of inhibition and kass were performed as previously described [37]. Thermal melts were performed in a quartz cuvette with a pathlength of 1 mm, at a protein concentration of 6 lM, as previously described [38]. Spectral studies. Fluorescence spectra were obtained on a Perkin– Elmer LS-50B Luminescence Spectrophotometer at 25 C. Proteins were at a protein concentration of 500 nM and incubated in a range of concentrations of either guanidine hydrochloride (GdnHCl), or guanidine thiocyanate (GdnSCN). Spectra were obtained with slit widths of 3 nm, each spectrum representing the average of 5 scans between 300 and 400 nm, exciting with a wavelength of 280 nm at 25 C. CD spectra were obtained on a Jasco 810 spectropolarimeter at 25 C. Each protein was at a concentration of 4 lM in 50 mM Tris/HCl, pH 7.4, except those unfolded samples which also contained 4 M GdnHCl. GdnSCN could not be used for CD measurements, as the signal to noise ratio was too low. CD spectra represent the average of 5 scans between 250 and 195 nm, at a scan speed of 20 nm/min. Fluorescence lifetimes. Fluorescence lifetime measurements were obtained using a time-correlated single photon counting technique, on a Photon Technology International TimeMaster Fluorescence Lifetimes Spectrometer. Samples containing 5 lM protein in buffer containing 0, 6 M GdnHCl, and 5 M GdnSCN were thermoregulated at 20 C, and L -tryptophan was used in the same buffer systems as a control. The excitation source was a nitrogen/dye laser, the output from which was frequency doubled to produce an excitation wavelength of 290 nm. Fluorescence measurements were obtained at 330 nm, with each acquisition comprised of an average of three complete scans. Refolding studies. Each protein was incubated in 6 M GdnHCl or 5 M GdnSCN at room temperature for 30 min. The proteins were then refolded by diluting an unfolded sample in 50 mM Tris/HCl, 50 mM NaCl, pH 7.4, followed by incubation at room temperature for 30 min. The final denaturant concentrations were 60.1 M. To assess the recovery of active material, each protein was incubated with chymotrypsin at a 1:1.5 mol:mol ratio for 15 min at 37 C, before the substrate pNA (SucAAPF-p-nitroanilide) was added and the absorbance change at 405 nm was recorded for over 10 min. The percentage of active material recovered was calculated as a percentage of the inhibitory activity before and after refolding.
Materials and methods Site-directed mutagenesis. Mutagenesis of ACT was performed using the Stratagene site-directed mutagenesis kit and verified using DNA sequencing. Production of the mutant form of ACT studied here, W276 ACT, involved replacing the other two intrinsic tryptophan (trp) residues with phenylalanine residues. Protein purification. a1-AT was expressed as inclusion bodies in BL21 (DE3) Escherichia coli cells and purified as previously described [36]. ACT was expressed in SG130009 E. coli cells. Following sonication (30 s on/30 s off) and centrifugation (18,000 rpm for 45 min), the supernatant was rocked at 4 C with 10 ml Ni–NTA resin for 2 h. ACT was eluted with 250 mM imidazole, 25 mM sodium phosphate, pH 8.0. Eluant was checked for inhibitory activity against chymotrypsin and pooled, and then diluted with 10 mM Mes, pH 6.8, to three times the
Results Properties of W276 ACT In this study we wanted to introduce a site-specific probe for residual structure within ACT. To accomplish this we replaced two (W194 and W215) of the three tryptophan residues within ACT with phenylalanine. Following mutagenesis and purification W276 ACT was characterised to determine whether the mutations introduced had any adverse effects on structure or
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Table 1 Biochemical and biophysical characterization of ACT, W276 ACT, and a1-AT SI kass Tm (C) % refolded (GuHCl) % refolded (GuSCN)
wt ACT
W276 ACT
a1-AT
1.3 3.35 · 105 61 32 ± 0.02 10 ± 0.03
1.3 3.00 · 105 62 — —
1.1 [26] 4.8 · 105 [26] 59.6 [26] 80 ± 1 <5 ± 0.5
function of the protein. The SI for W276 ACT with chymotrypsin was the same as wild type at 1.3 (Table 1). Association rates between both ACT proteins and chymotrypsin were determined to be 3.35 · 105 M 1 s 1 for wild type ACT and 3.00 · 105 M 1 s 1 for W276 ACT, indicative of there being very little difference between the functional properties of both proteins. Thermal melts were performed to determine whether there were any thermostability differences between the two proteins. A 1 C difference in Tm was observed between the two proteins, suggesting little difference in stability of the proteins (Table 1). Cumulatively these data show that the replacement of W194 with phenylalanine has little effect on the stability and function of the protein. Spectral evidence for residual structure within ACT Fluorescence spectroscopy has previously been used to identify the residual structure within denatured a1AT [29]. Here we attempted to determine whether the structurally related protein ACT also possesses such a structure. The tryptophan at position 276 is located within the B/C barrel of native ACT (Fig. 1). Previous studies have shown that 6 M GuHCl is sufficient to unfold ACT such that no further conformational change is detected using both far-UV CD and fluorescence spectroscopy [21]. This GuHCl concentration was therefore chosen initially to represent the unfolded state. Native wild type ACT had a fluorescence emission spectrum with a maximum at 334 nm (Table 2). As the concentration of GdnHCl was increased, the emission maximum gradually shifted to 347 nm, which remained stable between 4 and 6 M GdnHCl, indicative of increased solvent exposure of the tryptophan residues (Fig. 2A). As a comparison we placed L -tryptophan in 6 M GuHCl buffer, which resulted in an emission maximum centred around 356 nm (Fig. 2E and Table 2). These data therefore indicate that there is a significant degree of tryptophan burial within the denatured state of ACT. When incubated in increasing concentrations of GdnSCN, a stronger denaturant, the emission maximum shifted to 355 nm and remained there in denaturant concentrations between 4 and 5 M (Fig. 2C). These data are indicative of complete solvent exposure of the tryptophan residues.
Fig. 1. A schematic representation of ACT. The A b-sheet is present in red, the B b-sheet in green, and the C b-sheet in yellow. The location of W276 is represented by its Van der Waals spheres (orange). This image was produced using MOLSCRIPT [48]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.) Table 2 Emission maxima for ACT, W276 ACT, and L -Trp in various buffer conditions Protein
kmax (nm) Tris
6 M GuHCl
5 M GuSCN
wtACT W276 ACT L-Trp
335a 331 355
347 344 356
355 354 357
a The emission maxima for each protein represent the average of determinations from at least three different protein preparations.
The emission maximum for W276 ACT in buffer was 331 nm, indicative of a buried residue. Increasing the concentration of GdnHCl resulted in a shift in the emission maximum, however this value only ever reached 344 nm (Fig. 2B and Table 2). No further movement was detected
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Fig. 2. Fluorescence emission and far-UV CD spectra of ACT and L -tryptophan. Fluorescence emission spectra were obtained of wtACT (A) and W276 ACT (B) in increasing concentrations of GdnHCl (native spectra (filled squares), 1 M (filled triangles), 2 M (filled diamonds), 3 M (filled circles), 4 M (empty squares), 5 M (empty triangles), and 6 M (empty circles) denaturant). The vertical line at 355 nm represents the emission maxima of L -tryptophan in water. Fluorescence emission spectra were also obtained for wtACT (C) and W276 ACT (D) in GdnSCN, symbols as described above. (E) Spectra of L -tryptophan in Tris/HCl (solid line), GdnHCl (dashed line), and GdnSCN (dotted line) conditions. (E) Far-UV CD spectra of wtACT (squares) and W276 ACT (circles) in their folded and GdnHCl-unfolded states.
in GdnHCl concentrations greater than 4 M (Fig. 2B). These data suggest that at high GdnHCl concentrations, some structure persists around this residue. Incubating W276 ACT in increasing concentrations of GdnSCN resulted in a blue shift of the peak to 356 nm with no further shift at denaturant concentrations higher than 4 M (Fig. 2D). The emission maximum of the tryptophan residue at position 276 under these conditions was comparable to that of L -tryptophan when completely solvated, demonstrating full unfolding of this region (Fig. 2D). Fluorescence quenching experiments were attempted in order to assess the accessibility of the tryptophan residue in each state, however, due to the quenching effect of the GdnSCN it was not possible to perform these experiments. Far-UV CD spectra were also obtained for wild type ACT and W276 ACT in the folded and GdnHCl-un-
folded states (Fig. 2F). Spectra could not be obtained for the proteins in GdnSCN, as the signal-to-noise ratio under these conditions was too low. Both the wild type and mutant proteins appeared to have very similar secondary structure present, in either the folded or GdnHCl-unfolded states. The residual structure apparently reported by Trp fluorescence can be seen in the GdnHCl-unfolded states of both proteins (Fig. 2F), indicated by a CD signal less than zero. Fluorescence lifetimes Both the wild type and mutant ACT proteins possess tryptophan residues, three in the former and only one in the latter. To determine whether there was indeed structure surrounding W276 in ACT under GdnHCl-denaturing conditions, fluorescence lifetimes were assessed for
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the tryptophan fluorophores within the proteins. The lifetimes of the tryptophan residues were assessed for each protein when in Tris/HCl buffer, 6 M GdnHCl, and 5 M GdnSCN. To ensure that any differences in lifetimes were attributed to residual structure and not buffer effects the lifetimes of L -tryptophan in each buffer condition were also determined. Representative traces of the fluorescence decays for both wild type ACT and W276 ACT in their folded states can be seen in Fig. 3. Table 3 shows the lifetimes observed for wild type ACT, W276 ACT, and L -tryptophan in the various buffer conditions. For each protein and condition the v2 value for the fits was close to 1. A small change in fluorescence lifetime could be seen for L -tryptophan between the various buffer conditions assessed; therefore, lifetimes for the proteins were compared with those obtained for L -tryptophan under the same conditions to determine whether there was structure remaining. Wild type ACT data were difficult to interpret, as the presence of three tryptophan residues significantly complicated the lifetimes. Therefore, the focus was on W276 ACT, as this mutant possessed only the one tryptophan residue. In Tris/HCl buffer, W276 ACT
Fig. 3. Fluorescence lifetime analysis of ACT. Fluorescence intensity decays of wtACT (squares) and W276 ACT (circles) in 50 mM Tris/ HCl, pH 7.8. Each trace represents the average of three traces.
Table 3 Fluorescence lifetimes for ACT, W276 ACT, and various buffer conditions
L -tryptophan
in
Protein
Lifetimes (ns) Tris/HCl
6 M GuHCl
5 M GuSCN
WtACT
6.06 ± 0.50 3.55 ± 0.14 0.89 ± 0.14
3.33 ± 0.10 1.01 ± 0.10
3.59 ± 0.14 1.48 ± 0.05
W276 ACT
4.17 ± 0.10
3.67 ± 0.30 1.18 ± 0.30
2.25 ± 0.02 0.19 ± 0.02
L -Tryptophan
3.12 ± 0.10 0.59 ± 0.16
2.89 ± 0.08 0.50 ± 0.24
2.06 ± 0.08 0.62 ± 0.11
Each lifetime represents the average of three readings.
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had one lifetime of 4.17 ns, which was elevated in comparison to the two lifetimes seen for L -tryptophan under the same conditions (3.12 and 0.59 ns). This correlates with a more buried environment for the tryptophan in the protein in comparison to the free amino acid in solution. When in 6 M GdnHCl, L -tryptophan had lifetimes of 2.89 and 0.50 ns, which were still appreciably lower than those seen for W276 ACT at 3.37 and 1.18 ns, again indicative of structure still remaining around W276. When fully unfolded in 5 M GdnSCN, W276 ACT had lifetimes of 2.25 and 0.19 ns, similar to those obtained for L -tryptophan under the same conditions (2.06 and 0.62 ns), indicating that the structure surrounding W276 had been completely abolished. Disruption of the residual structure affects protein refolding Wild type ACT was denatured in both 6 M GdnHCl and 5 M GdnSCN, and then refolded by dilution to determine whether the different starting conformations effected the yield of active protein. For comparison, a1-AT was also denatured and refolded. Inhibitory activity was used as an indicator of the refolded conformation of the protein. Following refolding the denaturant concentrations were less than 0.1 M. Control experiments showed that this had no effect on the interaction between ACT or a1-AT with chymotrypsin (data not shown). When refolded from GdnHCl wild type ACT regained over 30% of its original inhibitory activity (Table 1). This was dramatically reduced to approximately 10% when refolded from GdnSCN. A similar pattern was seen for a1-AT, where material refolded from GdnHCl regained 80% of its original activity, however incubation in GdnSCN led to an almost complete loss of recoverable inhibitory activity (<5%).
Discussion How large proteins fold and misfold is of great biochemical and biomedical importance. A number of mechanisms have been proposed for small single domain/model proteins which account for the fact that many proteins reliably fold to the native state, despite the seemingly endless number of possible conformations [39]. A common theme of these mechanisms revolves around the idea that the polypeptide chain collapses. This reduces the number of possible conformations and interactions an amino acid can adopt, thereby reducing the search through conformational space, and increasing the stability of the species formed. Recent data indicate that nucleation sites form early in the folding process, which guide the molecule to the native state [40,41]. These sites have also been identified in equilib-
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rium studies using techniques such as NMR, H/H2 exchange, and far- and near-UV CD. These sites commonly consist of hydrophobic clusters and often tryptophan residues are found in their centre. In this study we have been able to identify a region of residual structure in the serpin ACT using intrinsic fluorescence spectroscopy [29]. The location of residual structure is similar to that within a1-AT, which we previously identified. This highly conserved region consists of the B and C b-sheets along with the G and H helices [29]. The folding nucleus of ACT involves interactions between W276, located on the G-helix, and other residues. Tryptophan is conserved at this position in over 60% of all serpins [42]. In the native state the indole ring of this residue is buried within the body of the protein, packed against strands 2 and 3 of the large B b-sheet (Fig. 1). Our fluorescence emission spectra and lifetime data support the idea that residual structure persists around W276 in denatured ACT. In combination with our previous data on a1-AT, this strengthens our hypothesis that this region, known as the B/C barrel, is conserved for folding of the serpin molecule. There are a number of lines of additional evidence that also support the idea that the B/C barrel helps direct folding. First, residues within this region of the serpin are highly conserved, especially those that are hydrophobic [42]. A number of computational and biophysical studies have shown that folding nuclei are hydrophobic in character and conserved within protein families [43,44]. Second, we identified a number of serpins within the Caenorhabditis elegans genome, four of which contained truncated sequences that apparently contain the B and C b-sheets, and the G and H helices [45]. Third, a comparison of the native, cleaved, and latent serpin structures reveals that this core is rigid when compared with the rest of the molecule [46]. These facts suggest that the residual structure may possess an enhanced stability compared to the rest of the structure, which would allow this region to act as a platform for the dynamic conformational changes required for proteinase inhibition. An interesting finding from this study was the difference in refolded yield for both ACT and a1-AT from the different strength denaturants. When residual structure was present, as in 6 M GdnHCl, a significantly higher yield of active protein was obtained than when this structure was disrupted. A simplistic interpretation of these data would imply that this region can act as a scaffold for the rest of the protein to productively fold upon, and when it is disrupted there is more competition from off-pathway reactions. In reality, serpins can fold to their native state, even when this structure is disrupted. This can most likely be attributed to the presence of chaperones in vivo which may allow the B/C barrel to form, thus allowing the rest of the molecule to fold around this. Several serpin mutations have been identified within this region that lead to deficiency of the
effected protein, and in each case the problem arises from a folding defect [47]. This information supports the argument that the region is paramount to directing the serpin to the correct fold. In conclusion, serpins are large single domain proteins that are particularly prone to misfolding and aggregation. It is possible that the evolutionary adaptation of this region of residual structure helps prevent misfolding events, and therefore serpin deficiencies.
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