Oxidative Folding and Structural Analyses of a Kunitz-Related Inhibitor and Its Disulfide Intermediates: Functional Implications

doi:10.1016/j.jmb.2011.10.018

J. Mol. Biol. (2011) 414, 427–441 Contents lists available at www.sciencedirect.com

Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b

Oxidative Folding and Structural Analyses of a Kunitz-Related Inhibitor and Its Disulfide Intermediates: Functional Implications Sílvia Bronsoms 1 †, David Pantoja-Uceda 2 †, Dusica Gabrijelcic-Geiger 3 , Laura Sanglas 1 , Francesc X. Aviles 1 , Jorge Santoro 2 , Christian P. Sommerhoff 3 and Joan L. Arolas 4 ⁎ 1

Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Mòdul B Parc de Recerca, E-08193 Bellaterra, Spain 2 Departamento de Química Físca Biológica, Instituto de Química Física Rocasolano, Consejo Superior de Investigaciones Científicas, c/ Serrano 119, E-28006 Madrid, Spain 3 Abteilung Klinische Chemie und Klinische Biochemie, Klinikumsstandort Innenstadt der Ludwig-MaximiliansUniversität, Nußbaumstraße 20, D-80336 München, Germany 4 Department of Structural Biology, Molecular Biology Institute of Barcelona, Consejo Superior de Investigaciones Científicas, Barcelona Science Park, c/ Baldiri Reixac 15, E-08028 Barcelona, Spain Received 8 August 2011; received in revised form 18 September 2011; accepted 12 October 2011 Available online 18 October 2011 Edited by K. Kuwajima Keywords: Kunitz inhibitor; oxidative folding; disulfide intermediate; NMR structure; tryptase inhibition

Tick-derived protease inhibitor (TdPI) is a tight-binding Kunitz-related inhibitor of human tryptase β with a unique structure and disulfide-bond pattern. Here we analyzed its oxidative folding and reductive unfolding by chromatographic and disulfide analyses of acid-trapped intermediates. TdPI folds through a stepwise generation of heterogeneous populations of one-disulfide, two-disulfide, and three-disulfide intermediates, with a major accumulation of the nonnative three-disulfide species IIIa. The ratelimiting step of the process is disulfide reshuffling within the three-disulfide population towards a productive intermediate that oxidizes directly into the native four-disulfide protein. TdPI unfolds through a major accumulation of the native three-disulfide species IIIb and the subsequent formation of twodisulfide and one-disulfide intermediates. NMR characterization of the acid-trapped and further isolated IIIa intermediate revealed a highly disordered conformation that is maintained by the presence of the disulfide bonds. Conversely, the NMR structure of IIIb showed a native-like conformation, with three native disulfide bonds and increased flexibility only around the two free cysteines, thus providing a molecular basis for its role as a productive intermediate. Comparison of TdPI with a shortened variant lacking the flexible prehead and posthead segments revealed that these regions do not contribute to the protein conformational stability or the inhibition of trypsin but are important for both the initial steps of the folding

*Corresponding author. E-mail address: [email protected]. † S.B. and D.P.-U. contributed equally to this study. Abbreviations used: TdPI, tick-derived protease inhibitor; BPTI, bovine pancreatic trypsin inhibitor; TAP, tick anticoagulant peptide; RNase A, ribonuclease A; LDTI, leech-derived tryptase inhibitor; RP-HPLC, reversed-phase highperformance liquid chromatography; MS, mass spectrometry; GSH, reduced glutathione; GSSG, oxidized glutathione; TCEP, Tris(2-carboxyethyl)phosphine; GdnHCl, guanidine hydrochloride; GdnSCN, guanidine thiocyanate; NOE, nuclear Overhauser enhancement; RT, room temperature; TFA, trifluoroacetic acid; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; NOESY, NOE spectroscopy. 0022-2836/$ - see front matter © 2011 Elsevier Ltd. All rights reserved.

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reaction and the inhibition of tryptase β. Taken together, the results provide insights into the mechanism of oxidative folding of Kunitz inhibitors and pave the way for the design of TdPI variants with improved properties for biomedical applications. © 2011 Elsevier Ltd. All rights reserved.

Introduction Kunitz-type inhibitors (family I2 according to the MEROPS database) are small disulfide-rich proteins that inhibit serine proteases such as trypsin, chymotrypsin, kallikreins, and plasmin. 1 Bovine pancreatic trypsin inhibitor (BPTI) is the prototype of this family. It is synthesized as a precursor protein that is subsequently cleaved, giving rise to a mature inhibitor consisting of 58 residues with three disulfide bonds. 2,3 Besides its use in medicine to reduce bleeding during surgery, BPTI is one of the most thoroughly studied proteins in terms of structural biology, mutagenesis, and folding. 4–6 Pioneering work in 1980 and in the early 1990s revealed that, under oxidative conditions, the mature inhibitor folds towards the native state through the accumulation of a few one-disulfide

and two-disulfide intermediates. 7–10 These species contain native disulfide bonds and evince nativelike structures. Another Kunitz inhibitor whose oxidative folding has been extensively studied is tick anti-coagulant peptide (TAP; 60 residues and three disulfide bonds). 11–13 Although BPTI and TAP show close levels of structural homology and similar disulfide-bond patterns, 14,15 TAP folds through a far more heterogeneous population of intermediates. 16 This population contains both native and nonnative disulfide-bonded species, with the latter including three-disulfide scrambled isomers that act as major kinetic traps, slowing the last steps of the folding reaction. Tick-derived protease inhibitor (TdPI) is a potent inhibitor of trypsin that is selectively produced by the salivary glands of adult ticks. 17 These hematophagous parasites use saliva compounds to

Fig. 1. Structures of the Kunitz domain of BPTI and TdPI. Richardson plot of the BPTI (a) and TdPI (b) heads. The Nterminus and the C-terminus, the regular secondary structure elements, and the disulfide bonds are labeled. The canonical lysine residue involved in trypsin inhibition is shown as a stick and labeled. This figure was prepared with PyMOL using Protein Data Bank codes 1PIT and 2UUX. (c) C α structure representation of the superposition of the BPTI (green) and TdPI (orange) heads. Orientation as in (a) and (b). Major structural differences between both molecules (in loops L1 and L2) are denoted by arrows. (d) Amino acid sequence of BPTI and TdPI (numbering based on Paesen et al. 17 and Creighton et al. 22). The structure elements and native disulfide-bond pairings are represented. The prehead and posthead regions are indicated.

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TdPI Folding, Unfolding, and Inhibitory Activity

Fig. 2. Oxidative folding of TdPIlong. RP-HPLC traces of acid-trapped intermediates that accumulate during the oxidative folding of TdPI-long. The reactions were carried out in Tris–HCl (pH 8.5) in the absence (Control −) and in the presence (Control +) of 0.25 mM 2-mercaptoethanol, 0.5 mM GSSG, or a mixture of 0.5 mM/1 mM GSSG/GSH. The retention times of the native (N) and fully reduced/unfolded (R) forms are indicated. XS stands for an ensemble of species with X number of disulfide bonds. IIIa is a major three-disulfide intermediate comprising nonnative disulfide bonds.

interfere with host defense mechanisms during feeding. 18 Importantly, TdPI is one of only two proteins identified so far that strongly inhibit human tryptase β, a mast-cell-derived tetrameric serine protease implicated in inflammation and allergy. 19–21 This inhibitor consists of 97 residues and contains a Kunitz/BPTI-related domain (55 residues crosslinked by four disulfide bonds) that is surrounded by prehead and posthead segments of 20 and 22 residues, respectively (Fig. 1). X-ray crystallography revealed that the TdPI head, despite possessing canonical structural elements typical of the Kunitz family, has a modified shape (comprising a shortened loop connecting the anti-parallel β-sheet to the Cterminal α-helix) and an altered disulfide-bond pattern. 17 Its structure resembles a triangular arrow with a sharp apex and a broad base, which enable it to penetrate the central oval pore of the tetrameric structure of tryptase β. 23–25 Due to its unique fold and disulfide-bond pairings (four disulfide bonds versus three disulfide bonds in BPTI and TAP), TdPI is an attractive model among Kunitz inhibitors for studying oxidative folding. Its four-disulfide nature is reminiscent of that of ribonuclease A (RNase A), an outstanding model protein that has been studied in terms of oxidative folding over the last three decades. 26–28 In addition, as TdPI is the second proteinaceous human tryptase β inhibitor to be reported, its functional characterization may shed light on the interaction between inhibitors and this biomedically relevant protease. We have previously investigated the structure and oxidative folding of leech-derived tryptase inhibitor (LDTI; 46 residues and three disulfide bonds; the first tryptase β inhibitor to be identified), which belongs to the Kazal-type family (family I1 according to the MEROPS database). 29–33 Here we analyzed the oxidative folding, reductive unfolding, conformational stability, and inhibitory activity of TdPI (hereafter referred to as TdPI-long) and its

Kunitz head (TdPI-short). In addition, we characterized the NMR structures of TdPI-short and its major folding intermediates to elucidate the molecular determinants that govern the folding and activity of this protein.

Results Oxidative folding of TdPI-long and TdPI-short Fully reduced and unfolded TdPI-long and TdPIshort were allowed to refold in Tris–HCl (pH 8.5) in the absence and in the presence of different redox agents. The intermediates that appeared during the folding reaction were trapped by acidification at selected time points and analyzed by reversedphase high-performance liquid chromatography (RP-HPLC). TdPI-long folds through heterogeneous intermediates with similar elution profiles both in the absence (Control −) and in the presence (Control +) of 2-mercaptoethanol (Fig. 2). Mass spectrometry (MS) analysis of the acid-trapped intermediates after derivatization with vinylpyridine revealed the sequential accumulation of one-disulfide, twodisulfide, and three-disulfide species and the absence of four-disulfide scrambled isomers. The three-disulfide population is highly heterogeneous and contains a major intermediate, termed IIIa, that elutes close to the native form. The folding reaction was accelerated by the presence of a reducing agent [2-mercaptoethanol or reduced glutathione (GSH)] that promotes disulfide reshuffling within the different disulfide populations. The slowest folding step was thus the generation of a productive three-disulfide intermediate that evolves to the native four-disulfide protein through oxidation of its two last free cysteines (see the text

430

TdPI Folding, Unfolding, and Inhibitory Activity Fig. 3. Oxidative folding of TdPI-short. RP-HPLC traces of acid-trapped intermediates that accumulate during the oxidative folding of TdPI-short. The reactions were carried out in Tris–HCl (pH 8.5) in the absence (Control −) and in the presence (Control +) of 0.25 mM 2-mercaptoethanol, 0.5 mM GSSG, or a mixture of 0.5 mM/1 mM GSSG/GSH. The retention times of the native (N) and fully reduced/ unfolded (R) forms are indicated. XS stands for an ensemble of species with X number of disulfide bonds. IIIa is a major three-disulfide intermediate comprising nonnative disulfide bonds.

below). Nonetheless, the overall folding process was efficient, with almost complete recovery of the protein in native conformation after 5 h of reaction (Control − and Control +). As expected, the addition of an oxidizing agent [oxidized glutathione (GSSG)] favored the formation of one-disulfide, two-disulfide, and three-disulfide intermediates, thereby strongly accelerating the folding reaction (15 min versus 5 h). Interestingly, it also changed the folding landscape, preventing the accumulation of the IIIa intermediate. TdPI-short also folds through the sequential formation of one-disulfide, two-disulfide, and three-disulfide intermediates that finally give rise to the native form. The chromatographic profile of the intermediates is reminiscent of that of TdPI-long, although the amounts are somewhat smaller (Figs. 2 and 3). However, the folding process of the short variant was less efficient than that of TdPI-long and required more than 7 h to reach completion (Control − and Control +). The slowest step of the folding reaction was the formation of intermediates that lead to the three-disulfide population (i.e., the initial steps of folding). Accordingly, the presence of an oxidizing agent in the buffer strongly accelerated the formation of the native protein (30 min versus 7 h) by promoting oxidation within the different disulfide populations. In addition, it led to a larger accumulation of IIIa as compared to the folding of TdPI-long under the same conditions. As with the long variant, the presence of a reducing agent accelerated the folding process by promoting disulfide rearrangement within the one-disulfide, two-disulfide, and three-disulfide populations. Reductive unfolding of TdPI-long and TdPI-short Native TdPI-long and TdPI-short were reduced in Tris–HCl (pH 8.5), using dithiothreitol (DTT), or in sodium acetate (pH 4.5), using Tris(2-carboxyethyl) phosphine (TCEP). The intermediates that accumu-

lated during the unfolding reaction were trapped by acidification in a time-course manner and analyzed by RP-HPLC. TdPI-long and TdPI-short showed similar chromatographic profiles under both reducing conditions (Fig. 4a and b) (i.e., the use of different reducing agents and buffers had no major effect on the pattern of accumulating intermediates). The most noticeable fractions of intermediates that arose during the unfolding reactions were derivatized with vinylpyridine and analyzed by MS. The analysis of both TdPI variants revealed the successive accumulation of three-disulfide, two-disulfide, and one-disulfide species before the fully reduced and unfolded form is achieved. Only small amounts of one-disulfide intermediates were detected. Importantly, the major three-disulfide intermediate that was generated during the reductive unfolding of both TdPI variants, termed IIIb, was distinct from the intermediate that accumulated during oxidative folding (IIIa), although their elution times in RPHPLC were similar. IIIb appeared during reduction at pH 8.5 and pH 4.5, with the latter condition preventing disulfide reshuffling, and may therefore comprise three native disulfide bonds (see the text below). In the reductive unfolding experiments, TdPI-short was about twice as resistant to reduction as TdPI-long. Disulfide scrambling of TdPI-long and TdPI-short Native TdPI-long and TdPI-short were incubated in Tris–HCl (pH 8.5) in the presence of 2-mercaptoethanol (as thiol initiator) and increasing concentrations of guanidine hydrochloride (GdnHCl) or guanidine thiocyanate (GdnSCN). The protein mixtures were allowed to reach equilibrium, trapped by acidification, and analyzed by RP-HPLC. In this approach, the unfolding promoted by the denaturant is reflected by the extent of conversion of the native protein into scrambled isomers. A similar population of scrambled isomers was observed for

TdPI Folding, Unfolding, and Inhibitory Activity

431

Fig. 4. Reductive unfolding and disulfide scrambling of TdPI-long and TdPI-short. RP-HPLC traces of acid-trapped intermediates that accumulate during the reductive unfolding of TdPI-long (a) and TdPI-short (b). The native protein was reduced with increasing concentrations of DTT in Tris–HCl (pH 8.5) or with increasing concentrations of TCEP in sodium acetate (pH 4.5). N and R stand for the native and fully reduced/unfolded forms, respectively. XS represents fractions of species with X number of disulfide bonds. IIIb is a major three-disulfide intermediate comprising only native disulfide bonds. RP-HPLC traces of the acid-trapped intermediates of TdPI-long (c) and TdPI-short (d) generated by disulfide scrambling. The native protein was denatured by incubation for 20 h in Tris–HCl (pH 8.5) containing 0.25 mM 2mercaptoethanol (as thiol initiator) and the indicated concentration of GdnSCN. The retention times of the native form (N) and the scrambled population (4S) are indicated.

both TdPI variants (Fig. 4c and d). In addition, both variants showed a similar midpoint denaturant concentration of approximately 4.2 M and 1.2 M for GdnHCl and GdnSCN, respectively. Stop/Go folding of the major intermediates of TdPI-long and TdPI-short The IIIa and IIIb intermediates of TdPI-long and TdPI-short were isolated and allowed to resume folding in Tris–HCl (pH 8.5) in the absence and in the presence of different redox agents. The timecourse RP-HPLC analysis of the reactions showed the formation of the native protein from both

intermediates, with similar efficiencies (Fig. 5a and b). As expected, RP-HPLC did not clarify whether the formation of the native protein is direct because both intermediates elute similarly. However, the use of an oxidizing agent (i.e., conditions that prevent disulfide reshuffling) indicated that IIIb is a productive intermediate because of its immediate conversion into the native form, which is much faster than the conversion of IIIa. The intermediates of the long and short variants behave equally (data not shown), suggesting that IIIb is a productive intermediate in the folding of both TdPI-long and TdPI-short. The finding of a native-like disulfidebond pattern for IIIb (see NMR Characterization of

Fig. 5. Stop/Go folding of the major intermediates of TdPI-long. The IIIa (a) and IIIb (b) intermediates of TdPI-long were isolated, lyophilized, and dissolved in Tris–HCl (pH 8.5) in the absence (Control −) and in the presence (Control +) of 0.25 mM 2-mercaptoethanol or 0.5 mM GSSG to reinitiate the folding reaction. Folding intermediates were subsequently trapped by acidification over time and analyzed by RP-HPLC. N denotes the native form.

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TdPI Folding, Unfolding, and Inhibitory Activity

Fig. 6. One-dimensional 1 H NMR spectra of the native TdPIshort and its major intermediates. The spectra of the native (N) and intermediate (IIIa and IIIb) forms of TdPI-short were recorded in H2O or D2O at pH 2.0 and 298 K.

the Major Folding Intermediates of TdPI-Short) is consistent with these results and further supports the role of this species as a productive intermediate. In contrast, all the experiments indicate that IIIa has a nonnative disulfide-bond pattern and is thus a nonproductive intermediate. NMR characterization of the major folding intermediates of TdPI-short The equivalence between the intermediates from TdPI-long folding and the intermediates from TdPIshort folding prompted the NMR analysis of the IIIa and IIIb intermediates of the short variant. Onedimensional 1H spectra were collected in H2O or D2O for the native TdPI-short and its two intermediates, revealing good peak dispersion for the native protein and IIIb, as well as a profile more characteristic of an unstructured protein for IIIa (Fig. 6). The strong similarity of the spectra of the native protein and IIIb indicates that both forms display a similar fold. The one-dimensional analysis also shows that the peak corresponding to the proton H α of residue Glu56 in the spectrum of the native TdPI-short has

no correspondence in the spectrum of IIIb, suggesting structural differences around this region. Furthermore, the variation of H α chemical shifts between the native protein and IIIb reveals that the major differences are close to residues Cys38 and Cys58 (data not shown). Several intercysteine nuclear Overhauser enhancement (NOE) connectivities confirm the disulfide-bond pairings previously reported for native TdPI (Fig. 1d): Cys24(H β)-Cys51 (H β ) (Ia), Cys47(H β )-Cys73(H β ,H α ) (Ib), Cys38 (H β )-Cys58(H β ) (II), and Cys52(H β )-Cys69(H β ) (III). In addition, the absence of NOE cross-peaks between Cys38 and Cys58 and surrounding residues demonstrates the lack of this disulfide bond in IIIb. These results suggest that the native protein and the IIIb intermediate have a similar structure, with differences around Cys38 and Cys58, and that IIIa has a highly disordered conformation that prevents the determination of its structure. The structure of the native TdPI-short in solution was first determined as a reference under acidic conditions, where the disulfide intermediates are stable. The structure of the IIIb intermediate was further determined by taking advantage of the

433

TdPI Folding, Unfolding, and Inhibitory Activity Table 1. Structural statistics of the 20 best NMR structures of the native TdPI-short and its IIIb folding intermediate NOE distance constraints Short-range distances (i − j) ≥ 1 Medium-range distances (i − j) b 5 Long-range distances (i − j) ≥ 5 Total Final CYANA target function valuea (Å2) AMBER energy (kcal/mol) Maximum violation (Å) Violations N0.25 Å Bond lengths (Å) Bond angles (°) RMSD to mean coordinates (Å) Backbone N, Cα, C′ All heavy atoms Ramachandran plot statisticsb Most favorable regions (%) Additionally allowed regions (%) Generously allowed regions (%) Disallowed regions (%) a b

Native

IIIb

403 119 189 711 0.10 ± 0.03

404 83 110 597 0.37 ± 0.06

−2228 ± 13 −2165 ± 12 0.19 0.22 0/20 0/20 0.0103 0.0105 ± 0.0001 ± 0.0001 1.90 ± 0.04 1.96 ± 0.04 (residues 23–74) 0.77 1.63 1.18 2.19 78.2 20.9 0.9 0.0

76.6 22.4 1.0 0.0

Average values over the 20 final CYANA conformers. Calculated with PROCHECK-NMR.34

assignment on the native protein. Data on the quality and precision of the 20 energy-minimized conformers representing both structures are summarized in Table 1. About 13 NOE distance constraints per residue were used in the final structure calculations of the native protein, and about 11 constraints were used for IIIb. The ensembles of the NMR structures are consistent with the experimental data, with no violations of NOE distance constraints greater than 0.25 Å. The precision of the structures is characterized by the low root-mean-square deviations (RMSDs) (0.77 Å and 1.63 Å for the native protein and IIIb, respectively), and the quality of the structures is reflected by the high percentage of (Φ,Ψ) backbone torsion-angle pairs found in the most favored regions of the Ramachandran plot (78.2% and 76.6% for the native protein and IIIb, respectively). The structure of the native TdPI-short shows the same overall fold as in the crystal structure, 17 with an RMSD of 0.8 Å for C α atoms. The structure resembles an arrowhead consisting of an initial long loop (L1), followed by an anti-parallel β-sheet (strands β1 and β2) that is connected to the Cterminal α-helix (α1) by another loop (L2) (Fig. 7a and c). The conformation adopted by the canonical Lys39 is similar to that found in the crystal structure. Notably, the structure of the native TdPI-short shows two main differences compared with the typical Kunitz domain: (i) the N-terminus is farther away from the C-terminal α-helix, which widens the base of the protein and contributes to the flatness of the beginning of loop L1; and (ii) loop L2 is

significantly shortened (Fig. 1c). The superposition of the crystal structure of the native protein and the 20 best NMR structures of the IIIb intermediate shows an RMSD of 1.8 Å for C α atoms (Fig. 7b and c). Although the structure of IIIb maintains a nativelike conformation with three native disulfide bonds, it shows significant differences around the two free cysteines (Cys38 and Cys58) that belong to the trypsin-binding region: loop L1 and strand β2 are farther apart, so the overall arrowhead shape of the molecule is lost. The deviation angle between the β2 strand of the native protein's crystal structure and IIIb's solution structure is 54 ± 14°. Consistently, Lys39 adopts different conformations in each of the 20 calculated NMR structures, indicating increased flexibility in this region. Inhibitory activity of TdPI-long and TdPI-short The inhibitory properties of the TdPI variants were examined using bovine trypsin and human tryptase β. Titration experiments performed at high trypsin concentrations (200 nM; [E] NN Ki) showed that both recombinant TdPI-long and TdPI-short are fully active as inhibitors (N 95% of theoretical value), thereby verifying that their fold was functional. Steady-state kinetics revealed that the inhibitory properties of both variants towards trypsin are identical, with equilibrium dissociation constants Ki in the nanomolar range (Table 2). In contrast, the affinity of TdPI-short for tryptase β was much lower than that of TdPI-long, with Ki values of 1300 nM and 0.02 nM, respectively. Even at high concentrations, a residual tryptase β activity of 25–30% was observed for both variants (Fig. 8).

Discussion Groundbreaking work using the model proteins BPTI, RNase A, and hirudin laid the foundation for the study of the oxidative folding of small disulfiderich proteins. 36 The folding pathways of the numerous disulfide-containing proteins that have since been analyzed are surprisingly diverse, 37,38 greatly hampering the prediction of folding reactions and motivating further work in the field. Here we applied the methods developed by the laboratories of Creighton, Scheraga, and Chang to comprehensively examine the oxidative folding of TdPI (TdPI-long) and its Kunitz head (TdPI-short). Both variants fold through the stepwise generation of the heterogeneous populations of one-disulfide, two-disulfide, and three-disulfide intermediates that finally give rise to the native form (see schematic diagram in Fig. 9). The conversion of the mixture of three-disulfide species into a productive intermediate constitutes a major rate-limiting step in the folding of both TdPI variants. This step resembles

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TdPI Folding, Unfolding, and Inhibitory Activity

Fig. 7. NMR structures of the native TdPI-short and its IIIb intermediate. Stereo-view superposition of (a) the crystal structure of TdPI (black; PDB code: 2UUX) and the 20 best NMR structures of the native TdPI-short (dark blue); (b) the crystal structure of TdPI (black) and the 20 best NMR structures of the IIIb folding intermediate (light blue); and (c) the crystal structure of TdPI (black) and the 20 best NMR structures of both the native TdPIshort (dark blue) and the IIIb folding intermediate (light blue). The N-terminus and the C-terminus, the regular secondary structure elements, and the canonical lysine residue (red or orange) are labeled. The cysteine residues are shown in dark or light green and labeled according to the following disulfide-bond patterns: Cys24Cys51 (Ia), Cys47-Cys73 (Ib), Cys38-Cys58 (II), and Cys52-Cys69 (III).

TdPI Folding, Unfolding, and Inhibitory Activity

435

Table 2. Inhibition constants of TdPI-long and TdPI-short Tryptase β

Trypsin

TdPI-long TdPI-short

Ki (nM)

Ki (nM)

Residual activity(%)

0.3 ± 0.02 0.3 ± 0.01

0.02 ± 0.009 1300 ± 150

30 ± 2 25 ± 3

The equilibrium dissociation constant Ki of the complex with trypsin and tryptase β and the residual tryptase β activity at high inhibitor concentrations are given as mean ± SEM (n ≥ 3).

the disulfide reshuffling that takes place within the scrambled population in the folding of TAP and hirudin. 11 Indeed, the mechanism of “trial and error” observed in the folding of TdPI-long and TdPI-short is reminiscent of that of the two aforementioned proteins. This contrasts the folding mechanism of BPTI and LDTI, where noncovalent interactions funnel the reaction towards the formation of a few native-like intermediates. 10,31 Despite this quasi-stochastic formation of intermediates, the folding of TdPI-long and TdPI-short is fast and efficient. This suggests that the lack of specific noncovalent interactions is compensated for by the fast formation and rearrangement of disulfide bonds within the different populations of intermediates and by the absence of scrambled isomers, which usually delay folding reactions. 39 Comparison of the oxidative folding of both TdPI variants reveals that the flexible prehead and posthead segments play an important role in the initial folding steps, favoring the recovery of the native protein. Their presence, however, does not affect the generation of the native protein from the three-

Fig. 8. Inhibition of trypsin and tryptase β by TdPI-long and TdPI-short. Trypsin (open symbols) and tryptase β (filled symbols) were incubated with increasing concentrations of TdPI-long (circles) or TdPI-short (squares), and residual activity was measured using a fluorogenic substrate. Data are presented as mean ± SEM (n ≥ 3). The inhibition curves were generated by fitting the data to an equation describing tight-binding inhibition (modified from Morrison 35).

Fig. 9. Oxidative folding of selected small disulfide-rich proteins. The major pathways for the oxidative folding (continuous arrows) and reductive unfolding (broken arrows) of BPTI, LDTI, TAP, hirudin, RNase A, and TdPI are represented schematically. R and N indicate the fully reduced/unfolded and native forms, respectively. The disulfide-bond pairings of folding/unfolding intermediates are shown inside parentheses; the missing disulfides are shown inside square brackets. 1S, 2S, 3S, and 4S are ensembles of molecules with the corresponding number of disulfide bonds. Asterisks refer to the ratelimiting steps of the folding reactions.

disulfide population (i.e., the subsequent step of disulfide reshuffling). In contrast, the related BPTI and the potato carboxypeptidase inhibitor fold at approximately the same rate, both in the absence and in the presence of the prehead region. 22,40 In TdPI, these flexible segments may influence the first stages of folding by promoting noncovalent interactions between N-terminal/C-terminal residues and the TdPI head. These interactions would be responsible for the larger accumulation of onedisulfide and two-disulfide intermediates in the folding of the long variant. For TdPI-short, the presence of oxidizing agents accelerates the overall folding reaction, rendering the native protein much faster but following the same folding pathway. In contrast, the presence of oxidizing agents during the folding of TdPI-long seems to change the overall folding pattern, with a larger amount of onedisulfide and two-disulfide intermediates that could directly lead to the formation of a productive three-disulfide intermediate without a significant accumulation of IIIa. Stop/Go folding and disulfidebond analyses support the hypothesis that IIIb is a productive intermediate while IIIa requires disulfide reshuffling through other three-disulfide species to achieve the native conformation. The attainment of

436 the native state via the formation of a productive intermediate is in line with the folding mechanism of BPTI, LDTI, and RNase A (Fig. 9). The folding of BPTI and LDTI proceeds through the formation of a productive two-disulfide species and, unlike TAP and hirudin, does not show an accumulation of scrambled isomers. Interestingly, the folding of TdPI and RNase A shares the presence of two major threedisulfide intermediates. In the case of TdPI, only one of them is a productive species, whereas in the case of RNase A, both of them are productive forms and lead to the formation of the native protein. 41–44 Pioneering work on BPTI and RNase A shed light on the structures of disulfide folding intermediates. The NMR structures of such intermediates were deduced from analogues in which the free cysteines had been mutated to alanines or serines. 36 Using NMR spectrometry, we characterized the acidtrapped and further isolated IIIa and IIIb intermediates from TdPI-short folding (i.e., we analyzed genuine intermediates). The NMR structure of IIIb reveals a highly native-like conformation, with the same secondary structure elements as the native state and with three native disulfide bonds. The major structural differences are found around the two free cysteines (Cys38 and Cys58), which show increased flexibility. This would allow them to be eventually in close proximity and thus oxidize and directly convert IIIb into the native protein. These data are in accordance with the NMR structures of the analogs of the two productive intermediates of RNase A and the genuine productive intermediate of a cyclic cystine knot protein. 45–47 Reductive unfolding and molecular dynamics simulations suggested that the last disulfide to be formed in these proteins is thermodynamically less stable than the other disulfide bonds. 45,48 This is also the case with TdPI because the missing disulfide bond in IIIb is the first one to be reduced in the native protein. In contrast, the analysis of IIIa evinces a highly disordered conformation, which is probably only sustained by the presence of the disulfide bonds, as reported for scrambled isomers. 39 Previous NMR studies on genuine nonproductive folding intermediates of leech carboxypeptidase inhibitor and LDTI also showed the presence of disordered regions, which accounted for the inability of these forms to convert directly into the native protein. 31,49 However, despite their accumulation, these intermediates are not dead-end species that are trapped indefinitely; they can reshuffle their solvent-accessible disulfide bonds and reenter the folding reaction. TdPI requires its unique disulfide-bond pattern to fold properly. Accordingly, a TdPI-short double mutant (Cys47Ala/Cys51Ala) constructed to mimic a typical three-disulfide Kunitz/BPTI head (see Fig. 1d) failed to reach a stable native-like conformation and aggregated during purification after expression in Escherichia coli (data not shown). This result

TdPI Folding, Unfolding, and Inhibitory Activity

highlights the difficulties in designing small disulfide-rich proteins, even in well-known families that share similar folds and disulfide-bond pairings. Although a simplified BPTI variant has been constructed, 50 modulating the folding rate and stability of such proteins is still a challenge, as is well illustrated by the extensive mutagenesis study performed on leech carboxypeptidase inhibitor. 51 TdPI itself is another example: TdPI-long folds faster than TdPI-short, but its disulfide stability is lower. The reduction of disulfide bonds in TdPI is reminiscent of that of BPTI, LDTI, and RNase A, and supports a correlation between the mechanism of oxidative folding and the mechanism of reductive unfolding: the accumulation of productive nativelike intermediates is associated with a sequential reduction of the native disulfide bonds (Fig. 9). In contrast, as found in TAP and hirudin, the accumulation of productive scrambled isomers correlates with the simultaneous reduction of the disulfides. On the other hand, disulfide scrambling experiments demonstrate that the flexible prehead and posthead segments do not contribute to protein stabilization, as the resistance of both TdPI variants to denaturing agents is similar. In this regard, TdPI is a highly stable protein, with midpoint denaturant concentrations very similar to those of TAP but significantly lower than those of BPTI. 52 This high resistance to denaturation, which may reflect an adaptation to resist harsh environmental conditions, has been observed for most of the small disulfiderich protease inhibitors analyzed to date. 37 Besides affecting folding and disulfide stability, the prehead and posthead segments of TdPI strongly influence the inhibitory properties of this molecule. TdPI-long and TdPI-short show similar inhibitory activity and affinity towards trypsin, as expected, since both variants share the same inhibitory loop and overall fold of the central Kunitz domain. In contrast, they exhibit strikingly different affinities towards human tryptase β: TdPI-long inhibits tryptase β with a Ki in the subnanomolar range, whereas the affinity of TdPI-short is more than 60,000-fold lower and within the micromolar range. The prehead and posthead segments of TdPI thus strongly contribute to the affinity towards tryptase β, most likely due to additional interactions with the protease, as suggested by modeling and truncation experiments. 17 Inhibition of tryptase β by both variants results in 25–30% residual activity towards peptide substrates even at the highest concentrations used, suggesting that the inhibitors affect only three of the four monomers of the active tryptase β tetramer. This unusual interaction, which is also observed for the inhibition of tryptase β by LDTI (the other known proteinaceous tryptase inhibitor), reflects steric hindrance in the accessibility of the monomers' active sites, which are located within the narrow central pore of the tetramer. 17,24,25,32

437

TdPI Folding, Unfolding, and Inhibitory Activity

In both BPTI and TdPI, the conserved disulfide bond located outside the protein core and involved in the trypsin-binding region is the last one to be formed during the folding process and the first one to be reduced during the unfolding reaction (Cys14Cys38 in BPTI and Cys38-Cys58 in TdPI) (Figs. 1 and 9). Studies using a BPTI Cys14Ser/Cys38Ser double mutant revealed that, in the absence of this disulfide bond, the inhibitor maintains its structure but is cleaved by trypsin much more rapidly. 53 This finding indicates that this disulfide bond greatly contributes to wild-type BPTI's high resistance to hydrolysis, thus preventing temporary inhibition as seen in other standard mechanism inhibitors. 54,55 A similar phenomenon is observed in the Kazal-type inhibitor LDTI. Its productive intermediate also shows an overall native-like structure, but the absence of a disulfide bond that tightens the reactive-site loop significantly increases the rate at which it is inactivated by trypsin. 31 Taken together, these results suggest a direct correlation between oxidative folding and inhibitory activity in both Kunitz and Kazal-type inhibitors, which are already active after the formation of native internal disulfide bonds but require the formation of the last external disulfide bond for long-term stability against proteolysis. An in-depth understanding of the folding of such serine protease inhibitors may thus foster the design of variants with improved properties that target, for example, human tryptase β and could alleviate allergic and inflammatory disorders such as asthma and rheumatoid arthritis. 21,56

Materials and Methods Protein expression and purification Genes coding for TdPI-long (UniProt code: Q1EG59; Asn1-Pro97; numbering does not take into account the 21residue signal peptide) and TdPI-short (Lys21-Gly75, with Asp added N-terminally) were synthesized by GENEART and cloned into a modified pET-32a vector. This vector attaches an N-terminal thioredoxin–His6 fusion tag, followed by a tobacco etch virus protease recognition site. Both proteins were produced by heterologous overexpression in E. coli Origami2 (DE3) cells (Novagen), which were grown at 37 °C in Luria–Bertani medium containing 100 μg/ml ampicillin and 10 μg/ml tetracycline. Each culture was induced at an OD550 (optical density at 550 nm) of 0.8 with 0.5 mM isopropyl-β-Dthiogalactopyranoside and incubated overnight at 18 °C. After centrifugation at 7000g for 30 min, the pellet was washed twice in 50 mM Tris–HCl and 500 mM NaCl (pH 8.0) and resuspended in the same buffer containing 10 mM imidazole, EDTA-Free Protease Inhibitor Cocktail Tablets (Roche Diagnostics), and DNase I (Roche Diagnostics). Cells were lysed at 4 °C with a cell disrupter (Constant Systems) at a pressure of 1.35 kbar, and cell debris was removed by centrifugation at 50,000g for 1 h at 4 °C. The supernatant was incubated with nickel-

nitrilotriacetic acid resin (Invitrogen) previously equilibrated with 50 mM Tris–HCl, 500 mM NaCl, and 10 mM imidazole (pH 8.0), and the fusion protein was subsequently eluted using the same buffer containing 500 mM imidazole. The sample was then dialyzed overnight at room temperature (RT) against 50 mM Tris–HCl and 250 mM NaCl (pH 8.0) in the presence of 0.5 mM/3 mM GSSG/GSH and His6-tagged tobacco etch virus protease at an enzyme/substrate ratio of 1:50 (wt/wt). After dialysis, the digested sample, comprising a glycine residue at the N-terminus, was passed through nickel-nitrilotriacetic acid resin several times to remove His6-tagged molecules. The flow-through was collected and further purified to homogeneity by RP-HPLC in a Waters Alliance apparatus using a 4.6-mm Jupiter C4 column (Phenomenex) and a linear gradient of 20–35% acetonitrile with 0.1% trifluoroacetic acid (TFA; Sigma) at a flow rate of 0.75 ml/min for 25 min. Protein identity and purity were assessed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS in a Bruker ultrafleXtreme mass spectrometer and by automated Edman degradation in an Applied Biosystems Procise 492 protein sequencer. The concentrations of TdPI-long and TdPIshort in solution were determined by measuring the absorbance at 280 nm considering theoretic absorption coefficients E0.1% of 1.83 and 2.13, respectively. The proteins were lyophilized and then stored. Oxidative folding Of the native protein, 0.5 mg was incubated in 0.5 M Tris–HCl (pH 8.5) containing 6 M GdnSCN and 200 mM DTT for 2–3 h at RT. To initiate folding, we loaded the fully reduced/unfolded protein onto a HiTrap desalting column connected to an ÄKTA Purifier System (GE Healthcare) that was previously equilibrated with 0.1 M Tris–HCl (pH 8.5). The protein was eluted in 1 ml of equilibration buffer (protein concentration ∼ 0.5 mg/ml) and incubated at RT both in the absence (Control −) and in the presence of redox agents: 0.25 mM 2-mercaptoethanol (Control +), 0.5 mM GSSG, or 0.5 mM/1 mM GSSG/GSH. To monitor folding, we removed the aliquots at various time points and quenched the reaction with 4% TFA. Acidtrapped intermediates were subsequently analyzed by RPHPLC, as detailed in Protein Expression and Purification. Stop/Go folding Acid-trapped intermediates were isolated by RP-HPLC, lyophilized, and allowed to reinitiate folding at 0.5 mg/ml in 0.1 M Tris–HCl (pH 8.5) at RT in the absence (Control −) and in the presence of redox agents: 0.25 mM 2mercaptoethanol (Control +), 0.5 mM GSSG, or 0.5 mM/1 mM GSSG/GSH. To monitor the Stop/Go reaction, we trapped the time-course aliquots of the samples with 4% TFA and analyzed them by RP-HPLC, as detailed in Protein Expression and Purification. Reductive unfolding The native protein (0.5 mg/ml) was dissolved at RT in either 0.1 M Tris–HCl (pH 8.5) or 0.1 M sodium acetate

438 (pH 4.5) containing increasing concentrations (0.5–100 mM) of DTT or TCEP (Sigma), respectively. To monitor the unfolding reaction, we trapped the time-course aliquots of the samples with 4% TFA and analyzed them by RP-HPLC, as detailed in Protein Expression and Purification. Disulfide scrambling The native protein (0.5 mg/ml) was dissolved in 0.1 M Tris–HCl (pH 8.5) containing 0.25 mM 2-mercaptoethanol and increasing concentrations of GdnHCl (0–8 M) or GdnSCN (0–6 M) and kept for 20 h at RT. The samples were then quenched with 4% TFA and analyzed by RPHPLC, as detailed in Protein Expression and Purification. Disulfide-bond content To count the disulfide bonds, we purified the acidtrapped intermediates by RP-HPLC under the conditions described in Protein Expression and Purification and lyophilized them further. Each sample was alkylated with 0.1 M Tris–HCl (pH 8.5) containing 0.1 M 4-vinylpyridine (Sigma) for 45 min at RT in the dark. The derivatized protein was then freed from reagents by RP-HPLC and analyzed by MALDI-TOF MS. Each free cysteine incorporates a vinylpyridine with a 105.1-Da increase in molecular mass. Samples were mixed (1:1, vol/vol) with a matrix solution of 10 mg/ml 2,6-dihydroxyacetophenone (Sigma) dissolved in 30% acetonitrile containing 20 mM dibasic ammonium citrate (pH 5.5). Then 0.5 μl of the mixture was spotted onto the MALDI-TOF MS plate using the dried-droplet method. Mass spectra were acquired in linear-mode geometry with N 1000 laser shots. The instrument was calibrated with a protein mixture ranging from 4 kDa to 20 kDa (Protein Calibration Standard I; Bruker Daltonics). NMR spectroscopy Samples for the native TdPI-short and the intermediates (IIIa and IIIb) were prepared at 0.5–1 mM protein concentration by dissolving the lyophilized material in 0.1% TFA containing either 10% or 100% D2O (pH 2.0) in standard 3-mm or 5-mm NMR tubes. The analysis was carried out under acidic conditions to prevent the oxidation of the free cysteines of the intermediates. For sequencespecific polypeptide backbone chemical shift assignments and structure calculations, homonuclear two-dimensional correlated spectroscopy, total correlated spectroscopy (mixing time, 80 ms), and NOE spectroscopy (NOESY) (mixing time, 120 ms) spectra were acquired at 298 K on a Bruker AV 800 spectrometer equipped with a 5-mm TCI tripleresonance cryoprobe. The number of data points was 2048 × 1024 in the direct and indirect dimensions, respectively, and the sweep width was 9615 Hz in both proton dimensions. All spectra were processed with the program TOPSPIN (Bruker Biospin) and analyzed using the program NMRView.57 One-dimensional spectra collected before and after the two-dimensional experiments were compared to verify the stability of the native and intermediate proteins over the NMR measurements. A strategy based only on homonuclear two-dimensional NMR spectroscopy 58 was used to assign the 1H NMR of the native TdPI-short and its

TdPI Folding, Unfolding, and Inhibitory Activity

IIIb intermediate. The distance constraints used for the structure calculations were derived from the homonuclear two-dimensional NOESY spectra. Although the IIIb intermediate was stable under the experimental conditions (pH 2.0), two sets of peaks were observed for some residues: one set from the intermediate and another much weaker set from the native form. In these cases, only peaks corresponding to the intermediate were used for structure calculations. The three-dimensional structures were determined by combined automated NOESY cross-peak assignment and structure calculation with torsion-angle dynamics, as implemented in the program CYANA. 59 A set of upper and lower distance limits was introduced for each pair of cysteine residues involved in disulfide bonds: 2.1/2.0 Å for S γ(i)–S γ(j) and 3.1/3.0 Å for C β(i)–S γ(j) and S γ(i)–C β(j). The compatibility of the disulfide-bond pattern identified in each protein 17 with the structures from the initial rounds of automated calculation was evaluated before these restraints were introduced in the subsequent structure calculations. Several intercysteine NOE connectivities further confirmed the accuracy of the disulfide-bond parings. Finally, a 2000step energy minimization with NMR distance constraints using a generalized Born solvent model was applied to the 20 conformers with the lowest values of the final CYANA target function, using the AMBER 9.0 program. 60 The 20 minimized conformers were used to validate the final structure with the program PROCHECK-NMR. 34 The program MOLMOL 61 was used to visualize the structures and to prepare the figures. Inhibition kinetics The concentrations of the inhibitory active TdPI variants and the equilibrium dissociation constants for the complexes with bovine trypsin and human tryptase β were determined essentially as described previously. 32,33,62 Briefly, bovine pancreatic trypsin (treated with N-p-tosylL-phenylalanine chloromethyl ketone) was standardized by titration with p-nitrophenyl-p′-guanidinobenzoate. 63 The TdPI variants were titrated with trypsin (200 nM), and the concentration was calculated by assuming a 1:1 interaction. Equilibrium dissociation constants Ki for the complexes of the inhibitors with trypsin and tryptase β (in the presence of 0.1 μg/ml heparin to stabilize the tetrameric enzyme) were determined using protease concentrations near or below Ki. 64 Initial velocities derived from three to six separate experiments were fitted to an equation describing “tight-binding” inhibition that was modified to accommodate residual enzymatic activity 32,35 with Prism (GraphPad). Accession numbers The atomic coordinates of the native TdPI-short and the IIIb intermediate have been deposited in the Protein Data Bank‡ under accession codes 2LFK and 2LFL, respectively. The NMR chemical shifts have been deposited in the BioMagResBank§ under accession numbers 17763 and 17779. ‡ www.pdb.org § www.bmrb.wisc.edu

TdPI Folding, Unfolding, and Inhibitory Activity

Acknowledgements We are grateful to Prof. F. Xavier Gomis-Rüth for the availability of laboratory facilities and helpful advice, to Sabine Streicher for excellent technical assistance, and to Robin Rycroft for language assistance. We also thank the Proteomics and Bioinformatics Facility of the Institut de Biotecnologia i de Biomedicina, a member of the ProteoRed-ISCIII network. This study was supported, in part, by projects FEDER UNAB08-4E-002 and CTQ2008-00080 of the Spanish Ministry of Science and Innovation, and by project SO 249/1-1 of the DFG Priority Program 1394 “Mast Cells— Promotors of Health and Modulators of Disease.” J.L.A. is beneficiary of a JAE postdoctoral contract from the Consejo Superior de Investigaciones Científicas.

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