Role of disulfide bonds in conformational stability and folding of 5′-deoxy-5′-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus

Biochimica et Biophysica Acta 1824 (2012) 1136–1143

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Role of disulfide bonds in conformational stability and folding of 5′-deoxy-5′-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus Giovanna Cacciapuoti a, b,⁎, Francesca Fuccio a, Luigi Petraccone c, Pompea Del Vecchio c, Marina Porcelli a, b a b c

Dipartimento di Biochimica e Biofisica “F. Cedrangolo”, Seconda Università di Napoli, Via Costantinopoli 16, 80138 Napoli, Italy Consorzio Interuniversitario Biostrutture e Biosistemi “INBB”, Viale Medaglie d'Oro 305, Roma, Italy Dipartimento di Chimica “Paolo Corradini”, Università di Napoli “Federico II”, Via Cintia, 80126 Napoli, Italy

a r t i c l e

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Article history: Received 14 March 2012 Received in revised form 1 June 2012 Accepted 21 June 2012 Available online 29 June 2012 Keywords: 5′-deoxy-5′-methylthioadenosine phosphorylase II from Sulfolobus solfataricus Hyperthermophilic protein Reversible chemical and thermal unfolding Conformational stability Disulfide bond

a b s t r a c t Sulfolobus solfataricus 5′-deoxy-5′-melthylthioadenosine phosphorylase II (SsMTAPII), is a hyperthermophilic hexameric protein with two intrasubunit disulfide bonds (C138–C205 and C200–C262) and a CXC motif (C259–C261). To get information on the role played by these covalent links in stability and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S mutants was studied by thermal and guanidinium chloride (GdmCl)-induced unfolding and analyzed by fluorescence spectroscopy, circular dichroism, and SDS-PAGE. No thermal unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while in the presence of the reducing agent Tris-(2-carboxyethyl) phosphine (TCEP), a Tm of 100 °C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Different from the wild-type, C262S and C259S/C261S show complete thermal denaturation curves with sigmoidal transitions centered at 102 °C and 99 °C respectively. Under reducing conditions these values decrease by 4 °C and 8 °C respectively, highlighting the important role exerted by the CXC disulfide on enzyme thermostability. The contribution of disulfide bonds to the conformational stability of SsMTAPII was further assessed by GdmCl-induced unfolding experiments carried out under reducing and nonreducing conditions. Thermal unfolding was found to be reversible if the protein was heated in the presence of TCEP up to 90 °C but irreversible above this temperature because of aggregation. In analogy, only chemical unfolding carried out in the presence of reducing agents resulted in a reversible process suggesting that disulfide bonds play a role in enzyme denaturation. Thermal and chemical unfolding of SsMTAPII occur with dissociation of the native hexameric state into denatured monomers, as indicated by SDS-PAGE. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Proteins from hyperthermophilic organisms that grow optimally at temperatures between 80 °C and 100 °C, offer a unique opportunity for the study and understanding of the mechanisms governing protein stability and thermodynamics [1–6]. Although these proteins have nearly identical sequences and overall structures compared to their mesophilic counterpart, they are much more resistant to thermal

Abbreviations: MTAP, 5′-deoxy-5′-methylthioadenosine phosphorylase; SsMTAPII, 5′-deoxy-5′-methylthioadenosine phosphorylase II from Sulfolobus solfataricus; PNP, purine nucleoside phosphorylase; MTA, 5′-deoxy-5′-methylthioadenosine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CD, circular dichroism; [θ], molar ellipticity per mean residue; GdmCl, guanidinium chloride; PIPES buffer, piperazine-N,N′-bis(ethanesulfonic acid); TCEP, Tris-(2-carboxyethyl) phosphine ⁎ Corresponding author at: Dipartimento di Biochimica e Biofisica “F. Cedrangolo”, Seconda Università di Napoli, Via Costantinopoli 16, 80138, Napoli Italy. Tel.: +39 081 5667519; fax: +39 081 5667519. E-mail address: [email protected] (G. Cacciapuoti). 1570-9639/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2012.06.014

denaturation and inactivation. Based on the elucidation of a large number of three-dimensional structures of hyperthermophilic proteins, several strategies of thermal stabilization have been proposed [1–4,7–10]. However, although many theoretical and experimental studies have been carried out in the past, no general mechanism for increased thermostability was established to date and the molecular basis of protein thermostability remains rather elusive. It has been recently reported that some proteins from hyperthermophiles are characterized by remarkably slow unfolding rates and it has been suggested that a kinetic barrier towards unfolding may be a common strategy used by many proteins to stand against extreme conditions [11,12]. In recent years computational, structural, and biochemical studies have highlighted the critical role of disulfide bonds in the stabilization of intracellular proteins in some hyperthermophilic Archaea and Bacteria [13–20]. In this work, 5′-deoxy-5′-methylthioadenosine phosphorylase II from the archaeon Sulfolobus solfataricus (SsMTAPII), a hyperthermophilic protein with multiple intrasubunit disulfide bonds, on the basis

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of its exceptionally thermal properties, has been used as model to study the mechanisms of how nature adapts the structure of proteins to survive in the extreme environmental conditions keeping the delicate balance between stability and conformational flexibility. SsMTAPII (EC 2.4.2.28), catalyzes the reversible phosphorolysis of 5′-deoxy-5′-methylthioadenosine (MTA), a sulfur-containing nucleoside generated from S-adenosylmethionine mainly through polyamine biosynthesis [21]. SsMTAPII is a member of the hexameric class of purine nucleoside phosphorylases (PNP), ubiquitous enzymes of purine metabolism that function in the salvage pathway [22,23]. SsMTAPII, whose tridimensional structure in complex with MTA and sulfate has recently been solved to 1.45 Ǻ resolution (PDB entry code 2A8Y) is a tightly packed protein of 180 kDa [19]. The crystal structure reveals that SsMTAPII is a hexamer containing six identical subunits which can be described as a dimer-of-trimers with one active site per monomer. The overall fold of SsMTAPII consists of a single α/β domain. Viewing down the 3-fold axis, the bottom trimer rotates anti clockwise with respect the top trimer by about 55 °C. The oligomeric assembly of the trimer and the monomer topology of SsMTAPII are almost identical with trimeric human 5′-deoxy-5′-methylthioadenosine phosphorylase (MTAP) [24]. Therefore, SsMTAPII can be considered the first reported hexamer in the trimeric class of PNPs from Archaea. There are seven cysteine residues per monomer. Four cysteine residues form two pairs of intrasubunit disulfide bridges, Cys138–Cys205 and Cys200–Cys262, near the C-terminus of the protein. In each monomer, the disulfide bridge formed by C138–C205 is located at >13 Ǻ of distance from the intersubunit interfaces while the disulfide bridge C200–C262 is less than 10 Ǻ away from the intersubunit interface between two adjacent monomers composing the trimer. Several residues right before Cys262, including Cys259 and Cys261, are disordered and invisible in the electron density map. The seventh cysteine, Cys164, is located near the protein surface but not directly exposed to the solvent. Each subunit contains in its C-terminal region a CXC motif (i.e. a couple of cysteine residues separated by one neighboring amino acid X) as a typical feature. This motif is very close to the two pairs of disulfide bridges that are separated at a distance of 8.5 Ǻ from the centers of disulfide bonds. By enzymatic activity measurements it has been demonstrated that the substitution of these cysteine residues (Cys259 and Cys261) with serine results in a significant lowering of thermal stability parameters of SsMTAPII [18] and that this unusual CXC disulfide is necessary to obtain a complete reversibility from the unfolded state of the protein [25]. SsMTAPII shares 51% identity with human MTAP but, unlike this enzyme which is highly specific for MTA, it also accepts adenosine as a substrate [18]. The biochemical characterization of SsMTAPII has highlighted features of exceptional thermophilicity and thermostability. SsMTAPII is highly thermoactive with an optimum temperature of 120 °C. Short term kinetics of thermal denaturation and the kinetics after prolonged incubation at high temperatures evidenced the extreme thermostability of this enzyme with an apparent melting temperature at 112 °C and a notably high stability toward irreversible thermal inactivation processes. Moreover, SsMTAPII possess a remarkable resistance to SDS, proteolytic cleavage, and guanidinium chloride (GdmCl)-induced unfolding [18]. To elucidate the stabilization mechanisms of hyperthermophilic proteins, the stability in solution should be quantitatively determined and the energetic aspects stabilizing the proteins from a thermodynamic point of view should be elucidated. The measurement of parameters associated with protein thermodynamic stability requires the protein unfolding be fully reversible. Hyperthermostable proteins, however, unfold irreversibly under most in vitro conditions [26–28] as a consequence of formation of insoluble aggregates due to the sticky, hydrophobic nature of the exposed surfaces of the unfolded protein. Therefore, only few of such proteins, mainly the small single-domain proteins, have been characterized in terms of their thermodynamic stability profiles [29].

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In this study, the conformational stability of SsMTAPII has been investigated, and the role of protein disulfide bonds was highlighted by a comparative analysis of the chemical and thermal stability of the wild-type enzyme and its C262S and C259S/C261S mutants. Moreover, the reversible GdmCl- and temperature-induced unfolding of SsMTAPII was analyzed by far-UV circular dichroism (CD) spectroscopy and by activity measurements and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This is the first report, to the best of our knowledge, on the reversible unfolding of a hyperthermostable oligomeric protein with multiple disulfide bonds. 2. Experimental procedures 2.1. Materials Dithiothreitol, GdmCl 8 M, and Tris-(2-carboxyethyl) phosphine (TCEP) 0.5 M solutions were from Sigma-Aldrich. PIPES was supplied by Applichem (Darmstadt, Germany). [methyl- 14C] S-adenosylmethionine (50–60 mCi/mmol) was supplied by the Radiochemical Centre (Amersham Bioscience, U.K.). MTA and 5′-deoxy-5′-[methyl- 14C] MTA were prepared from unlabeled and labeled S-adenosylmethionine [30]. All buffers and solutions were prepared with ultra-high quality water. All reagents were of the purest commercial grade. 2.2. Enzyme preparation and assay Recombinant SsMTAPII and its C262S and C259S/C261S mutant forms were expressed in E. coli BL21 (lDE3) cells and purified with heat-treatment and MTA-Sepharose chromatography, according to Cacciapuoti et al. [18]. Manipulations of DNA and E. coli were carried out using standard protocols [18,31]. Protein was analyzed by SDS-PAGE and quantified with the Bradford assay [32] or estimated spectrophotometrically using the theoretical sequence-based coefficient of 33,265 M − 1 cm − 1 calculated at 280 nm for the hexameric protein [33]. SDS-PAGE was carried out as described by Weber et al. [34]. MTAP activity was determined by measuring the formation of [methyl- 14C]5-methylthioribose-1-phosphate from 5′-[methyl- 14C] MTA [18]. In all enzymatic assays the amount of the protein was adjusted so that no more than 10% of the substrate was converted to product and the reaction rate was strictly linear as a function of time and protein concentration. 2.3. Circular dichroism spectroscopy CD spectra were recorded with a Jasco J-715 spectropolarimeter equipped with a Peltier type temperature control system (Model PTC-348WI). The spectropolarimeter was calibrated with an aqueous solution of d-10-(+)-camphorsulfonic acid at 290 nm [35]. Molar ellipticity per mean residue, [θ] in deg·cm2·dmol −1, was calculated from the equation: [θ] = [θ]obs·mrw·(10·l·C) −1, where [θ]obs is the ellipticity measured in degrees, mrw is the mean residue molecular weight (111.6 Da), C is the protein concentration in g/ml and l is the optical path length of the cell in cm. CD spectra in the far-UV region were recorded from 190 nm to 250 nm in a 0.1 cm path length cell. A protein stock solution of each protein (0.5 mg/ml) in piperazine-N, N′-bis(ethanesulfonic acid) (PIPES buffer) 10 mM, pH 7.4, was diluted into buffer to achieve a normalized starting ellipticity of −13.0, using a final protein concentration of 0.2 mg/ml. Near-UV CD spectra were recorded from 250 nm to 320 nm using 2 mg/ml protein samples and 0.5 cm path length cells. CD spectra were recorded at 25 °C with a time constant of 4 s, a 2 nm bandwidth and a scan rate of 20 nm/min; the signal was averaged over at least three scans and baseline corrected by subtracting a buffer spectrum. Spectra were analyzed for secondary structure amount according to the Selcon method [36] using the on-line

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Circular Dichroism Website DICHROWEB [37]. The GdmCl-induced denaturation curves, at fixed constant temperature of 25 °C, were obtained by recording the CD signal at 222 nm for the samples containing increasing amounts of GdmCl. Thermal unfolding curves were recorded in the temperature mode, over the range 50–108 °C, in PIPES buffer, pH 7.4, by following the change of the CD signal at 222 nm, with a scanning rate of 1.0 °C min−1. Reduction of protein disulfide bonds within SsMTAPII and its mutants was achieved via incubation with TCEP, that was the reducing agent utilized in these studies because of its general compatibility with spectroscopic measurements. 2.4. Fluorescence spectroscopy Steady-state fluorescence measurements were made with a JASCO FP-750 apparatus equipped with a circulating water bath to keep the cell holders at constant temperature of 25 °C. Fluorescence emission spectra were recorded between 300 and 450 nm (1 nm sampling interval) with the excitation wavelength set at 295 nm or at 280 nm. The experiments were performed by using a 1 cm sealed cell and a 5 nm emission slit width and corrected for background signal. The protein concentration ranged from 0.05 to 0.1 mg/ml. 2.5. Chemical denaturation and renaturation Stock solutions of SsMTAPII and its mutants were prepared in PIPES buffer 10 mM, pH 7.4. A commercial 8 M GdmCl solution from Sigma was utilized as denaturant solvent. For equilibrium transition studies stock solutions of GdmCl, in different amounts were mixed with protein solutions to give a constant final value of the protein concentration. The final concentration of GdmCl was in the range 0–6 M. Each sample was incubated at 25 °C for 22 h in the presence or in the absence of 15 mM dithiothreitol. Longer incubation times led to identical spectroscopic signals. The GdmCl-induced unfolding curves, at a fixed constant temperature of 25 °C, were obtained by recording the CD signal at 222 nm, the shift in fluorescence maximum wavelength after excitation at 295 nm, the quenching of fluorescence intensity at 329 nm after excitation at 295 nm, the quenching of fluorescence intensity at 328 nm after excitation at 280 nm, and the shift in fluorescence maximum wavelength after excitation at 280 nm. To test the reversibility of GdmCl-induced unfolding process, the protein (final concentration 0.125 mg/ml) was unfolded by incubation for 22 h at 25 °C in the presence of GdmCl 6 M concentration in PIPES buffer 10 mM, pH 7.4, containing 15 mM dithiothreitol. The unfolding was probed by recording the intrinsic fluorescence emission upon excitation at 290 nm. The refolding was started by 20-fold dilution of the unfolding mixture in PIPES buffer 10 mM, pH 7.4 at 25 °C. The final concentration of GdmCl in the renaturation mixture was 0.3 M whereas the protein concentration was about 6 μg/ml. The refolded enzyme, after extensive dialysis against PIPES buffer 10 mM, pH 7.4 until complete removal of GdmCl, was analyzed by far-UV CD spectra, intrinsic fluorescence emission, catalytic activity measurements under standard conditions, and SDS-PAGE analysis. CD and fluorescence spectra of the native and renatured protein were monitored using equal concentration of both samples. 2.6. Thermal unfolding/refolding Reversible thermal unfolding experiments were carried out in a Jasco J-810 spectropolarimeter. For thermal scans, the protein samples, 0.2 mg/ml in PIPES buffer 10 mM, pH 7.4, containing TCEP 20 mM were heated from 25 °C to 90 °C and subsequently cooled to 25 °C with heating/cooling rate of 1 °C/min by a Jasco programmable Peltier element. The values of denaturation temperature are calculated as the midpoint of each sigmoidal denaturation curve. Denaturation was monitored by far-UV CD spectra (190–250 nm) and SDS-PAGE analysis.

Renaturation was induced by extensive dialysis of the protein against PIPES buffer 10 mM, pH 7.4 and monitored by comparing the CD spectrum and the SDS-PAGE of the protein after dialysis with a protein sample before denaturation. 3. Results and discussion 3.1. Spectroscopic characterization The structural properties of SsMTAPII and its C262S and C259S/C261S mutants, lacking the disulfide Cys200–Cys262 and the CXC motif respectively, have been assessed by various spectroscopic techniques. In Fig. 1A, the far-UV CD spectra of SsMTAPII and its mutant forms recorded in PIPES buffer 10 mM, pH 7.4 at 25 °C are reported. The positive band at 195 nm and the broad negative band centered at 222 nm are indicative of the presence of both α- and β-secondary structure elements, respectively. Analysis of the far-UV CD data resulted in a secondary structure determination of 25% α-helix, 20% β-sheet and 55% of unordered segments. These values agree with the three-dimensional crystal structure of the wild-type protein [19]. Moreover, the far-UV CD spectra of native enzyme and its mutants were indistinguishable, having the same overall shape, thus indicating that the three proteins shared the same secondary structure. In order to detect any tertiary structural differences among wild-type SsMTAPII and its mutants, near-UV CD spectra were recorded in PIPES buffer 10 mM, pH 7.4 at 25 °C. CD bands in the near-UV region (250–320 nm) originate from aromatic amino acids. Disulfide group also contributes significant ellipticity in this region, showing two transitions that give broad absorption bands between 250 and 260 nm [38]. The wavelength at which the maximum absorption of the disulfide chromophore occurs is strongly conformation-dependent. Moreover, the sign of the disulfide CD band can be related to the chirality of the disulfide bridge [38]. As shown in Fig. 1B, the near-UV CD spectra are dominated by a strong negative signal at 259 nm possibly ascribed to the effect of protein disulfides as chromophores [39,40]. The almost complete overlap of the three spectra indicates that the tertiary structure of SsMTAPII monomers was maintained at pH 7.4 in the wild-type as well as in its mutants suggesting that the three proteins are isomorphous in their native states and that the removal of the disulfide bond causes no significant change in their native three-dimensional structure as also indicated by the observations that the mutant forms are fully active under native conditions. Fluorescence spectra of SsMTAPII and the two mutant forms, recorded after excitation at 295 nm, show a maximum at 329 nm (Fig. 1C). In the presence of 4 M GdmCl a significant quenching of fluorescence intensity is observable and the maximum shifts around 353 nm for SsMTAPII. If the excitation is done at 280 nm to include the contribution of Tyr residues, the results are very similar: the maximum shifts from 328 nm to around 353 nm with a significant quenching of fluorescence intensity (data not shown). These findings indicate that: (a) the three Trp residues present in each subunit at positions 77 (α1), 86 (β5), and 179 (α3), are well buried in the native structure and become solvent-exposed upon GdmCl-induced unfolding. This result is in agreement with the three-dimensional structure of SsMTAPII, showing that all Trp residues have an accessibility to the solvent less than 10% of their global volume [19]; (b) there is a significant excitation energy transfer between the eleven Tyr residues and the three Trp residues present in each subunit of the hexameric structure [41]. 3.2. Thermal stability A striking structural feature of SsMTAPII that accounts for its extraordinary thermoactivity and thermostability is the presence of an unusual CXC motif and two pairs of intrasubunit disulfide bridges near the C-terminus in each subunit. Disulfide bridges are critical

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posttranslational modifications of proteins. These covalent links not only stabilize protein structures by lowering the entropy of the unfolded polypeptide, but also are required for the proper folding and biological activity of several proteins. Biochemical and genomic analysis demonstrates that disulfides are common in the intracellular proteins of many hyperthermophilic organisms, where their frequencies correlate with increased growth temperatures [42], suggesting a contribution to protein thermostability. Disulfide bridges were shown to play a crucial role for the stability of SsMTAPII as their removal by reduction or mutation results in a stability loss [18]. To analyze the stability of SsMTAPII and its mutants against thermal denaturation, far-UV CD spectra in the 190–250 nm region were recorded and the effect of temperature on the secondary structure was studied by monitoring the variation of the observed ellipticity at 222 nm in a temperature range from 50 °C to 108 °C in the presence and in the absence of the reducing agent TCEP. The numeric values of the calculated denaturation temperatures are reported in Table 1. As shown in Fig. 2A, the temperature-induced unfolding transition of SsMTAPII is not completed up to 108 °C, the maximum operative temperature of our CD instrument. This result is in line with the extreme thermostability of SsMTAPII inferred from enzymatic activity measurements [18]. At 108 °C the molar ellipticity of SsMTAPII is close to 0 (see inset in Fig. 2A) suggesting that most of the protein is aggregated. In fact, after cooling, the solution becomes not transparent. In order to shift the midpoint of the thermal transition to lower temperatures, we tried to destabilize the native state of SsMTAPII by reducing its multiple disulfide bridges. Therefore, the experiment was carried out in the presence of 10 mM TCEP. As shown in Fig. 2A, under these experimental conditions, we were able to measure a Tm of 100 °C. It is interesting to note that the far-UV CD spectrum of the protein recorded at 108 °C under reducing conditions shows features of a denatured protein with a marked loss of secondary structure elements, even though it does not correspond to that of a random coil (inset in Fig. 2A). This result confirms that the conformational stability of SsMTAPII depends upon its multiple disulfide bridges and suggests that, in the presence of these covalent links, the protein retains its conformation until extremely high temperatures where denaturation and degradation become concomitant processes. As also reported in the inset in Fig. 2A, the far-UV CD spectra of SsMTAPII recorded at 25 °C in the presence and in the absence of TCEP appear superimposable, indicating that SsMTAPII maintains a folded conformation in the presence of the reducing agent at this temperature. This result confirms the literature data reporting that SsMTAPII remains fully stable and active after preincubation with extremely high concentrations of dithiothreitol up to 50 °C [18]. Differently from the wild-type, the two mutants are characterized by complete thermal denaturation curves, whose profiles show a sharp sigmoidal transition centered at 102 °C for the C262S mutant and at 99 °C for C259S/C261S mutant (Fig. 2B). It is interesting to note that this result is in agreement with the data from kinetic measurements of catalytic activity after short time incubation as a function of temperature, demonstrating that the single and the double mutation cause an increasing destabilization for the folded structure of SsMTAPII by lowering the apparent Tm value by 6 °C and 10 °C respectively [18]. As expected, the presence of 10 mM TCEP causes a further destabilizing effect (Fig. 2B). The temperature denaturation values, indeed, remarkably decrease, from 102 °C to 98 °C for C262S mutant and from 99 °C to 91 °C for C259S/C261S mutant indicating that the double mutant, i.e. the mutant lacking the structural CXC motif, has more impact on the thermostability of SsMTAPII than the single mutant.

Fig. 1. Far-UV CD (panel A) and near-UV CD (panel B) spectra of SsMTAPII (▲) and the mutants C262S (◆) and C259S/C261S (■) in PIPES buffer 10 mM, pH 7.4 at 25 °C. Fluorescence spectra after excitation at 295 nm (panel C) of SsMTAPII (▲) C262S (◆), C259S/C261S (■) and SsMTAPII in the presence of 4 M GdmCl (○).

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Table 1 Values of the denaturation temperature for SsMTAPII and the two mutant forms, C262S and C259S–C261S, determined by recording the molar ellipticity at 222 nm at protein concentration of 0.065 mg/ml in (a) PIPES buffer 10 mM, pH 7.4 and (b) PIPES buffer 10 mM, pH 7.4, containing TCEP 10 mM. Enzyme

a Td (°C)

b Td (°C)

SsMTAPII C262S C259S/C261S

Not measurable 102 99

100 98 91

The errors on Td are ±1 °C.

The stabilizing role of CXC disulfide appears to be rather intriguing. One would expect, in fact, that disulfide bonds contribute to the thermostability when they join residues far apart in primary structure. In SsMTAPII, instead, Cys259 and Cys261 are separated by a single residue of small size, i.e. serine. Therefore, the stability imparted to the native protein structure could be almost marginal. It is interesting to note, however, that the two cysteine residues in the CXC sequence form a strained 11-membered disulfide ring that represents a good redox

Fig. 2. (A) Thermal denaturation curves of SsMTAPII in the absence (■) and in the presence (□) of TCEP 10 mM. The curves were obtained by recording the changes in the molar ellipticity at 222 nm as a function of temperature. In the inset, the far-UV CD spectra of SsMTAPII recorded at 25 °C in the absence (■) and in the presence (□) of TCEP 10 mM and at 108 °C in the absence (△) and in the presence (▲) of TCEP 10 mM are reported. (B) Thermal denaturation curves of C262S in the absence (□) and in the presence (■) of TCEP 10 mM and C259S/C261S in the absence (△) and in the presence (▲) of TCEP 10 mM. The curves were obtained by recording the changes in the molar ellipticity at 222 nm as a function of temperature. The enzymes were at fixed concentration of 0.065 mg/ml in PIPES buffer 10 mM, pH 7.4.

agent [43]. Following these observations, it has been recently demonstrated that the CXC motif plays a specific functional role in stabilizing disulfide-containing proteins, such as SsMTAPII, by acting as a redox reagent in restoring the native state of the other reduced or scrambled structural disulfides of the enzyme [25]. 3.3. Chemical stability To analyze the chemical stability of SsMTAPII and to point out the role of disulfide bonds we performed equilibrium transition studies by incubating the enzyme and its disulfide-lacking mutants at increasing GdmCl concentrations in PIPES buffer 10 mM, pH 7.4. Fig. 3 shows the GdmCl-induced unfolding curves of the wild-type and the two mutant proteins obtained by recording the CD signal at 222 nm (Fig. 3A), and the shift in the wavelength of the maximum of fluorescence spectrum after excitation at 295 nm (Fig. 3B). For comparing the denaturant-induced unfolding curves, the experimental values were normalized reporting the fraction of unfolded protein (fU) as a function of the concentration of the denaturing agent. The GdmCl-induced unfolding curves of SsMTAPII and its mutant forms have also been constructed by recording the quenching of fluorescence intensity at 329 nm after excitation at 295 nm, the quenching of fluorescence intensity at 328 nm after excitation at 280 nm, and the shift of the wavelength corresponding to the maximum of fluorescence spectrum after excitation at 280 nm (data not shown). All together these probes should allow us to monitor the unfolding of the secondary and tertiary structures of SsMTAPII and its mutant forms. The results obtained indicate that the experimental GdmCl-induced unfolding curves show, in all cases, a sharp sigmoidal shape indicative of a cooperative unfolding process, thus suggesting that the destruction of the different structure levels occurs in a simultaneous manner. The values of GdmCl concentration at half completion of the unfolding transition are collected in Table 2. A quick glance to the numbers emphasizes that: i) the unfolding transitions of the mutant proteins occurred at lower concentrations of GdmCl with respect to the wild-type suggesting the involvement of disulfide bridges in the stabilization of the enzyme; ii) the double mutant Cys259Ser/Cys261Ser shows even weaker resistance towards chemical denaturation compared to the single mutant, indicating a specific role played by this unusual CXC disulfide; iii) the [GdmCl]1/2 values are practically identical regardless of the experimental probe used, suggesting that the unfolding process is so cooperative to be represented as a two-state N6 ⇆ 6D transition, where N6 is the native hexameric structure and D represents the unfolded subunit. The contribution of disulfide bonds to the structural stability of SsMTAPII was further assessed by means of unfolding experiments with GdmCl as denaturant in the presence of TCEP 10 mM. In fact, as shown in Fig. 3A, under reducing conditions the apparent midpoint of the transition for GdmCl shifts from 3.2 M to 2.6 M. It is interesting to note that this last value is identical to that of the C259S/C261S mutant measured under nonreducing conditions. This result resembles that obtained from thermal unfolding experiments. Also in this case, indeed, the Tm value measured for SsMTAPII in the presence of TCEP 10 mM (100 °C) is very close to that determined for the C259S/C261S mutant under nonreducing conditions (99 °C), highlighting a significant role of the unusual C259–C261 disulfide on the stability of SsMTAPII. 3.4. Thermal and chemical unfolding require reducing agents for reversibility As demonstrated by thermal stability experiments, the temperatureinduced unfolding transition of SsMTAPII is not completed up to 108 °C and is irreversible, as deduced by a pronounced aggregation of the unfolded state. Since the presence of reducing agents has proven to be able to decrease the thermal melting temperature of the enzyme thus

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prolonged dialysis of the sample, closely matched that of the native protein, indicating the reversibility of the process (Fig. 4). The reversibility of the thermal unfolding was also probed by SDS-PAGE. The inset in Fig. 4 compares the electrophoretic pattern of the native, denatured, and refolded enzyme. The samples were subjected to SDS-PAGE without boiling and under nonreducing conditions in order to obtain a picture of the protein species present. After heating until 90 °C in the presence of TCEP, only the monomer of SsMTAPII is observable (lane 2) suggesting that the reduction of the protein disulfide bonds has made possible the unfolding of the monomers and their dissociation. After refolding (lane 3), the enzyme migrates as a single band of 180 kDa corresponding to the molecular mass of the hexameric SsMTAPII (lane 1) thus indicating the reversibility of the process. The re-heating of a sample previously heated gave a superimposable melting profile thus confirming the reversibility of the process. In analogy with the temperature-induced denaturation, only the GdmCl-induced unfolding carried out under reducing conditions proves to be reversible. The unfolded protein sample, in fact, regains all the spectroscopic features of the folded structure after suitable dilution and prolonged dialysis and gives rise to GdmCl-induced unfolding curves superimposable to those of the native enzyme (data not shown). Also in this case, owing to the stability of SsMTAPII towards SDS 2% at room temperature, it has been possible to monitor the recovery of the native structure by SDS-PAGE. 4. Conclusions

Fig. 3. GdmCl-induced unfolding of SsMTAPII and its mutant forms. The curves were obtained by recording: A) the changes in the molar ellipticity at 222 nm as a function of GdmCl concentration of SsMTAPII in the absence (●) and in the presence (○) of TCEP 10 mM, C262S (■), and C259S/C261S (▲); B) the shift in the wavelength of the maximum of fluorescence spectrum after excitation at 295 nm as a function of GdmCl concentration of SsMTAPII (●), C262S (■), and C259S/C261S (▲). The enzymes were at a fixed concentration of 0.15 mg/ml in PIPES buffer 10 mM, pH 7.4. Three independent measurements were performed for each experimental condition. The reported data are the mean values from such measurements. The uncertainties are within 5% of the reported number.

avoiding the overlap between denaturation and inactivation at high temperatures, we try to ascertain the reversibility of the thermal unfolding of SsMTAPII in the presence of TCEP. Under these experimental conditions, when the temperature was raised above 90 °C, the protein was irreversibly denatured as indicated by aggregation observed when the temperature was slowly lowered down to 25 °C. On the contrary, when refolding was carried out starting from 90 °C, the solution remained transparent and the CD spectrum, registered at 25 °C after

SsMTAPII is a hexameric hyperthermophilic and hyperthermostable protein. Inspection of the three-dimensional structure reveals two striking structural features that may account for the extraordinary thermoactivity and thermostability of this enzyme: the unique dimerof-trimers quaternary structure, characterized by more than 50% buried surface area for each subunit, and the presence of a CXC motif and two pairs of intrasubunit disulfide bridges at the C-terminus [19]. Three important considerations can be derived from the spectroscopic characterization of SsMTAPII and its disulfide-lacking mutants and from thermal and GdmCl-induced unfolding/refolding experiments: i) the almost complete overlap of near- and far-UV CD spectra of the three proteins together with previous investigations demonstrating that disulfide bonds are not required for SsMTAPII activity [18], suggest that disulfide bridges are probably not essential for the correct folding into the catalytically active conformation but, instead, they are crucial for the structural stability of the enzyme; ii) in the absence of the C259–C261 disulfide, SsMTAPII behaves like the full-reduced enzyme. This confirms previous results demonstrating that CXC motif is needed to maintain the protein disulfide bridges in their native state [25] by acting as a functional mimic of protein disulfide isomerase, the most efficient known catalyst of oxidative protein folding [44]. It is interesting to note that a CGC motif in thiol oxidase Erv2p from yeast [45], which is located on a flexible C-terminal segment was found to be involved in the exchange of the de novo synthesized disulfide bridge with substrate protein [45]. The observation that in analogy with Erv2, the CXC motif in SsMTAPII is part of a flexible C-terminal segment and is very close to the two pairs of disulfide bridges [18,19] further strengthens this hypothesis. iii) Chemical and thermal unfolding of

Table 2 Values of the GdmCl concentration in molar units at half-completion of the unfolding transition for SsMTAPII and the two mutant forms, C262S and C259S–C261S, determined by CD and fluorescence measurements at 25 °C in PIPES buffer 10 mM, pH 7.4. Enzyme

[θ]222nm

I329nm λexc = 295nm

λmax λexc = 295nm

I328nm λexc = 280nm

λmax λexc = 280nm

SsMTAPII C262S C259S/C261S

3.2 3.0 2.6

3.2 3.0 2.6

3.2 3.0 2.5

3.2 3.0 2.6

3.2 3.0 2.6

Three independent measurements were performed for each experimental condition. The reported data are the mean values from such measurements. The uncertainties are within 5% of the reported number.

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Fig. 4. Far-UV CD spectrum of SsMTAPII (○) is compared to that of the protein thermally denatured up to 90 °C in PIPES buffer 10 mM, pH 7.4 containing TCEP 20 mM and renatured by slowly cooling to room temperature and prolonged dialysis against PIPES buffer 10 mM, pH 7.4 (●). The inset shows the SDS-PAGE pattern of native, unfolded, and refolded SsMTAPII. The samples (5 μg) were subjected to SDS-PAGE without boiling and under nonreducing conditions. Lane 1, SsMTAPII; lane 2, thermally unfolded SsMTAPII; lane 3, refolded SsMTAPII; M, molecular mass markers.

SsMTAPII are irreversible processes. Nevertheless, when unfolding experiments were carried out under reducing conditions, we were able to highlight the reversible dissociation of the hexamer to monomers and to detect the reversibly unfolded species by SDS-PAGE (Fig. 4). In conclusion, SsMTAPII could serve a good model to study the refolding process of a hexameric enzyme, as only few oligomeric proteins regain completely both structure and activity upon refolding. The reported results provide convincing evidence that disulfide bonds play a key role in the conformational stability of SsMTAPII. In fact, the loss of these covalent links by reduction or mutation, weakening the tightly compact structure of the enzyme, allows the reversible transition from the native to the denatured state before covalent modifications, induced by high temperatures and chemical denaturants, may cause the irreversible protein degradation. Experiments aimed to quantify the thermodynamic contribution of disulfide bonds to the conformational stability of SsMTAPII are in progress in our laboratory. Acknowledgements This research was supported by grants from “Ministero dell'Istruzione, dell'Università e della Ricerca”, PRIN 2008 and from “Regione Campania”, L.R. n. 5/2002. References [1] S. Kumar, S. Nussinov, How do thermophilic proteins deal with heat? Cell Mol. Life Sci. 58 (2001) 1216–1233. [2] R. Ladenstein, G. Antranikian, Proteins from hyperthermophiles: stability and enzymatic catalysis close to the boiling point of water, Adv. Biochem. Eng. Biotechnol. 612 (1998) 37–85. [3] R. Sterner, W. Liebl, Thermophilic adaptation of proteins, Crit. Rev. Biochem. Mol. Biol. 36 (2001) 39–106. [4] C. Vieille, G.J. Zeikus, Hyperthermophilic enzymes: sources, uses and molecular mechanisms for thermostability, Microbiol. Mol. Biol. Rev. 65 (2001) 1–43. [5] A. Mukaiyama, K. Takano, Slow unfolding of monomeric proteins from hyperthermophiles with raversible unfolding, Int. J. Mol. Sci. 10 (2009) 1369–1385. [6] A. Razvi, J.M. Scholtz, Lessons in stability from thermophilic proteins, Protein Sci. 15 (2006) 1560–1578. [7] S. Kumar, C.J. Tsai, R. Nussinov, Factors enhancing protein thermostability, Protein Eng. 13 (2000) 179–191. [8] G. Vogt, S. Woell, P. Argos, Protein thermal stability, hydrogen bonds and ion pairs, J. Mol. Biol. 269 (1977) 631–643. [9] L. Xiao, B. Honig, Electrostatic contributions to the stability of hyperthermophilic proteins, J. Mol. Biol. 289 (1999) 1435–1444.

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