Báo cáo khoa học: Purine nucleoside phosphorylases from hyperthermophilic Archaea require a CXC motif for stability and folding pot

Purine nucleoside phosphorylases from hyperthermophilic Archaea require a CXC motif for stability and folding Giovanna Cacciapuoti, Iolanda Peluso, Francesca Fuccio and Marina Porcelli Department of Biochemistry and Biophysics ‘F. Cedrangolo’, Second University of Naples, Italy Introduction In the fascinating field of protein biochemistry, ther- mostability and folding comprise important factors that are currently gaining wide attention. Disulfide bonds represent an important structural feature of many proteins, especially extracellular ones. They 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. Disulfide bond formation occurs in the endoplasmic reticulum and mitochondrial inter- membrane space of eukaryotes and in the periplasm of prokaryotes [1]. Disulfide bonds are a typical feature of secretory proteins and are considered to contribute significantly to their overall stability [2]. By contrast, in intracellular proteins from well-known organisms, and as a result of the reductive chemical environment inside the cells [3], the presence of these covalent links is limited to proteins involved in the mechanism of the response to redox stress [4] or to proteins catalyz- ing oxidation–reduction processes [1,5]. Despite this classical view, recent computational, structural and biochemical studies have highlighted the critical role of Keywords 5¢-deoxy-5¢-methylthioadenosine phosphorylase; CXC motif and oxidative protein folding; disulfide bonds; hyperthermophilic proteins; purine nucleoside phosphorylase Correspondence G. Cacciapuoti, Dipartimento di Biochimica e Biofisica ‘F. Cedrangolo’, Seconda Universita ` di Napoli, Via Costantinopoli 16, 80138 Napoli, Italy Fax: +39 081 5667519 Tel: +39 081 5667519 E-mail: giovanna.cacciapuoti@unina2.it (Received 5 June 2009, revised 22 July 2009, accepted 29 July 2009) doi:10.1111/j.1742-4658.2009.07247.x 5¢-Deoxy-5¢-methylthioadenosine phosphorylase II from Sulfolobus solfatari- cus (SsMTAPII) and purine nucleoside phosphorylase from Pyrococcus furiosus (PfPNP) are hyperthermophilic purine nucleoside phosphorylases stabilized by intrasubunit disulfide bonds. In their C-terminus, both enzymes harbour a CXC motif analogous to the CXXC motif present at the active site of eukaryotic protein disulfide isomerase. By monitoring the refolding of SsMTAPII, PfPNP and their mutants lacking the C-terminal cysteine pair after guanidine-induced unfolding, we demonstrated that the CXC motif is required for the folding of these enzymes. Moreover, two synthesized CXC-containing peptides with the same amino acid sequences present in the C-terminal regions of SsMTAPII and PfPNP were found to act as in vitro catalysts of oxidative folding. These small peptides are involved in the folding of partially refolded SsMTAPII, PfPNP and their CXC-lacking mutants, with a concomitant recovery of their catalytic activ- ity, thus indicating that the CXC motif is necessary to obtain complete reversibility from the unfolded state of the two proteins. The two CXC- containing peptides are also able to reactivate scrambled RNaseA. The data obtained in the present study represent the first example of how the CXC motif improves both stability and folding in hyperthermophilic proteins with disulfide bonds. Abbreviations AdoMet, S-adenosylmethionine; GdnCl, guanidinium chloride; GSH, glutathione; GSSG, glutathione disulfide; MTA, 5¢-deoxy-5¢- methylthioadenosine; MTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase; PDI, protein disulfide isomerase; PfCGC, NH 2 -RRCGCKD- COOH; PfPNP, purine nucleoside phosphorylase from Pyrococcus furiosus; PNP, purine nucleoside phosphorylase; sRNaseA, scrambled RNaseA; SsCSC, NH 2 -GSCSCCN-COOH; SsMTAPII, 5¢-deoxy-5¢-methylthioadenosine phosphorylase II from Sulfolobus solfataricus. FEBS Journal 276 (2009) 5799–5805 ª 2009 The Authors Journal compilation ª 2009 FEBS 5799 these covalent links in the structural stabilization of intracellular proteins in some hyperthermophilic Archaea and Bacteria [6–12]. The abundance of disulfides observed across hyper- thermophilic organisms also has stimulated research into the identification of the biochemical mechanisms related to disulfide maintenance. It was recently dem- onstrated that specific protein disulfide oxidoreducta- ses, which are structurally and functionally related to protein disulfide isomerase (PDI) [13], play a key role in intracellular disulfide shuffling in hyperthermophilic proteins [14]. Two enzymes, 5¢-deoxy-5¢-methylthioadenosine pho- sphorylase II from Sulfolobus solfataricus (SsMTAPII) [10,11] and purine nucleoside phosphorylase from Pyrococccus furiosus (PfPNP) [12], have been isolated and characterized from hyperthermophilic Archaea. These enzymes are members of purine nucleoside phos- phorylases (PNP), comprising ubiquitous enzymes of purine metabolism that function in the salvage path- way of the cells [15]. SsMTAPII is a homohexamer (subunit 30 kDa) characterized by extremely high affinity towards 5¢-deoxy-5¢-methylthioadenosine (MTA), a natural sulfur-containing nucleoside formed by S-adenosylmethionine (AdoMet) mainly through polyamine biosynthesis [16]. SsMTAPII shares 51% identity with human 5¢-deoxy-5¢-methylthioadenosine phosphorylase (MTAP) and is able to recognize adeno- sine [10], in contrast to human MTAP which is strictly specific for MTA [17]. PfPNP displays a much higher similarity with MTAP than with PNP family members [12]. Similar to human PNP [18], PfPNP shows an absolute specificity for inosine and guanosine [12]. SsMTAPII and PfPNP show features of exceptional thermophilicity and thermostability and are character- ized by stabilizing disulfide bonds. Two pairs of intra- subunit disulfide bridges have been revealed by the crystal structure of SsMTAPII [11] and three pairs of intrasubunit disulfide bridges have been assigned to PfPNP by integrating biochemical methodologies with MS [12]. It is interesting to note that SsMTAPII and PfPNP contain, in their C-terminal region, an unusual CXC motif (i.e. cysteines separated by one neighbouring amino acid X) as a typical feature, and that the substi- tution of these two cysteines with serines significantly affects both their thermodynamic and kinetic stability [10,12], suggesting the involvement of the cysteine pair in the thermal stabilization of both enzymes. In the present study, we demonstrate that the CXC motif of SsMTAPII and PfPNP plays an important functional role in the oxidative folding of these enzymes and that two short CXC-containing peptides with amino acid sequences corresponding to those present in the C-terminal region of SsMTAPII and PfPNP, respectively, act in vitro as functional mimics of PDI. The presence of a CXC motif in hyperthermo- philic proteins with multiple disulfide bonds, such as SsMTAPII and PfPNP, represents the first example of a new molecular strategy adopted by these enzymes to improve their stability and to preserve their folded state under extreme conditions. Results and Discussion SsMTAPII and PfPNP require a CXC motif to preserve their folded state SsMTAPII and PfPNP are highly thermoactive, with an optimum temperature of 120 °C, and extremely thermostable, with apparent T m values of 112 °C and 110 °C, respectively. These enzymes are also character- ized by a remarkable kinetic stability and resistance to many chemicals, including SDS and guanidinium chlo- ride (GdnCl) [10,12]. As demonstrated by structural and biochemical studies, SsMTAPII and PfPNP utilize multiple intrasubunit disulfide bridges as a principal mechanism to achieve superior levels of stability [11,12]. A striking structural feature of these two enzymes is the presence in their C-terminal region of a cysteine pair, organized in an unusual CXC sequence motif. This structural motif plays an important role in thermal stability because the substitution of cysteine with serine results in a significant lowering of the thermodynamic and kinetic stability parameters of SsMTAPII and PfPNP [10,12]. The stabilizing role of the CXC disulfide appears almost intriguing. Indeed, disulfide bonds are expected to make a higher contri- bution to thermostability when they join residues far apart in the primary structure. Instead, in SsMTAPII and PfPNP, they are separated by a single residue of small size (i.e. serine and glycine, respectively). Therefore, the stability imparted to the native protein structure could be almost marginal. On the basis of these observations, it is possible to hypothesize that CXC plays an important functional role in stabilizing the protein through an oxidative folding mechanism involving the other structural disulfides of the enzyme. Although rare in nature, few examples of oxidized CXC have been reported in the literature, including CSC in the Mengo virus coat protein [19], CSC in the L40C mutant of NADH peroxidase [20], CDC in AK1 protease from Bacillus sp. [21], CTC in chaperone Hsp33 from Escherichia coli [22], CPC in redox-regu- lated import receptor Mia40 [23,24] and CGC in MTAP from P. furiosus [9], which is highly homo- CXC and oxidative protein folding G. Cacciapuoti et al. 5800 FEBS Journal 276 (2009) 5799–5805 ª 2009 The Authors Journal compilation ª 2009 FEBS logous to SsMTAPII and PfPNP. It is interesting to note that a CGC motif at the C-terminus of the yeast thiol oxidase Erv2p was found to be involved in the exchange of the de novo synthesized disulfide bridge with substrate protein [25]. Moreover, it was demon- strated that a synthesized CGC peptide and a CGC motif in a mutant of E. coli thioredoxin were func- tional mimics of PDI [26]. Taken together, these data allowed us to speculate that the CXC motif in SsM- TAPII and PfPNP could act as a redox reagent and exert its stabilizing role by rescuing, in analogy with PDI, any possible damage of the other disulfide bonds of the protein. To demonstrate the active role of CXC motif in the oxidative folding process, we carried out the unfolding of SsMTAPII, PfPNP and their CXC- lacking mutants by incubation for 22 h at 25 °C with 6 m GdnCl in 20 mm Tris ⁄ HCl (pH 7.4), containing 30 mm dithiothreitol. The reversibility of the GdnCl- induced unfolding was then examined by assaying the catalytic activity after complete removal of the denaturant. As shown in Fig. 1, SsMTAPII and PfPNP are able to refold with a recovery of catalytic activity of 59% and 90%, respectively, compared to their control enzymes. By contrast, lower values of reactivation were observed for SsMTAPIIC259S ⁄ C261S and PfPNPC 254S ⁄ C256S, the two CXC-lacking mutants (25% and 46% activity, respectively). These results demonstrate that the CXC motif is necessary to obtain almost com- plete reversibility from the unfolded state, and suggest that the cysteine pair is able to act as redox reagent in the rearrangement of scrambled disulfide bonds, thus contributing to the recovery of native and biologically active enzyme. The observation that, in analogy with Erv2p [25], the CXC motif is part of a flexible C-termi- nal segment in SsMTAPII and in PfPNP [10,12], and that the CXC motif is very close to the two pairs of disulfide bridges in SsMTAPII [11], further strengthens this hypothesis. The data obtained in the present study represent the first example of how the CXC motif improves stability and folding in hyperthermophilic disulfide-containing proteins and could provide useful information with respect to engineering stable proteins and enzymes for therapeutic and industrial applications. From the sequence comparison of the C-terminal region of several PNPs present in databases, it appears that, despite their remarkable amino acid sequence identity, the CXC motif is conserved only in hyper- thermophilic enzymes, whereas it is absent in their mesophilic counterparts (Fig. 2). This observation suggests a specific role of this structural motif in the stability of hyperthermophilic PNPs against extreme temperature and allows the hypothesis that the CXC disulfide could represent an additional aspect (i.e. besides protein disulfide oxidoreductase protein) of the complex system involved in disulfide bond mainte- nance in hyperthermophilic organisms. NH 2 -GSCSCCN-COOH (SsCSC) and NH 2 -RRCGCKD-COOH (PfCGC) act as in vitro catalysts of oxidative protein folding PDI, the most efficient known catalyst of oxidative folding, is a multifunctional eukaryotic enzyme that utilizes the active site motif CGHC to catalyze the for- mation of native disulfides and the rearrangement of incorrect disulfide bonds, especially those within kineti- cally trapped, structured folding intermediates [13]. In recent years, interest in protein folding in vitro has expanded rapidly, mainly focusing on the production, in bacteria, of disulfide-containing proteins with poten- tial biotechnological applications. Therefore, increased attention has been paid to the design and synthesis of novel, small-molecule reagents that could improve the efficiency of the oxidative folding process. Recently, on the basis of the physical properties of PDI, a variety of CXXC peptides have been synthesized and assayed [27]. The active site of PDI has also been modeled as a SsMTAPII SsMTAPII C259S/C261S PfPNP PfPNP C254S/C256S 100 60 80 SsMTAPII C259S/C261S PfPNP C254S/C256S 0 20 40 Relative activity (%) Fig. 1. Refolding of SsMTAPII, PfPNP and their CXC-lacking mutants after Gdn-induced unfolding. The reversibility of Gdn- induced unfolding of MTAPII, PfPNP and their respective mutants was started by a 20-fold dilution of the unfolding mixture and extensive dialysis until complete removal of GdnCl. Refolding was analyzed by catalytic activity measurements performed under stan- dard conditions. U ⁄ R indicates SsMTAPII, PfPNP and their mutants refolded after GdnCl-induced unfolding. The activity of control enzymes was expressed as 100%. Each value is the average of three separate experiments. G. Cacciapuoti et al. CXC and oxidative protein folding FEBS Journal 276 (2009) 5799–5805 ª 2009 The Authors Journal compilation ª 2009 FEBS 5801 CGC peptide, a molecule that, upon oxidation, forms a strained 11-membered disulfide ring, representing a good oxidizing agent [26]. This peptide shows a disul- fide reduction potential close to that of PDI and its first thiol pK a is less than that of the natural redox reagent glutathione. Therefore, this CXC peptide is able to function as an efficient catalyst of disulfide isomerization [26]. On the basis of these observations, and in analogy with PDI, it is possible to hypothesize a nucleophilic attack of the thiolate from CXC on an incorrect protein disulfide followed by a thiol–disulfide interchange within the substrate, leading in turn to a native disulfide. Two CXC-containing peptides, namely SsCSC and PfCGC, whose amino acid sequences are identical to those present in the C-terminal region of SsMTAPII and PfPNP, respectively, have been synthe- sized and their disulfide isomerase activity has been assayed utilizing the partially refolded forms of SsMTAPII, PfPNP and their CXC-lacking mutants as substrates. As shown in Fig. 3, both peptides are involved in the oxidative folding of these enzymes with a concomitant recovery of their catalytic activity. Indeed, after 22 h of incubation in the presence of SsCSC and PfCGC, the enzymatic activity of SsMTAPII and its mutant, expressed as a percentage of their control enzymes, reaches 86.8% and 51.8%, and 68% and 49.3%, respectively (Fig. 3A). Similar results were obtained when the unfolded ⁄ refolded forms of PfPNP and its mutant were assayed under the same experimental conditions (Fig. 3B). It is inter- esting to note that, although PfPNP and its mutant show a higher reactivation values than SsMTAPII and its mutant (Fig. 3), the ratio of these values with respect of their control enzymes is similar, thus indicat- ing that the efficiency of the process is comparable. These data demonstrate that the CXC motif of SsM- TAPII and PfPNP is active, even when isolated from the proteins, and that it is able to induce the in vitro oxidative folding of these enzymes. To further confirm the ability of SsCSC and PfCGC to function as efficient catalysts of oxidative protein folding, the disulfide isomerase activity of these CXC- containing peptides was assayed by monitoring the reactivation of scrambled RNaseA (sRNaseA). As shown in Fig. 4, after 210 min of incubation in the presence of SsCSC and PfCGC, inactive sRNaseA shows a 5.5-fold and 3.6-fold activation, respectively, compared to the 8.9-fold activation observed in the presence of PDI. These results demonstrate that the two CXC-containing peptides can act in the same way Fig. 2. Multiple sequence alignment of C-terminal regions of hyperthermophilic and mesophilic PNPs. The CXC motif is shown in white lettering on a black background. 100 AB SsMTAPII SsMTAPII C259S/C261S PfPNP PfPNP C254S/C256S 20 40 60 80 0 U|R U/R + PDI U/R + SsCSC U/R + PfCGC U|R U/R + PDI U/R + SsCS C U/R + PfCGC Relative activity (%) Fig. 3. Effect of CXC-containing peptides on the reactivation of refolded SsMTAPII, PfPNP and their mutants. SsMTAPII, PfPNP and their mutants, partially refolded after GdnCl-induced unfolding, (U ⁄ R), were incu- bated for 22 h at 30 °C in the presence of various oxidative folding catalysts (reactiva- tion assay). The catalytic activity of (A) SsM- TAPII and SsMTAPIIC259S ⁄ C261S and (B) PfPNP and PfPNPC254S ⁄ C256S was then measured under standard assay conditions. The activity of control enzymes was expressed as 100%. Each value is the average of three separate experiments. CXC and oxidative protein folding G. Cacciapuoti et al. 5802 FEBS Journal 276 (2009) 5799–5805 ª 2009 The Authors Journal compilation ª 2009 FEBS as PDI, catalyzing the rearrangement of incorrect disulfide bonds in a protein substrate. It is interesting to note that SsCSC displays a higher oxidative folding activity than PfCGC (Figs 3 and 4), suggesting that the presence in SsCSC of a third thiol in the sequence CXCC could most likely enhance the reactivity toward disulfide bonds. In conclusion, the results obtained in the present study provide insight into the variety of molecular mechanisms utilized for stabilizing folded proteins under extreme thermal environments and allow us to speculate that disulfide bonds and the CXC motif could combine together to provide a novel stabilization strategy. Experimental procedures Materials Glutathione disulfide (GSSG), glutathione (GSH), bovine liver PDI and bovine pancreatic sRNaseA were obtained from Sigma. GdnCl and dithiothreitol were obtained from Applichem (Darmstadt, Germany). [methyl- 14 C]AdoMet (50–60 mCiÆmmol )1 was supplied by the Radiochemical Centre (Amersham Bioscience, Little Chalfont, UK). MTA and 5¢-[methyl- 14 C]MTA were prepared from unlabeled and labeled AdoMet [10]. Specifically synthesized CXC-contain- ing peptides, PfCGC and SsCSC, were obtained from PRIMM (Milan, Italy). All reagents were of the purest commercial grade. Expression and purification of SsMTAPII, PfPNP and their mutant forms SsMTAPII, PfPNP and their CXC-lacking mutants (i.e. SsMTAPIIC259S ⁄ C261S and PfPNPC254S ⁄ C256S) utilized for these studies were expressed and purified as previously described [10,12]. Manipulations of DNA and E. coli were carried out using standard protocols [10,12,28]. Protein concentration was determined by the Bradford assay [29]. Assays of enzyme activity PNP activity was determined by monitoring the formation of purine base from the corresponding nucleoside by HPLC using a Beckman System Gold apparatus (Beckman Coul- ter, Fullerton, CA, USA). The assay was carried out as described previously [12]. MTAP activity was determined by monitoring the forma- tion of [methyl- 14 C]5-methylthioribose-1-phosphate from 5¢-[methyl- 14 C]MTA [10]. 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. GdnCl-induced unfolding and refolding SsMTAPII, PfPNP and their respective CXC-lacking mutants (final concentration 0.4 mgÆmL )1 ) were incubated for 22 h at 25 °C in the presence of 6 m GdnCl in 20 mm Tris ⁄ HCl (pH 7.4) containing 30 mm dithiothreitol. Unfold- ing was probed by recording the intrinsic fluorescence emis- sion. To test the reversibility of the process, the refolding was started by a 20-fold dilution of the unfolding mixture in Tris ⁄ HCl 20 mm (pH 7.4). The refolded enzyme, after extensive dialysis against Tris ⁄ HCl 20 mm (pH 7.4) until complete removal of GdnCl, was analyzed by catalytic activity measurements performed under standard condi- tions. Reactivation assay of SsMTAPII, PfPNP and their CXC-lacking mutants The activity of SsCSC and PfCGC as catalysts of oxidative protein folding was tested by the ability to reactivate SsMTAPII, PfPNP, SsMTAPIIC259S ⁄ C261S and PfPNPC254S ⁄ C256S after GdnCl-induced unfolding and refolding. SsCSC and ⁄ or PfCGC were first reduced with a five-fold excess of dithiothreitol in 50 mm Tris ⁄ HCl (pH 7.4) for 10 min at 30 °C and then incubated at 30 °C for 22 h in a reactivation mixture containing (in a final volume of 50 lL): 50 mm Tris ⁄ HCL (pH 7.4), 2 mm EDTA, a gluta- thione redox buffer (1 mm GSH, 0.2 mm GSSG), 540 lm CXC peptide, and the protein to be reactivated (3 lg; final concentration 2 lm). After the incubation, SsMTAPII or PfPNP activity was measured under standard conditions. The activity of control enzymes was expressed as 100%. The positive control was represented by the reactivation of 0.8 0.4 0 0 70 140 210 Absorbance 296 Time (min) Fig. 4. Time course for the reactivation of sRNaseA by various oxi- dative folding catalysts. - - -, None; , PDI; d, SsCSC; , PfCGC. Each value is the average of three separate experiments. G. Cacciapuoti et al. CXC and oxidative protein folding FEBS Journal 276 (2009) 5799–5805 ª 2009 The Authors Journal compilation ª 2009 FEBS 5803 the enzymes catalyzed by PDI (final concentration 0.11 lm) under the same experimental conditions. Reactivation of sRNaseA Disulfide isomerase activity was assayed as described previ- ously [30] by monitoring the reactivation of sRNaseA, a fully oxidized protein containing a random distribution of its four disulfide bonds. 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Biochemistry 30, 619–625. G. Cacciapuoti et al. CXC and oxidative protein folding FEBS Journal 276 (2009) 5799–5805 ª 2009 The Authors Journal compilation ª 2009 FEBS 5805 . Purine nucleoside phosphorylases from hyperthermophilic Archaea require a CXC motif for stability and folding Giovanna Cacciapuoti, Iolanda Peluso, Francesca. removal of GdnCl, was analyzed by catalytic activity measurements performed under standard condi- tions. Reactivation assay of SsMTAPII, PfPNP and their CXC- lacking