Báo cáo khoa học: Structural stability of the cofactor binding site in Escherichia coli serine hydroxymethyltransferase – the role of evolutionarily conserved hydrophobic contacts potx

Structural stability of the cofactor binding site in Escherichia coli serine hydroxymethyltransferase – the role of evolutionarily conserved hydrophobic contacts Rita Florio 1 , Roberta Chiaraluce 1 , Valerio Consalvi 1 , Alessandro Paiardini 1 , Bruno Catacchio 1,2 , Francesco Bossa 1,3 and Roberto Contestabile 1 1 Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’, ‘Sapienza’ Universita ` di Roma, Italy 2 CNR, Istituto di Biologia e Patologia Molecolari, ‘Sapienza’ Universita ` di Roma, Italy 3 Centro di Eccellenza di Biologia e Medicina Molecolare (BEMM), ‘Sapienza’ Universita ` di Roma, Italy Introduction Pyridoxal 5¢-phosphate (PLP)-dependent enzymes comprise a vast and highly diversified group of cata- lysts, whose action is required in a large number of cellular processes. It is generally accepted that PLP-dependent enzymes originated very early on in evolution, before the three biological kingdoms Keywords cofactor binding site; conserved hydrophobic contacts; pyridoxal phosphate; serine hydroxymethyltransferase; urea- induced denaturation Correspondence R. Contestabile, Dipartimento di Scienze Biochimiche, ‘Sapienza’ Universita ` di Roma, Piazzale Aldo Moro 5, 00185, Roma, Italy Fax: +39 0649917566 Tel: +39 0649917569 E-mail: roberto.contestabile@uniroma1.it Website: http://w3.uniroma1.it/bio_chem/ sito_biochimica/EN/index.html (Received 3 September 2009, revised 12 October 2009, accepted 16 October 2009) doi:10.1111/j.1742-4658.2009.07442.x According to their fold, pyridoxal 5¢-phosphate-dependent enzymes are grouped into five superfamilies. Fold Type I easily comprises the largest and most investigated group. The enzymes of this group have very similar 3D structures. Remarkably, the location of the cofactor in the active site, between the two domains that form a single subunit, is almost identical in all members of the group. Nonetheless, Fold Type I enzymes show very lit- tle sequence identity, raising the question as to which structural features determine the common fold. An important fold determinant appears to be the presence of three evolutionarily conserved clusters of hydrophobic con- tacts. A previous investigation, which used Escherichia coli serine hydrox- ymethyltransferase, a well characterized Fold Type I member, demonstrated the involvement of one of these clusters in the stability of the quaternary structure. The present study focuses on the role of the same cluster in the stability of the cofactor binding site. The investigation was carried out by equilibrium denaturation experiments on serine hydroxymethyltransferase forms in which the hydrophobic contact area of the cluster under study was reduced by site-directed mutagenesis. The results obtained show that the mutations clearly affected the process of pyridoxal 5¢-phosphate dissociation induced by urea, reducing the stability of the cofactor binding site. We sug- gest that the third cluster promotes the formation of a bridging structural region that stabilizes the overall protein structure by connecting the two domains, shaping the cofactor binding site and participating in the forma- tion of the quaternary structure. Structured digital abstract l MINT-7293394, MINT-7293405, MINT-7293418: eSHMT (uniprotkb:P0A825) and eSHMT (uniprotkb: P0A825) bind (MI:0407)bycosedimentation in solution (MI:0028) Abbreviations CHCs, conserved hydrophobic contacts; eSHMT, Escherichia coli serine hydroxymethyltransferase; PLP, pyridoxal 5¢-phosphate. FEBS Journal 276 (2009) 7319–7328 ª 2009 The Authors Journal compilation ª 2009 FEBS 7319 diverged, from different protein ancestors that gener- ated five independent families, corresponding to as many different Fold Types [1,2]. The Fold Type I, or aspartate aminotransferase family, is the largest, functionally most diverse and best characterized. Its members are made of dimers or multiple of dimers, whose subunits are formed by a large domain and a small domain. Despite the poor sequence similarity among many Fold Type I enzymes, all members of the family share the same basic protein architecture, and are assembled with 17 structurally conserved regions that form the heart of the domains [3]. The presence of three clusters of evolutionarily conserved hydrophobic contacts (CHCs; Fig. 1A) appears to be one important structural feature determining the native fold of Fold Type I enzymes [3]. Although two of these clusters are located in the central cores of the domains and presumably stabilize their scaf- fold, the role of the third cluster is much less clear. This cluster forms a hinge between two conserved a-helices (which correspond to two structurally con- served regions), located respectively at the beginning and at the end of the large domain. Examination of the contact network shows that, in this cluster, the CHCs lie along one side of each helix, forming a buried spine at positions i, i + 4 and i +7. In a previous study investigating dimeric Escherichia coli serine hydroxymethyltransferase (eSHMT; EC 2.1.2.1), a well characterized Fold Type I member, we reported a site-directed mutagenesis study in which the third cluster of CHCs was destabilized, reducing its hydrophobic contact area [4]. The characterization of the enzyme mutant forms (L85A, L276A and L85A ⁄ L276A) under native conditions indicated that the stability of the cluster is essential for the correct quaternary assembly of the enzyme and is increased by the binding of PLP and substrates. Indeed, the two helices that form the cluster interact with the N-terminal a-helix of the other subunit in the dimer and are contiguous with two polypeptide loops, which, in all Fold Type I enzymes, mediate the interactions between the subunits and are involved in cofactor binding, substrate binding and catalysis (Fig. 1). On the other hand, the mutations did not affect either the capability to bind the cofactor or the catalytic activity of the enzyme. The monomeric form of the enzyme (resulting from the double L85A⁄ L276A mutation) binds PLP with comparable affinity with respect to the dimeric wild-type form, suggesting that the subunit structure of the monomer is more or less the same as that in the dimer. By contrast with the CHCs located in the core of the large and small domains, the third hydrophobic A B Fig. 1. (A) Cartoon representation of the crystal structure of eSHMT, a Fold Type I enzyme, showing the residues involved in CHCs as spheres. Subunits of eSHMT ternary complex with glycine and 5-for- myl-tetrahydropteroylglutamate (Protein Data Bank code: 1dfo) are represented in cyan and salmon. Residues forming CHCs are shown only in one subunit. The residues of the clusters located in the large and small domains are shown in magenta and orange, respectively. The residues forming the third cluster are shown in red. The PLP–Gly complex is represented by yellow sticks, with the phosphorus atom depicted in orange, the oxygen atoms in red and the nitrogen atoms in blue. The helices forming the third cluster interact with the N-ter- minal helix of the other subunit in the dimer (shown in blue). Two polypeptide loops (in green), which are contiguous with the helices of the third cluster of CHCs, contribute residues that interact, at the active site of the other subunit, with PLP and substrates. (B) Mono- meric structure of eSHMT. Only the residues involved in the forma- tion of the third CHCs are shown as spheres and the mutated residues are indicated by arrows. The N-terminal tail (residues 1–61) of the protein is coloured in orange, the large domain (residues 62– 211) in salmon, the interdomain segment (residues 212–279) in green and the small domain in blue (for details, see Discussion). Stability of cofactor binding site in SHMT R. Florio et al. 7320 FEBS Journal 276 (2009) 7319–7328 ª 2009 The Authors Journal compilation ª 2009 FEBS cluster is not directly involved in the proper position- ing or stabilization of the active site or PLP-binding residues. Its location rather suggests a role in bridging different structural regions of the protein in order to stabilize its native overall fold. The present study investigated the role of the third cluster of CHCs with respect to the structural stability of the PLP-binding site by means of urea-induced denaturation experi- ments on eSHMT L85A, L276A and L85A ⁄ L276A mutants. These mutations were carried out to reduce the hydrophobic contact area in the cluster. Leu276 is the most conserved residue in the cluster, with its posi- tion being almost invariably occupied by a leucine resi- due in Fold Type I enzymes [3]. Leu85 was chosen because this residue shows the largest contact area with Leu276. Results The structural stability of holo-eSHMT wild-type and mutant forms was investigated by performing equilib- rium unfolding experiments, using urea as denaturing agent. The structural changes of the active site and overall protein induced by urea were monitored by measuring catalytic activity, the fraction of covalently bound cofactor, intrinsic fluorescence emission, far-UV CD and the sedimentation coefficient. The reversibility of the unfolding process was ana- lyzed by measuring the activity that denatured eSHMT samples (23 lm in 8 m urea at 20 °C) were able to recover after 4 h, subsequent to a ten-fold dilution with buffer at 20 °C. In agreement with a previous study [5], complete enzyme activity was recovered after refolding. In this respect, comparable results were obtained with all mutant forms. Activity and internal aldimine measurements Enzyme samples (2.3 lm) were incubated with increas- ing urea concentrations in 50 mm NaHepes buffer (pH 7.2), containing 200 lm dithiothreitol and 100 lm EDTA, at 20 °C for 15 h. The residual fractions of catalytic activity and covalently bound cofactor (inter- nal aldimine) were then measured and reported as a function of denaturant concentration (Fig. 2). With all enzyme forms, the loss of internal aldimine appears to take place according to a sigmoid process. The decrease in catalytic activity shows a more complex behaviour. At a urea concentration in the range 0–1 m, almost half of the activity is lost, apparently as a result of an hyperbolic process. The complete loss of activity, taking place at higher urea concentrations, appears to follow a sigmoid course. In 1 m urea, almost all the cofactor is bound to the enzyme as internal aldimine, indicating that the loss of activity does not result from the denaturation of the active site. These observations suggest that urea might act as an enzyme inhibitor. Indeed, experiments in which the kinetic parameters of the catalyzed reaction were determined in the absence or presence of 0.25, 0.50, 0.75 and 1 m urea clearly demonstrated that urea is responsible for a mixed-type Fig. 2. Dependence of catalytic activity and PLP covalent binding to eSHMT on the urea concentration. The fractions (f) of retained catalytic activity (open symbols) and internal aldimine (closed sym- bols) were measured as a function of the urea concentration. The reaction catalyzed by eSHMT was the aldol cleavage of L-threo-3- phenylserine into glycine and benzaldehyde. The profiles obtained with the L85A (triangles), L276A (squares) and L85A ⁄ L276A (dia- monds) mutants are compared in each panel with the profiles obtained with the wild-type enzyme (circles). The lines through the experimental points (dashed lines for wild-type and continuous lines for mutant enzymes) are those obtained from the global nonlinear least squares fitting of internal aldimine and activity data to Eqns (1,2). R. Florio et al. Stability of cofactor binding site in SHMT FEBS Journal 276 (2009) 7319–7328 ª 2009 The Authors Journal compilation ª 2009 FEBS 7321 inhibition. The inhibitory effect of urea on catalytic activity has been also reported for other enzymes and is well documented in the literature [6,7]. Figure 2 shows that the denaturation profiles of the L85A and L85A ⁄ L276A mutants are shifted toward lower urea concentrations with respect to those of the wild-type and L276A enzymes. To quantify this shift, the internal aldimine and activity data of each enzyme form were analyzed according to Eqn (1) (the equation of a sigmoid curve) and Eqn (2) (which takes in account both the inhibitory and denaturing effects of urea), respectively, in a global least squares minimiza- tion procedure in which the parameters of the sigmoid denaturation processes contained in both equations were shared (Fig. 2). The analysis gave a good global fit of the data and showed that the midpoints of the sigmoid transitions coincide and, in the case of the L85A and L85A ⁄ L276A mutants, are approximately 0.3 m lower compared to the wild-type (Table 1). Although the midpoint of the L276A mutant coincides with that of the wild-type enzyme, it is clear from the values of n given in Table 1 that the steepness of the sigmoid transitions is lower. This indicates that the mutation had the effect of lowering the cooperativity of the denaturation process. The same consideration can be made for the double L85A ⁄ L276A mutant. Spectroscopic measurements Intrinsic fluorescence emission and far-UV CD mea- surements on wild-type and mutant eSHMTs were car- ried out on protein samples (2.3 lm) incubated with increasing denaturant concentrations (from 0 to 7.9 m) in 50 mm NaHepes buffer (pH 7.2), containing 200 lm dithiothreitol and 100 lm EDTA at 20 °C for 15 h (Fig. 3). With the wild-type enzyme, a urea concentra- tion rising up to 2.3 m determines the increase in rela- tive fluorescence emission intensity (Fig. 3A). Part of this increase clearly takes place with negligible dissoci- ation of the cofactor (from 0 to 1 m urea) and appears to coincide with the drop of catalytic activity attribut- able to urea inhibition. The further increase of fluores- cence matches the complete loss of catalytic activity and the release of the cofactor, which relieves the quenching of tryptophan fluorescence [5]. As previ- ously shown, this represents the first of two steps in the denaturation mechanism of eSHMT. At a urea concentration of 2.3 m, the enzyme is known to exist in the form of a denaturation intermediate [5,8,9], which denatures completely and loses part of its fluo- rescence emission as the urea concentration is increased up to 7.9 m. The fluorescence profiles obtained with the mutant enzymes are similar in shape to that of wild-type eSHMT. However, at a urea concentration in the range 0–1 m, the relative fluorescence emission inten- sity of the mutant forms is higher than that of the wild-type enzyme. Moreover, with the L85A and L85A ⁄ L276A mutants, the increase of fluorescence emission that corresponds to the dissociation of the cofactor is shifted towards lower urea concentrations with respect to wild-type and L276A forms (Fig. 3). This observation is reminiscent of that noted for the internal aldimine and activity data, and indicates a shift of the equilibrium between the native and the intermediate forms in favour of the latter. At urea con- centrations above 2.3 m, and as the protein unfolds completely, the fluorescence emission profiles of all enzyme forms coincide, suggesting that the equilibrium between the denaturation intermediate and the unfolded protein is not affected by the mutations. The influence of the mutations on the far-UV CD and average lambda (for a definition, see Experimental procedures) profiles is not as clear as in the case of the activity and internal aldimine data (Fig. 3E, F). The CD signal and average lambda do not vary at a urea concentration in the range 0–1 m. At a urea concentra- tion of 2.3 m, approximately one-third of the far-UV CD signal is lost, although very little change of the average lambda is observed. This indicates that the loss of cofactor that takes place in the first denatur- ation step does not correspond to large structural changes. Sedimentation velocity measurements In a previous study [4], we reported that the L85A, L276A and L85A ⁄ L276A mutations affect the quater- nary structure stability of eSHMT. The wild-type enzyme is a dimer with molecular mass of approxi- mately 91 kDa and shows a single band in the sedimentation coefficient distribution with a maximum Table 1. Parameters obtained from the global best-fit of activity and internal aldimine data. Parameters are expressed as the mean ± SE determined by the global nonlinear least squares fitting of data to Eqns (1,2), as detailed in the text. K i represents the cal- culated inhibition constant, whereas cm 1 and n reflect, respectively, the urea concentration midpoints and the steepness of the sigmoi- dal curves. K i (M) cm 1 (M) n Wild-type 1.71 ± 0.05 1.88 ± 0.01 9.54 ± 0.47 L85A 1.30 ± 0.08 1.52 ± 0.01 11.19 ± 0.66 L276A 1.49 ± 0.05 1.88 ± 0.01 7.04 ± 0.34 L85A ⁄ L276A 1.38 ± 0.06 1.56 ± 0.01 6.84 ± 0.35 Stability of cofactor binding site in SHMT R. Florio et al. 7322 FEBS Journal 276 (2009) 7319–7328 ª 2009 The Authors Journal compilation ª 2009 FEBS at 5.5 S ( s 20,w ), which is the value expected for a hydrated dimer with an approximately spherical shape. The hydrophobic cluster mutations shifted the equilib- rium between dimeric and monomeric forms of the enzyme in favour of the latter, which shows a sedimen- tation coefficient of approximately 3 S. Interestingly, the monomeric form of the enzyme was shown to bind PLP. This observation is not unprecedented for Fold Type I enzymes [10]. In the present study, ultracentrifugation experiments were carried out aiming to analyse the effect of urea on the sedimentation properties of wild-type and mutant eSHMTs. Sedimentation velocity experiments were performed in the presence of either 1 or 2.3 m urea. These concentrations correspond to crucial events in the denaturation mechanism: exposure of the enzyme to 1 m urea results in the loss of half of the catalytic activity, although all the cofactor is retained at the active site as internal aldimine. In 2.3 m urea, the cofactor is lost as the denaturation intermediate is formed (Fig. 2). In Table 2, the results of the present ultracentrifuge analysis are compared with those obtained in the absence of urea [4]. In 1 m urea, the wild-type enzyme is dimeric, but becomes mainly monomeric when the urea concentration is increased up to 2.3 m, as indicated by a predominant band with a maximum at 3.1 S in the sedimentation coefficient distribution (Fig. 4). The frictional ratio (f ⁄ f 0 ) (i.e. the ratio between the experimentally calculated friction coefficient and the minimum friction coefficient of an anhydrous sphere) of monomeric wild-type eSHMT is Fig. 3. Spectroscopic analysis of urea- induced unfolding of wild-type (d), L85A (D), L276A (h) and L85A ⁄ L276A ()) enzyme forms. All spectroscopic measurements were carried out on enzyme samples at a concentration of 2.3 l M,in50mM NaHepes (pH 7.2), containing 200 l M dithiothreitol and 100 l M EDTA at 20 °C. Intrinsic fluores- cence emission measurements at 336 nm on wild-type and mutant forms were per- formed with a 1 cm quartz cuvette upon excitation at 295 nm. (A) Comparison among relative fluorescence emission (F r ) and retained fractions (f ) of activity (s) and internal aldimine (·) measured with wild-type eSHMT as a function of the urea concentration. (B–D) Relative fluorescence denaturation profiles obtained with wild-type and mutant enzymes. (E) Molar ellipticity at 222 nm ([h] 222 ) calculated from far-UV CD measurements carried out in a 0.2 cm quartz cuvette. (F) Average lambda (k) pro- files calculated from fluorescence emission spectra acquired from 320–500 nm, with excitation at 295 nm. Table 2. Sedimentation coefficients calculated from ultracentrifuge experiments on wild-type and mutant eSHMTs. The frictional ratio (f ⁄ f 0 ) is the ratio between the experimentally calculated friction coefficient and the minimum friction coefficient of an anhydrous sphere. s 20, w (S) No urea 1 M urea 2.3 M urea Wild-type 5.5 (f ⁄ f 0 = 1.2) 5.4 (f ⁄ f 0 = 1.2) 5.5 (17%) 3.1 (83%) L85A 5.5 (f ⁄ f 0 = 1.2) 5.3 (88%) 2.9 (12%) 6.4 (10%) 2.8 (90%) L276A 5.5 (f ⁄ f 0 = 1.2) 5.5 (77%) 3.1 (23%) 2.5 (f ⁄ f 0 = 1.5) L85A ⁄ L276A 5.5 (66%) 3.3 (34%) 5.5 (35%) 3.3 (65%) 2.5 (f ⁄ f 0 = 1.5) R. Florio et al. Stability of cofactor binding site in SHMT FEBS Journal 276 (2009) 7319–7328 ª 2009 The Authors Journal compilation ª 2009 FEBS 7323 equal to that of its dimeric form (1.2) and close to that of a spherical protein, suggesting that the subunit dissociation took place without any large structural denaturation. With all mutant forms, 1 m urea determines the partial dissociation of the dimer subunits (Fig. 4 and Table 2). The extent of dissociation is greater for the double mutant, which already exists as an equilibrium mixture of monomers and dimers in the absence of urea, and is smaller for the L85A mutant and interme- diate for the L276A mutant. Nothing may be con- cluded regarding the shape of the mutant monomeric forms in 1 m urea because the frictional ratio cannot be calculated when more than one sedimentation species is present at equilibrium. However, a sedimen- tation coefficient of approximately 3 S suggests that all monomers have an approximately spherical shape in 1 m urea. It should be noted that, with all mutant forms, the dissociation of subunits in 1 m urea takes place without any loss of cofactor (Fig. 2), in agree- ment with a substantial retention of the active site native structure. By contrast, with the wild-type enzyme, the dissociation of subunits is observed at a much higher urea concentration range (1–2.3 m), apparently in concomitance with the loss of cofactor. In 2.3 m urea, the L276A and L85A ⁄ L276A mutant forms exist as single sedimentation species with a coef- ficient of 2.5 S. The frictional ratio calculated for this species is 1.5, suggesting that the decrease in the sedi- mentation coefficient is determined by the loss of spherical shape as a result of a partial structural dena- turation of the monomer. The L85A mutant in 2.3 m urea is present as two sedimentation species with coef- ficients of 2.8 S and 6.4 S. The 6.4 S species most probably results from aggregation. The 2.8 S species, which shows a broad coefficient distribution, may also correspond to a partially denatured monomer. PLP-binding properties of the monomeric denaturation intermediate The cofactor binding properties of monomeric wild- type and mutant enzymes in 2.3 m urea (dissolved in the same buffer used in the previous experiments) were analysed in order to probe the structural features of the active site. The visible absorption spectrum of wild-type eSHMT shows a characteristic absorption band with maximum at 420 nm, as a result of the pres- ence of PLP bound at the active site as a protonated internal aldimine [11]. The absorption spectra of the enzymes (10 lm subunit concentration) in 2.3 m urea were recorded and subtracted from the absorption spectra acquired after 20 min subsequent to the addi- tion of 100 lm PLP, allowing sufficient time for the cofactor binding equilibrium to be reached (as indi- cated by the fact that the absorption spectrum stopped changing). The differential spectrum obtained with the wild-type enzyme (Fig. 5) clearly results from the development of a positive 420 nm absorbing band, demonstratring that the addition of PLP determines Fig. 4. Sedimentation velocity distributions obtained with wild-type and mutant eSHMTs. Sedimentation velocity measure- ments were performed at 116 480 g on 2.5 l M (subunit concentration) enzyme sam- ples kept at 20 °Cin50m M NaHepes buffer (pH 7.2) in the absence (——) or presence of 1 M (- - - -) and 2.3 M urea (ÆÆÆÆÆ). Stability of cofactor binding site in SHMT R. Florio et al. 7324 FEBS Journal 276 (2009) 7319–7328 ª 2009 The Authors Journal compilation ª 2009 FEBS the formation of a native-like internal aldimine. The differential spectra recorded with the mutant enzymes indicate the formation of a band with maximum absorbance at lower wavelengths (at approximately 390–400 nm). Evidently, in 2.3 m urea, PLP binds to the mutant enzymes, but without the formation of a native-like internal aldimine, suggesting a structural difference between the mutant active site and the wild-type. Discussion Previous studies [5,8,9] show that the urea-induced denaturation of wild-type eSHMT, from a native dimer to a fully denatured monomer, takes place according to a three-state mechanism (Scheme 1A), in which an intermediate catalytically inactive apo-form of the enzyme mostly accumulates at a urea concentra- tion of approximately 2.3 m. The data obtained in the present study show that, at this urea concentration, the enzyme has lost most of the cofactor (Fig. 2) and is in a monomeric form (Fig. 4 and Table 2). There- fore, the urea-induced loss of cofactor and dissociation of subunits appear to be simultaneous events of the first denaturation step (N 2 ¢ ÀPLP þPLP 2I in Scheme 1A). The dissociation of subunits apparently takes place without any large denaturation of the monomer structure. This is suggested by the fact that the enzyme monomers in 2.3 m urea retain a spherical shape, as indicated by the frictional ratio (f ⁄ f 0 ) and the sedimentation coefficient calculated from the ultracentrifuge analysis (Table 2). The minor change of the far-UV CD spectrum observed when the urea concentration is increased up to 2.3 m is therefore hardly attributable to a loss of secondary structure, and may instead result from a conformational change of the protein monomer. Upon PLP binding, the native apo-eSHMT is known to undergo a conformational change, which most proba- bly results from a shift of the equilibrium between the open and closed forms of the enzyme [12]. Urea might act to shift this equilibrium in favour of the open form, promoting PLP dissociation from the active site. The wild-type enzyme in 2.3 m urea, which is mostly monomeric and in the apo-form, maintains the capa- bility to form a native-like internal aldimine if a large excess of PLP is added (Fig. 5). Increasing the urea concentration from 2.3 to 8 m determines the complete unfolding of the enzyme (2I¢2U in Scheme 1A), with a complete loss of secondary structure, as reported in previous studies [5,8,9], and as can be deduced from the spectroscopic data shown in Fig. 3. In this higher urea concentration range, the denaturation profiles obtained with the mutant enzymes do not differ from those of wild-type eSHMT. Mutations clearly influence the apparent denatur- ation mechanism of the enzyme at a urea concentra- tion in the range 0–2.3 m. Comparison of data obtained from the ultracentrifuge analysis and from the measurement of covalently bound cofactor shows that, in contrast to that observed with the wild-type enzyme, with the mutant forms, the urea-induced sub- unit dissociation is detectable as a separate process with respect to the dissociation of PLP. With all three mutant forms, at a urea concentration of 1 m, subunit dissociation is clearly visible (N 2 ¢2N in Scheme 1B), whereas the loss of cofactor is negligible and becomes visible only at higher denaturant concentrations (2N ¢ ÀPLP þPLP 2I 0 ; Scheme 1B). This observation agrees with the previously published data, indicating that the monomeric form of the enzyme is able to bind PLP and that the third cluster of CHCs plays an important role in the stabilization of the eSHMT quaternary structure [4]. The extent of subunit dissociation in 1 m Fig. 5. Analysis of PLP-binding properties of wild-type and mutant enzymes in 2.3 M urea. Separate enzyme (20 lM) and PLP (200 lM) samples were kept in 2.3 M urea at 20 °C for 15 h. Equal volumes of enzyme and PLP samples were then mixed and absorption spec- tra recorded after 20 min. Absorption spectra of enzyme samples mixed with an equal volume of buffer containing 2.3 M urea were subtracted from spectra acquired in the presence of PLP, generat- ing the differential spectra shown: wild-type eSHMT (——), L85A (- - - -), L276A (ÆÆÆÆÆ) and L85A ⁄ L276A (ÆÆ-ÆÆ) mutants. A B Scheme 1. Presumed equilibrium denaturation mechanisms of wild-type (A) and mutant (B) eSHMTs. N and U represent, respec- tively, the native and the fully denatured forms of the enzyme subunits. The denaturation intermediate formed by the wild-type enzyme is indicated by I, whereas I¢ represents the partially dena- tured intermediate formed by the mutant enzymes. R. Florio et al. Stability of cofactor binding site in SHMT FEBS Journal 276 (2009) 7319–7328 ª 2009 The Authors Journal compilation ª 2009 FEBS 7325 urea is higher for the double L85A ⁄ L276A form, lower for the L85A form and intermediate for the L276A mutant. This situation again reflects what is observed for the same enzyme forms under native conditions [4]. A novel observation is that the mutations clearly affected the process of PLP dissociation induced by urea. The L85A and L85A ⁄ L276A mutations lowered the midpoint of this transition and the L276A muta- tion lowered its cooperativity (Table 1). Moreover, upon PLP dissociation in 2.3 m urea, the monomeric mutant enzymes appear to be partially denatured (I¢ in Scheme 1B), as indicated by the frictional ratio and the sedimentation coefficients calculated from the ultracentrifuge analysis (Table 2). The extent of dena- turation of this monomeric intermediate is not expected to be very large because the average lambda profiles of the wild-type and mutant enzymes are very similar (Fig. 3). Nevertheless, the addition of excess PLP to the partially denaturated monomer does not result in the formation of a native internal aldimine. Evidently, the concentration of the species at equilib- rium cannot be altered by the addition of PLP, con- firming that the mutant enzymes follow a different denaturation mechanism with respect to wild-type eSHMT. Taken together, these observations suggest that, in the mutant enzymes, the urea-induced dissocia- tion of PLP is favoured by the disruption of the third cluster of CHCs. The mutations introduced with the aim of reducing the hydrophobic surface contacts have instead reduced the stability of the PLP binding site. This conclusion points to a clear relationship between the formation of the cluster and the structural integrity of the active site. By contrast to the CHCs located in the cores of the large and small domains, the cluster under study is not directly involved in the positioning or stability of any active site or PLP-binding residue. Considering the studies on eSHMT folding, we suggest that this cluster may play a fundamental role in the folding mechanism of the enzyme, which may be divided into two phases [5,8,9,13]. In the first phase, the large and small domains rapidly assume their native state, forming a folding intermediate that has almost all of the native secondary and tertiary structure, but is unable to bind PLP. In this intermediate, the N-terminus and an inter-domain segment remain exposed to solvent [8] (Fig. 1B). In the second, slower phase, these structural elements fold into the native structure, conferring the enzyme with the capability of binding PLP. Because the interdomain segment comprises the a-helix contrib- uting L276 and the second a-helix (where L85 is located) of the cluster is part of the large domain, it follows that the last folding event may correspond to the formation of the third cluster of CHCs. The for- mation of the cluster presumably fastens together all the structural components of the protein and confers stability to the active site. The high degree of sequence and structural conservation of the third cluster of CHCs, as observed for the majority of fold-type I enzymes, suggests that this function, which is hypothe- sized for eSHMT, could be extended to the whole superfamily. Experimental procedures Materials Ingredients for bacterial growth were obtained from Sigma-Aldrich (St Louis, MO, USA). Chemicals for the purification of the enzymes were obtained from BDH (Poole, UK); DEAE-sepharose and phenyl-sepharose were obtained from GE Healthcare (Milwaukee, WI, USA). The L85A, L276A and L85A ⁄ L276A mutant forms of the E. coli glyA (SHMT encoding gene) were already available from a previous study [4]. Wild-type and mutant forms of eSHMT were purified as described previously [14]. PLP was added to protein samples during the purification procedure, but it was left out in the final dialysis step. All experiments were performed at 20 °Cin50mm NaHepes buffer at pH 7.2, containing 200 lm dithiothreitol and 100 lm EDTA. PLP was obtained from Sigma-Aldrich (98% pure). All other reagents were obtained from Sigma-Aldrich. Preparation of holoenzyme samples We noted that different batches of purified e SHMT samples contained variable holoenzyme ⁄ apoenzyme ratios. This observation was made with either wild-type or mutant forms of the enzyme. To carry out comparable experiments, it was mandatory to prepare enzyme samples that con- tained the same fraction of protein-bound PLP (possibly close to saturation) and, at the same time, were devoid of any excess of cofactor. Holoenzymes were then prepared from apoenzyme samples, by the addition of PLP at the concentration needed to obtain 98% saturation, as calcu- lated on the basis of the related dissociation constant of PLP binding equilibrium [4]. The subunit concentration of the holoenzyme was calculated according to a molar absorptivity value of e 280 = 44884 cm )1 Æm )1 [12]. Apoen- zyme samples were prepared as described previously [5]. Unfolding and refolding experiments Concentrated protein samples were diluted into urea solu- tions (0–7.9 m in 50 mm NaHepes, pH 7.2, containing 200 lm dithiothreitol and 100 lm EDTA, at 20 °C) to obtain a final protein concentration of 2.3 lm. Spectro- Stability of cofactor binding site in SHMT R. Florio et al. 7326 FEBS Journal 276 (2009) 7319–7328 ª 2009 The Authors Journal compilation ª 2009 FEBS scopic measurements, activity assays, measurements of internal aldimine and ultracentrifuge analyses were carried out after 15 h, which allows sufficient time to reach the equilibrium [5]. The l-threo-3-phenylserine cleavage activity of urea- incubated enzyme samples was measured after the direct addition of 7.5 mm substrate, which corresponds to approx- imately 16% saturation. The dilution of urea resulting from substrate addition was taken into account. The reversibility of the urea-induced unfolding was ana- lyzed by means of refolding experiments in which denatured enzyme samples (23 lm kept with 8 m urea in 50 mm NaHepes, pH 7.2, containing 200 lm dithiothreitol and 100 lm EDTA for 15 h at 20 °C) were diluted ten-fold with buffer at 20 °C. These conditions have been shown to give the highest yield of refolded enzyme [5]. Four hours later, after allowing sufficient time to complete the refolding pro- cess [5], the enzyme activity of the refolded sample was assayed upon a further 50-fold dilution of the sample into the assay reaction mixture (the final enzyme concentration in the activity assay was 0.05 lm). Ten-fold molar excess PLP was added to refolded samples immediately before dilution into the activity assay reaction mixture. Activity assays All assays were carried out at 20 °Cin50mm NaHepes (pH 7.2), containing 200 lm dithiothreitol and 100 lm EDTA. The rate of l-threo-3-phenylserine cleavage (3 lm enzyme samples) was obtained from spectroscopic measure- ment of benzaldehyde production at 279 nm, employing a molar absorptivity value of e 279 = 1400 cm )1 Æm )1 [15,16]. Spectroscopic measurements Fluorescence emission measurements were carried out with a LS50B spectrofluorimeter (Perkin Elmer Life Sciences, Waltham, MA, USA) using a 1 cm path length quartz cuvette. Fluorescence emission spectra were recorded in the range 320–500 nm (1 nm sampling interval; 5 nm emission slit), with the excitation wavelength set at 295 nm (3 nm excitation slit). Fluorescence measurements were expressed as relative fluorescence intensity and average lambda (aver- age lambda = R(I i · k i ) ⁄ R(I i ), where k i is the ith wave- length and I i is the corresponding relative fluorescence intensity). Far-UV (190–250 nm) CD spectra were measured using a 0.2 cm path length quartz cuvette and expressed as the mean residue ellipticity [Q], assuming a mean residue molecular mass of 110 per amino acid residue. UV-visible spectra were recorded with a double-beam Lambda 16 spectrophotometer (Perkin Elmer Life Sciences). Kinetic measurements in the activity assays were performed on a Hewlett-Packard 8453 diode-array spectrophotometer (Hewlett-Packard, Palo Alto, CA, USA). All spectroscopic measurements were carried out at 20 °Cin50mm NaHepes (pH 7.2), containing 200 lm dithiothreitol and 100 lm EDTA. Analytical ultracentrifugation analysis Sedimentation velocity experiments were carried out at 20 °Cin50mm NaHepes buffer pH 7.2, containing 200 lm dithiothreitol and 100 lm EDTA, on a Beckman XL-I ana- lytical ultracentrifuge (Beckman Coulter, Fullerton, CA, USA) equipped with absorbance optics and an An60-Ti rotor. In the sedimentation velocity experiments, performed at 116 480 g, the protein concentration was 2.5 lm . Data were collected at 277 nm at a spacing of 0.003 cm with three averages being obtained in a continuous scan mode. Sedimentation coefficients and integration of data were obtained using the software sedfit (provided by P. Schuck, National Institutes of Health, Bethesda, MD, USA). The values were reduced to water (S 20,w ) using standard proce- dures. The buffer density and viscosity were calculated using the software sednterp (http://www.jphilo.mailway. com). The ratio f ⁄ f 0 was calculated from the diffusion coef- ficient, which in turn is related to the spreading of the boundary, using the software sedfit. Internal aldimine measurements Borohydride (BH 4 ) ) is known to reduce imines and alde- hydes rapidly and efficiently [17]. When the internal aldi- mine (PLP-enzyme Schiff base) of PLP-dependent enzymes is reduced with BH 4 ) , the cofactor is irreversibly attached to the protein, giving an absorption spectrum with a band at approximately 330 nm [18]. NaBH 4 was prepared as a concentrated solution (5 m)in50mm NaOH and added to proteins samples (2.3 lm in 8 mL of 50 mm NaHepes buf- fer at pH 7.2, containing 200 lm dithiothreitol and 100 lm EDTA) incubated with urea, so that its final concentration was 83 mm. Thirty minutes after the addition of NaBH 4 , protein samples were concentrated in Amicon Ultra centri- fuge filters (30 kDa cut-off; Millipore, Billerica, MA, USA) and diluted with 50 mm NaHepes buffer repeatedly in order to eliminate low-molecular mass molecules, including the reduced free cofactor. In the final dilution step, the buffer was added to obtain a final volume of 2 mL and absorption spectra were recorded. The A 335 ⁄ A 280 ratio was calculated to normalize the absorbance of the reduced internal aldi- mine on the basis of protein concentration, and then divided by the ratio found for a native, PLP-saturate enzyme sample. The result obtained gave the fraction of PLP molecules bound per monomer of enzyme. Data analysis The equilibrium unfolding profiles obtained from the mea- surement of the internal aldimine and the catalytic activity R. Florio et al. Stability of cofactor binding site in SHMT FEBS Journal 276 (2009) 7319–7328 ª 2009 The Authors Journal compilation ª 2009 FEBS 7327 were expressed as fraction of the related measurements car- ried out in the absence of urea and analyzed, respectively, according to Eqns (1,2). Equation (1) describes a sigmoid curve and conforms to a two-state denaturation process. Equation (2) takes into account both the inhibitory and denaturing effects of urea on the catalytic activity of the enzyme. The enzyme in the presence of urea is assumed to exist as a mixture of free, active enzyme and urea-bound inactive enzyme at equilibrium. Both enzyme forms are also assumed to denature according to an identical sigmoid pro- cess (Eqn 1). In the equations, f represents the fraction of either internal aldimine (Eqn 1) or activity (Eqn 2), n reflects the steepness of the sigmoid transition, and c m is its urea concentration midpoint. In Eqn (2), K i is the inhibi- tion constant of the urea binding equilibrium. f ¼ 1 À urea½ n c n m þ urea½ n ð1Þ f ¼ 1 À urea½ n c n m þ urea½ n  Â 1 À urea½ urea½þK i  ð2Þ Internal aldimine and activity data were fitted in a global least squares minimization procedure in which the parame- ters of the sigmoid denaturation processes contained in Eqns (1,2) were shared, using the software prism (Graph- Pad Software Inc., La Jolla, CA, USA). Acknowledgements We thank Professor Verne Schirch for helpful discus- sions. This work was supported by grants from the Italian Ministero dell’Universita ` e della Ricerca. Rita Florio is the recipient of a fellowship from the Facolta ` di Scienze Matematiche, Fisiche e Naturali of ‘Sapien- za’ Universita ` di Roma, Italy. References 1 Mehta PK & Christen P (2000) The molecular evolution of pyridoxal-5¢-phosphate-dependent enzymes. 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Structural stability of the cofactor binding site in Escherichia coli serine hydroxymethyltransferase – the role of evolutionarily conserved hydrophobic contacts Rita Florio 1 ,. interactions between the subunits and are involved in cofactor binding, substrate binding and catalysis (Fig. 1). On the other hand, the mutations did not affect either the capability to bind the cofactor or the. a bridging structural region that stabilizes the overall protein structure by connecting the two domains, shaping the cofactor binding site and participating in the forma- tion of the quaternary