Báo cáo khoa học: The role of evolutionarily conserved hydrophobic contacts in the quaternary structure stability of Escherichia coli serine hydroxymethyltransferase pptx

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The role of evolutionarily conserved hydrophobic contactsin the quaternary structure stability of Escherichia coliserine hydroxymethyltransferaseRita Florio1, Roberta Chiaraluce1, Valerio Consalvi1, Alessandro Paiardini1, Bruno Catacchio1,2,Francesco Bossa1,3and Roberto Contestabile11 Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’, ‘Sapienza’ Universita`di Roma, Italy2 CNR, Istituto di Biologia e Patologia Molecolari, ‘Sapienza’ Universita`di Roma, Italy3 Centro di Eccellenza di Biologia e Medicina Molecolare (BEMM), ‘Sapienza’ Universita`di Roma, ItalyPyridoxal 5¢-phosphate (PLP)-dependent enzymes are alarge ensemble of biocatalysts that make use of thesame cofactor but have distinct evolutionary originsand protein architectures [1–3]. According to their 3Dstructure, PLP-dependent enzymes are grouped intofive evolutionarily unrelated superfamilies, correspond-ing to as many different folds (fold types) [4]. The foldtype I group, also referred to as the aspartate amino-transferase family [5], is the largest, functionally mostdiverse and best characterized. Its members are catal-ytically active as homodimers, although they mayassemble into higher order complexes. A single subunitfolds into two domains [6]. The central feature of theN-terminal, larger domain is a seven-stranded b-sheet.In some instances, the N-terminal tail does not partici-pate as a part of the large domain but comprises aseparate structural element. The small, C-terminaldomain, comprises a three- or four-stranded b-sheet,covered with helices on one side. The active site islocated at the interface of the domains and is delimitedby amino acid residues that are contributed byboth subunits of the catalytic dimer. Remarkably, theKeywordsconserved hydrophobic contacts; fold type Ienzymes; pyridoxal phosphate; quaternarystructure; serine hydroxymethyltransferaseCorrespondenceR. Contestabile, Dipartimento di ScienzeBiochimiche, ‘Sapienza’ Universita`di Roma,Piazzale Aldo Moro 5, 00185 Rome, ItalyFax: +39 0649 917566Tel: +39 0649 917569E-mail: roberto.contestabile@uniroma1.itWebsite: http://w3.uniroma1.it/bio_chem/sito_biochimica/EN/index.html(Received 18 September 2008, revised 23October 2008, accepted 27 October 2008)doi:10.1111/j.1742-4658.2008.06761.xPyridoxal 5¢-phosphate-dependent enzymes may be grouped into five struc-tural superfamilies of proteins, corresponding to as many fold types. Thefold type I is by far the largest and most investigated group. An importantfeature of this fold, which is characterized by the presence of two domains,appears to be the existence of three clusters of evolutionarily conservedhydrophobic contacts. Although two of these clusters are located in thecentral cores of the domains and presumably stabilize their scaffold, allow-ing the correct alignment of the residues involved in cofactor and substratebinding, the role of the third cluster is much less evident. A site-directedmutagenesis approach was used to carry out a model study on the impor-tance of the third cluster in the structure of a well characterized memberof the fold type I group, serine hydroxymethyltransferase fromEscherichia coli. The experimental results obtained indicated that the clus-ter plays a crucial role in the stabilization of the quaternary, native assem-bly of the enzyme, although it is not located at the subunit interface. Theanalysis of the crystal structure of serine hydromethyltransferase suggestedthat this stabilizing effect may be due to the strict structural relationbetween the cluster and two polypeptide loops, which, in fold type Ienzymes, mediate the interactions between the subunits and are involved incofactor binding, substrate binding and catalysis.AbbreviationsCHC, conserved hydrophobic contact; eSHMT, Escherichia coli serine hydroxymethyltransferase; H4PteGlu, tetrahydropteroylglutamate; PLP,pyridoxal 5¢-phosphate; SCR, structurally conserved region.132 FEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBSsuperimposition of fold type I enzymes reveals that thelocation of the cofactor in the active site is virtuallyidentical in all members of the group [7].Despite the high similarities of their 3D structures,many fold type I enzymes show very little sequenceidentity, highlighting the need to identify the structuralfeatures that determine the common fold. Accordingly,a computational analysis that made use of 23 nonre-dundant crystal structures and 921 sequences of foldtype I enzymes identified 17 structurally conservedregions (SCRs), which form the common cores of thelarge and small domains. Within these SCRs, there arethree clusters of evolutionarily conserved hydrophobiccontacts (CHCs) [8]. The first and second cluster arelocated in the cores of the large and small domains,respectively, and appear to stabilize their protein scaf-folds, allowing the proper positioning of the residuesinvolved in PLP binding, substrate binding and modu-lation of the cofactor’s catalytic properties. The thirdcluster forms a hinge between two conserved a-helices(which correspond to two SCRs), located at the begin-ning and at the end, respectively, of the large domain(Fig. 1). Examination of the contact network showsthat the CHCs lie along one side of each helix, forminga buried spine at positions i, i + 4, and i +7. Byapparent contrast to the two previously described clus-ters, the third cluster does not appear to be directlyinvolved in the proper positioning of any active siteresidue, suggesting that its high degree of evolutionaryconservation could be due to a merely structural,rather than functional role.In the present study, the importance of the thirdhydrophobic cluster as a structural determinant of theEscherichia coli serine hydroxymethyltransferase(eSHMT) overall native fold was investigated bydecreasing the hydrophobic contact area of the cluster,using a site-directed mutagenesis approach. The effectsof L85A, L276A and L85A⁄ L276A mutations on thenative structure of the enzyme were analyzed (Fig. 1).ResultsThe consequences of the mutations on the native struc-ture of eSHMT were evaluated by analyzing andcomparing the ultracentrifuge sedimentation, cofactorbinding, catalytic and spectral properties of wild-typeand mutant apo- and holoenzymes.Quaternary structure analysisThe subunit assembly of apo- and holo-forms of wild-type and mutant eSHMTs was characterized by analyt-ical ultracentrifugation. Table 1 shows the values ofsedimentation coefficient and dissociation constant(Kd) calculated from combined sedimentation velocityand equilibrium approaches. As established in theavailable literature [9,10], wild-type eSHMT exists as adimer in both apo- and holo-forms, with a molecularmass of approximately 91 kDa [9]. The ultracentrifuga-tion experiments confirmed that the depletion of thecofactor does not have any effect on the dimericassembly of the enzyme. The sedimentation velocityFig. 1. Schematic representation of the monomeric structure of eSHMT. Cartoon representation of a single subunit of the eSHMT ternarycomplex with glycine and 5-formyl H4PteGlu (Protein Data Bank: 1dfo) [14], showing the N-terminal tail (residues 1–61) colored in orange,the large domain (residues 62–211) in salmon, the interdomain segment (residues 212–279) in green and the small domain in blue. The PLP-Gly complex is shown as yellow sticks, with the phosphorus atom in orange, the oxygen atoms in red and the nitrogen atoms in blue. Thetwo a-helices involved in the formation of the third cluster of CHCs are enclosed in a circle. A magnified view of these helices shows theresidues that form the CHCs represented both as sticks and as transparent spheres. L85 and L276 are indicated by arrows.R. Florio et al. Role of hydrophobic contacts in serine hydroxymethyltransferaseFEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBS 133patterns of apo- and holo-forms are indeed almostsuperimposable, with a single symmetrical peak char-acterized by a sedimentation coefficient, S20,w, of 5.5S(Fig. 2), which is the value expected for a hydratedeSHMT dimer endowed with an approximately spheri-cal shape. The sedimentation equilibrium experimentsshowed that, in the 2.5–25 lm subunit concentrationrange, the wild-type eSHMT is a dimer, either in thepresence or absence of cofactor. In the same concen-tration range, the L85A and L276A mutant holoen-zymes are also dimers, with sedimentation coefficientsof 5.5S. Interestingly, the frictional ratio (f ⁄ f0, the ratiobetween the experimentally calculated friction coeffi-cient and the minimum friction coefficient of an anhy-drous sphere) of dimeric wild-type, L85A and L276AeSHMT holoenzymes is close to that of a sphericalprotein, namely 1.2–1.3, suggesting that the singlemutations did not result in significant changes in theshape of the dimeric protein.Depletion of the cofactor affected the quaternarystructure of both single mutants, which showed anextra sedimentation peak, at approximately 2.7S in thecase of L85A and at 3.1S with the L276A mutant(Fig. 2). The smaller sedimentation coefficient corre-sponds to that of monomeric eSHMT. Therefore, thesingle mutant apoenzymes exist as an equilibrium mix-ture of dimers and monomers, which interconvertslowly with respect to the period of elapsed time in thesedimentation velocity experiments. A comparison ofthe dissociation constants of the single mutant apo-forms, obtained by a fitting of the sedimentation equi-librium curves to a monomer-dimer model, indicatesthat the destabilizing effect of PLP depletion is greaterin L276A (Kd= 2.7 · 10)6m)1) than in L85A(Kd= 4.0 · 10)9m)1). When both mutations are pres-ent, as in the L85A ⁄ L276A double mutant, the apoen-zyme exists as a monomer in the range ofconcentrations tested (2.5–25 lm). The frictional ratioof this monomeric species was calculated to be approx-imately 1.2, suggesting that the dissociation determinedby the double mutation was not accompanied by largestructural changes.Compared to that observed with the single L85Aand L276A mutants, cofactor binding to theL85A ⁄ L276A double mutant apoenzyme did not shiftthe equilibrium completely in favor of the dimer. Inthe double mutant holoenzyme, obtained by addingPLP to 98% of saturation (as calculated from the dis-sociation constant of the related cofactor binding equi-librium; see below), a residual 35% fraction ofmonomer is in equilibrium with the dimer (Table 1and Fig. 2). A dissociation constant of 1.7 · 10)6m)1was calculated for this equilibrium. Because it isknown that PLP bound to eSHMT through a Schiffbase linkage to the active site lysine residue absorbslight maximally at 420 nm [9], a sedimentation velocityexperiment was performed on a double mutant holoen-zyme sample (33 lm), measuring absorbance at thiswavelength. The presence of a 3.1S peak in the sedi-mentation pattern indicated that the cofactor wascovalently bound to the monomeric form of theenzyme (Fig. 2). The lower percentage of monomerpresent in this sedimentation profile (12% instead of35%; Fig. 2 and Table 1) is accounted for by thehigher concentration of enzyme employed in the exper-iment, and as calculated by using the equation describ-Table 1. Sedimentation and dissociation constants calculated fromultracentrifuge experiments on apo- and holo-forms of wild-typeand mutant eSHMTs. Values are shown of the S20,wsedimentationcoefficient calculated in sedimentation velocity experiments onenzyme samples at 2.5 lM subunit concentration, in 50 mMNaHepes buffer (pH 7.2), containing 200 lM dithiothreitol and100 lM EDTA, at 20 °C. Percentages in parenthesis correspond tothe fraction of enzyme subunits that sediment with the relatedcoefficient and were calculated from an integration of the sedimen-tation profiles shown in Fig. 2. The dissociation constants of dimer–monomer equilibria (Kd) were determined from sedimentationequilibrium experiments carried out on enzyme samples in the2.5–25 lM subunit concentration range.S20, w(S) Kd(M)1)aHoloenzyme formsWT 5.5 NDL85A 5.5 NDL276A 5.5 NDL85A ⁄ L276A 5.5 (66%) 3.3 (34%)5.5 (88%)b3.1 (12%)b1.7 · 10)6Apoenzyme formsWT 5.5 NDL85A 5.5 (90%)c2.7 (8%)c4.0 · 10)9L276A 5.5 (65%) 3.1 (35%) 2.7 · 10)6L85A ⁄ L276A 3.1 NDaDissociation constants could not be calculated for wild-type andsingle mutant holoenzymes and for the double mutant apoenzymebecause these were completely either in the dimeric or monomericstate in the range of protein concentration used (ND, not deter-mined). However, the detection limit of the instrumentationemployed, which may be estimated to be approximately 1% (per-centage of detectable monomer in a dimeric sample or vice versa),restricts the Kdfor the dimeric holo-forms to values £ 8 · 10)9M)1and the Kdfor the monomeric double mutant apoenzyme to values‡ 5 · 10)4M)1(calculated on the basis of Eqn (1), assuming that1% of undetected dimer or monomer were present in the sedimen-tation velocity experiments carried out at a subunit concentration of2.5 lM).bCalculated on data collected at 420 nm with an enzymesample at a subunit concentration of 33 lM. Data of all other exper-iments were collected at 277 nm.cIn the sedimentation velocityexperiments on the L85A apoenzyme, approximately 2% of sub-units sedimented very slowly in the form of aggregates.Role of hydrophobic contacts in serine hydroxymethyltransferase R. Florio et al.134 FEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBSing the dissociation equilibrium (Eqn 1). A completeshift of the equilibrium in favor of the dimeric formwas obtained when l-serine (1 mm) was added to thedouble mutant holoenzyme (Fig. 2).PLP binding propertiesThe affinity of wild-type and mutant forms for thecofactor was measured to evaluate the impact of themutations on the structure of the PLP binding site.Because PLP binding to apo-eSHMT is known toquench the intrinsic fluorescence emission of theenzyme [10], without changing the wavelength of maxi-mum emission, the dissociation constant of the bindingequilibrium was calculated from saturation curvesobtained by measuring the fluorescence emission ofapoenzyme samples (26 nm subunit concentration) atincreasing PLP concentrations (Fig. 3). Table 2 showsthat the apparent Kdvalues calculated from leastsquare fitting of experimental data points to Eqn (2)are essentially the same for all enzyme forms. The cal-culated relative fluorescence intensities in the absenceof PLP (F0) or in the presence of saturating concentra-tions of cofactor (Finf) were: F0= 125 ± 1 andFinf=65 ± 1 for the wild-type enzyme; F0= 139 ± 1and Finf= 66 ± 1 for L85A; F0= 122 ± 1 andFinf= 63 ± 1 for L276; and F0= 103 ± 1 andFinf= 73 ± 1 for L85A ⁄ L276A. The higher fluores-cence with respect to wild-type observed with the L85Aapoenzyme may be explained by the presence of a smallpercentage of subunits present as aggregates (Table 1).A remarkable difference is noted with respect to theintrinsic fluorescence emission intensities of apo- andholo-forms of the L85A ⁄ L276A double mutant: therelative fluorescence emission of the double mutantapoenzyme is considerably lower than that of the otherapo-forms and PLP binding does not quench fluores-cence to the same extent it does with the other holo-forms, although the wavelength of maximum emissionis the same for all enzyme forms (Fig. 3, insets).In the light of the results obtained from the ultra-centrifuge experiments, it should be noted that, at aconcentration of 26 nm, the association state of subun-its is expected to vary among wild-type and mutantenzymes. Indeed, it may be calculated, using the disso-ciation constants showed in Table 1 and according toEqn (1), that, at this concentration, the apoenzymesubunits of wild-type eSHMT are mostly in thedimeric state (for a fraction ‡ 90%), the apo-L85A isapproximately 75% dimeric, whereas the apo-L276Aand the apo-L85A ⁄ L85A mutants are in the mono-meric state. The association state of the holoenzymescan be estimated to be ‡ 90% dimeric for all enzymeforms, except for the L85A ⁄ L276A double mutant,Fig. 2. Sedimentation velocity distributions obtained with apo- and holo-forms of wild-type and mutant eSHMTs. Sedimentation velocitymeasurements were performed at 116 480 g on 2.5 lM (subunit concentration) holoenzyme (—) and apoenzyme (- - -) samples kept at20 °Cin50mM NaHepes buffer (pH 7.2), containing 200 lM dithiothreitol and 100 lM EDTA. L-serine (1 mM) was added to a sample of theL85A ⁄ L276A double mutant holoenzyme (ÆÆÆÆÆ). Absorbance data were all collected at 277 nm, except in the case of a sedimentation experi-ment carried out on the double mutant holoenzyme at a subunit concentration of 33 lM, when the absorbance of protein-bound cofactorwas measured at 420 nm (Æ-Æ-Æ).R. Florio et al. Role of hydrophobic contacts in serine hydroxymethyltransferaseFEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBS 135which is expected to be mostly in the monomeric state.Therefore, the similar values of dissociation constantobtained with wild-type and mutant enzymes suggestthat the cofactor binds to monomeric and dimericforms with similar affinities.Catalytic propertiesSHMT catalyses the reversible transfer of the Cbofl-serine to tetrahydropteroylglutamate (H4PteGlu),with formation of glycine and 5,10-methylene-H4Pte-Glu. However, in the absence of H4PteGlu, it alsoaccelerates the cleavage of several different l-3-hydr-oxyamino acids to glycine and the correspondingaldehyde [11]. Both erithro and threo forms of l-3-phenylserine are rapidly cleaved to glycine and benzal-dehyde [12,13]. The serine hydroxymethyltransferaseand l-threo-phenylserine aldolase activities of alleSHMT forms were assayed using enzyme samples (at0.05 and 3 lm subunit concentrations, respectively)saturated with PLP. The calculated kinetic parametersof both reactions are shown in Table 2. The L85Amutation had virtually no effect on the catalytic prop-erties of the enzyme. Minor differences with respect toTable 2. Dissociation constants of PLP binding equilibrium and kinetic parameters determined with wild-type and mutant eSHMTs. Para-meters are expressed as the mean ± SD determined by nonlinear least squares fitting of data to the related equation (see Experimentalprocedures).KdPLPa(nM)aKmbSer(mM)aKmbH4PteGlu(lM)kcatSHMTc(min)1)Km/-Serd(mM)kcat/-Serd(min)1)Wild-type 5.11 ± 1.14 0.14 ± 0.01 7.03 ± 0.88 686.6 ± 21.7 36.3 ± 1.4 202.1 ± 4.2L85A 5.88 ± 0.73 0.15 ± 0.01 7.16 ± 1.57 647.4 ± 36.3 37.4 ± 2.9 257.8 ± 13.7L276A 5.01 ± 0.87 0.20 ± 0.01 4.35 ± 0.52 400.5 ± 10.8 42.6 ± 1.8 132.3 ± 3.2L85A ⁄ L276A 6.50 ± 1.69 0.20 ± 0.01 11.20 ± 1.34 400.0 ± 15.3 42.1 ± 4.5 173.9 ± 10.7aDissociation constant of PLP binding equilibrium.bApparent Kmof either L-serine or H4PteGlu in serine hydroxymethyltransferase reactionwhen the other substrate is at saturating concentration.cCatalitic constant of the serine hydroxymethyltransferase reaction.dKinetic param-eters of theL-threo-3-phenylserine cleavage reaction.Fig. 3. Comparison of PLP-binding saturation curves obtained with wild-type and mutant enzymes. Apoenzyme samples (26 nM) were mixedwith different concentrations of PLP (1–400 nM)in50mM NaHepes (pH 7.2), containing 200 lM dithiothreitol and 100 lM EDTA, at 20 °C.Fluorescence emission spectra were measured in a 1-cm quartz cuvette with excitation at 280 nm. The graphs report the relative fluores-cence intensity at 336 nm (Fr) as a function of the total PLP concentration (i.e. the concentration of free and enzyme-bound PLP). The contin-uous lines are those calculated from the least square fitting of experimental data to Eqn (2). Insets show comparisons between the intrinsicfluorescence emission spectra of holoenzyme (—) and apoenzyme (- - -) forms of wild-type (thick lines) and mutant (thin lines) eSHMT.Role of hydrophobic contacts in serine hydroxymethyltransferase R. Florio et al.136 FEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBSwild-type were observed with the L276A and theL85A ⁄ L276A mutants, which yielded similar kineticparameters overall: with both reactions tested, kcatvalues were decreased by up to two-thirds and theKmfor amino acid substrates were slightly increased.Given the dimerization effect observed in the ultracen-trifuge experiments upon addition of l-serine to thedouble mutant holoenzyme, it may be assumed thatthe slightly different kinetic parameters of L276A andL85A ⁄ L276A mutants do not depend on the oligomer-ization states of the enzymes. It may be inferred thatthe minor changes of catalytic properties observed withthe double mutant enzyme are determined by theL276A mutation.Far and near-UV circular dichroism spectraThe far-UV CD spectra of wild-type and mutant apo-and holoenzymes at a subunit concentration of 2.5 lmwere virtually identical (data not shown), indicatingthat the mutations did not alter the secondary structureof the enzyme. The tertiary structure of all apo- andholo-forms was analyzed measuring and comparingtheir near-UV CD spectra at a subunit concentrationof 35 lm. A substantial similarity was observed amongthe aromatic CD spectra of wild-type, L85A andL276A apo-eSHMTs, with respect to fine structure andrepresentative bands (Fig. 4). It may be estimated thatthe L276A mutant at this concentration is approxi-mately 80% dimeric, whereas the other apo-forms arecompletely dimeric. In the case of the L85A ⁄ L276Adouble mutant apoenzyme, the fine structure of thespectrum is markedly less resolved than it is with allother apo-forms, despite the similarity in overall ellipt-icty (Fig. 4). The loss of fine structure may beaccounted for by the fact that, at a concentration of35 lm, the enzyme exists mostly as a monomer (for afraction ‡ 90%). Because PLP binds to the monomericand dimeric apoenzyme with similar affinities, it maybe deduced that the structure of the monomer is analo-gous to that of the subunits in the dimeric enzyme.The protein-bound cofactor contributes to the CDspectra of all the holoenzymes with a broad positiveband centered at 420 nm and a negative band atapproximately 340 nm, which are similar in all enzymeforms (data not shown). The presence of the cofactornegative band determines a significant increase of theoverall negative ellipticity below 320 nm (Fig. 4). PLPbinding to the wild-type apoenzyme also determines anincrease of the aromatic 285 nm band contribution. Avery similar spectral change is observed with the singlemutants that, at a concentration of 35 lm and in theholo-form, are fully dimeric. PLP binding to the dou-ble mutant apoenzyme definitely improves the resolu-Fig. 4. Comparison of the near-UV CD spec-tra of apo- and holo-forms of mutant (contin-uous lines) and wild-type (dashed lines)eSHMTs. Enzymes samples (35 lM) weredissolved in 50 mM NaHepes (pH 7.2), con-taining 200 lM dithiothreitol and 100 lMEDTA, at 20 °C. CD spectra were measuredusing a 1 cm quartz cuvette.R. Florio et al. Role of hydrophobic contacts in serine hydroxymethyltransferaseFEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBS 137tion of the fine structure of the CD spectrum. How-ever, the CD spectrum of the double mutantholoenzyme (85% dimeric at a concentration of35 lm) shows lower negative ellipticity in the aromaticregion and a less pronounced 285 nm band (Fig. 4),indicating that the formation of the dimer induced byPLP binding is not accompanied by a complete recov-ery of the native tertiary structure.DiscussionThe decrease of hydrophobic contact area determinedby the mutations in the third cluster of CHCs caused ashift of the equilibrium between dimeric and monomericforms of eSHMT in favor of the latter. The extent towhich this happened was maximum for theL85A ⁄ L276A double mutation and minimum for theL85A mutation, whereas the L276A had an intermedi-ate effect. The alteration of the dimer–monomer equi-librium determined by the single L85A and L276Amutations was not accompanied by any significantchange of tertiary structure, as determined by analysisof the fluorescence and near-UV CD properties. On theother hand, the L85A ⁄ L276A double mutation had visi-ble effects on both the intrinsic fluorescence and near-UV CD properties of the enzyme. Both L276A andL85A ⁄ L276A apoenzymes are in the monomeric stateat the concentration used in the intrinsic fluorescencemeasurements. However, the fluorescence emission ofthe double mutant apoenzyme is much less intense thanthat of the L276A apoenzyme, for which the emissionspectrum, in turn, is very similar to that of the wild-typeapoenzyme (Fig. 3). This indicates that the presence ofboth mutations slightly perturbed the native structureof the monomer. When PLP binds to the monomericdouble mutant, the intrinsic fluorescence is quenched toa lesser extent than with the other enzyme forms. Thisdifference may be attributed to the fact that, upon PLPbinding, the double mutant holoenzyme stays in themonomeric state, whereas all the other forms becomedimeric, as revealed by analytical ultracentrifugation.Nevertheless, at a higher enzyme concentration, whenthe subunits of the double mutant associate into adimer, the aromatic CD spectrum of the double mutantindicates that minor structural changes with respect tothe wild-type enzyme are present.None of the introduced mutations had large conse-quences on the catalytic properties of the enzyme. Inthis respect, it should be noted that substrate bindingto the double mutant eSHMT was observed to stabi-lize the dimeric, catalytically competent form of theenzyme (Fig. 2). Therefore, it is expected that allenzyme forms were dimeric in the conditions used toassay their catalytic activity. It may be deduced thatthe structural alterations determined by the mutationshad a modest impact on the overall tertiary structureof the enzyme and were largely confined to the stabil-ity of the native quaternary assembly. Initially, theobserved effects of the mutations on the quaternarystructure may appear to be rather surprising becausethe residues replaced by site-directed mutagenesis arefar away from the subunit interface. Nevertheless, inSHMT, an important interaction between the subunitsis established between the a-helices of the third clusterof CHCs and the N-terminal a-helix of the adjacentsubunit (Fig. 5). This observation may be sufficient toexplain the increase of the dissociation constant of themonomer–dimer equilibrium determined by the muta-tions. The results obtained in the present study alsoshow that the monomeric form of the enzyme is ableto bind PLP and that this binding event counteractsthe effect of the mutations, stabilizing the dimericform. The stabilizing effect is even more pronounced ifl-serine binds to the cofactor at the active site, as isevident in the case of the double mutant enzyme. Thisis monomeric in the apo-form, exists as an equilibriummixture of monomers and dimers even when all activesites are occupied by PLP and is fully converted into adimer by the addition of l-serine. Evidently, the muta-tions caused a slight and indirect alteration of crucialinteractions at the subunit interface. The stabilizingeffect of PLP and l-serine on the dimeric assemblysuggests that these alterations involve regions at thesubunit interface that are contacted by cofactor andsubstrates, when these are bound to the active site ofthe adjacent subunit. Scrutiny of the eSHMT crystalstructure [14] reveals that two polypeptide loops, atthe N-terminal ends of the a-helices that form the thirdcluster of CHCs, are likely to be the relevant structuralregions (Fig. 5). One is the polypeptide section madeof residues 55¢–67¢ (where the primes indicate that theresidues are contributed by the other subunit). IneSHMT, Y55¢ interacts with the phosphate moiety ofPLP; E57¢ is crucial in the binding of the l-serinehydroxyl group [15] and in the catalysis of the hydrox-ymethyltransferase reaction [16]; and Y65¢ interactswith the carboxylate group of substrates and plays akey role in substrate binding [17]. An alignment among63 amino acid sequences of the enzyme from severaldifferent sources showed that all three residues areinvariant in SHMT [18]. The second polypeptide loop,comprising residues 258¢–264¢, is a very conservedregion which interacts with the phosphate moiety ofPLP. It is delimited by two invariant proline residues,P258¢ (which is in a cis configuration in all five knownstructures from mouse [19], human [20], rabbit [21],Role of hydrophobic contacts in serine hydroxymethyltransferase R. Florio et al.138 FEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBSE. coli [14] and Bacillus stearothermophilus [15]) andP264¢. The importance of this structural region is testi-fied by site-directed mutagenesis studies on P258¢ andP264¢, which showed how the native conformation ofthe loop is pivotal to PLP binding and catalysis [22].Given the above considerations, a series of possible,correlated outcomes of the mutations may be envis-aged. The decrease of the hydrophobic contact area inthe third cluster of CHCs is expected to alter the asso-ciation of the a-helices that form the cluster and,consequently, to weaken their interaction with theN-terminal a-helix. This can be imagined to be themain cause of subunit dissociation in apo-eSHMT. Atthe same time, the mutations may indirectly alter thestructure of the 55¢–67¢ and 258¢–264¢ loops. In holo-eSHMT, this alteration may compromise the interac-tions between the cofactor bound at the active site andthe loops of the adjoining subunit, promoting subunitdissociation. In the holoenzyme, effects on both theN-terminal helix and on the 55¢–67¢ and 258¢–264¢loops would be present. On the other hand, PLP bind-ing to the apoenzyme is expected to stabilize the struc-ture of the loops and, indirectly, the associationbetween the helices of the cluster. This, as a conse-quence, would reinforce the interaction among thehelices and the N-terminal helix of the other subunit,promoting dimerization. At this point, the observationthat the monomeric eSHMT binds PLP (Fig. 2) andthat all mutant forms show similar affinity for thecofactor (Table 2), although they exist as differentmixtures of monomers and dimers at equilibrium, israther puzzling. One possible explanation is that theaffinity of monomeric eSHMT for PLP is lower thanthat of the dimer, although not drastically different.This difference may not be detectable in the experi-ments performed in the present study.The high degree of sequence and structural conserva-tion of the third cluster of CHCs, observed for themajority of fold type I enzymes, suggests that its stabi-lizing function, hypothesized for eSHMT, could beextended to the whole superfamily. The extent to whichthe third hydrophobic cluster is involved in the stabil-ization of the dimer assembly might be differentamongst fold type I enzymes, depending on the sizeand orientation of the N-terminal arm. However,although the 55¢–67¢ loop of eSHMT is a highly diversi-fied region in fold type I enzymes, it invariably containsresidues involved in PLP and substrate binding andcatalysis [8,23]. Moreover, the second polypeptide loop(residues 258¢–264¢ in eSHMT) represents a very con-served region in this group of enzymes, interacting withthe phosphate moiety of PLP. Therefore, the capabilityof the third cluster of CHCs to confer stability to thepolypeptide loops of the active site is presumablyimportant in all members of the fold type I family.An additional clue on the structural importance of thethird cluster of CHCs in fold type I enzymes comes fromfolding studies on eSHMT. The folding mechanism ofeSHMT may be divided into two phases [10,22,24,25].In the first, relatively rapid phase, the two domains foldinto a native-like intermediate that has virtually all ofthe native secondary and tertiary structure, but is unableFig. 5. Schematic representation of thedimeric structure of eSHMT. Enzyme subun-its of the eSHMT•Gly•5-formyl H4PteGluternary complex (Protein Data Bank: 1dfo)[14] are shown in salmon and cyan,whereas the PLP-gly complex is shown asin Fig. 1. The a-helices involved in the for-mation of the third cluster of CHCs belongto the subunit shown in salmon. The firsta-helix and the 55¢–67¢ loop are colored inred; the second a-helix and the 258¢–264¢loop are shown in magenta. The N-terminalhelix of the cyan subunit is shown in blue.Residues mentioned in the text are labeled.R. Florio et al. Role of hydrophobic contacts in serine hydroxymethyltransferaseFEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBS 139to bind PLP. In this intermediate, the N-terminus andan inter-domain segment remain exposed to solvent. Inthe following, slow phase, these structural elementsassume their native conformation, completing the activesite that acquires the capability to bind PLP. Notice-ably, one helix of the third cluster of CHCs is part of theinter-domain segment and the other helix lines theboundary between the major domain and the N-termi-nus (Fig. 1). Therefore, the formation of the thirdcluster may represent a key event of the final phaseof folding, determining the assembly of eSHMT activesite and promoting dimerization.Experimental proceduresMaterialsIngredients for bacterial growth were obtained from Sigma-Aldrich (St Louis, MO, USA). Chemicals for the purifica-tion of the enzymes were obtained from BDH ⁄ Merck(Whitehouse Station, NJ, USA); DEAE-Sepharose andPhenyl-Sepharose were obtained from GE Healthcare (Mil-waukee, WI, USA). Wild-type and mutant forms ofeSHMT and methylenetetrahydrofolate dehydrogenase werepurified as previously described [26,27]. PLP was added toprotein samples during the purification procedure, but itwas left out in the final dialysis step. (6S)H4PteGlu, was agift from Eprova AG (Schaffhausen, Switzerland). PLP wasobtained from Sigma-Aldrich (98% pure). All otherreagents were obtained from Sigma-Aldrich.Preparation of apoenzyme and holoenzymesamplesApo-eSHMT was prepared using l-cysteine as previouslydescribed [10]. The apoenzyme, for which the subunit con-centration was calculated according to a molar absorptivityvalue of e280= 42790 cm)1Æm)1[28], was stored in 10%glycerol at )20 °C for no more than 3 days before use. Asmall, residual fraction (less than 5%) of holoenzyme, esti-mated with activity assays, was present in the apoenzymesamples. We noticed that different batches of purifiedeSHMT samples contained variable holoenzyme ⁄ apo-enzyme ratios. This observation was made with eitherwild-type or mutant forms of the enzyme. To carry outcomparable experiments with the holo-forms, it was man-datory to prepare enzyme samples containing the samefraction of protein-bound PLP (possibly close to saturation)and, at the same time, devoid of any excess of cofactor.Holoenzymes were prepared from apoenzyme samples, byaddition of PLP at the concentration needed to obtain a98% saturation, calculated on the basis of the related disso-ciation constant of PLP binding equilibrium (Table 2),using Eqn (2A). The subunit concentration of the holo-enzyme was calculated according to a molar absorptivityvalue of e280= 44884 cm)1Æm)1[28].Site-directed mutagenesisSite-directed mutagenesis of E. coli glyA (SHMT encodinggene) coding region was performed with the Quick-ChangeÔ kit from Stratagene (La Jolla, CA, USA), usingthe pBS::glyA plasmid as template [26] and two comple-mentary oligonucleotide primers containing the mutations,which were synthesized by MWG-Biotech AG (Anzinger,Germany). The L85A and L276A mutants were producedusing as primers 5¢-CGTGCAAAGAAGCGTTCGGCGC-3¢ and 5¢-GCGGTTGCTGCGAAAGAAGCG-3¢, respec-tively, and their complementary oligonucleotides (themutated bases are underlined). The L85 ⁄ L276A doublemutant was obtained introducing the L276A mutation intoa template pS::glyA plasmid that already contained theL85A mutation. E. coli DH5a cells were used to amplifythe mutated plasmids . Both strands of the coding region ofthe mutated genes were sequenced. The only differenceswith respect to wild-type nucleotide sequence were thosethat were intended. Enzyme expression was performedusing the GS1993 recA) strain of E. coli [26].Analytical ultracentrifugation analysesSedimentation velocity and equilibrium experiments werecarried out at 20 °Cin50mm NaHepes buffer (pH 7.2),containing 200 lm dithiothreitol, 100 lm EDTA, on aBeckman XL-I analytical ultracentrifuge equipped withabsorbance optics and an An60-Ti rotor (Beckman Coul-ter, Fullerton, CA, USA). In the sedimentation velocityexperiments, performed at 116 480 g the protein concentra-tion was 2.5 lm for both apo- and holo-forms. Data werecollected at 277 nm and at a spacing of 0.003 cm withthree averages in a continuous scan mode. In the case ofthe double mutant L85A ⁄ L276A holoenzyme the sedimen-tation velocity experiments were also performed at420 nm, aiming to evaluate the presence of PLP covalentlybound to the monomeric species as a Schiff base with theactive site lysine residue [9]. Sedimentation coefficients andintegration of data were obtained using the software sed-fit (provided by P. Schuck, National Institutes of Health,Bethesda, MD, USA). The values were reduced to water(S20,w) using standard procedures. The buffer density andviscosity were calculated by the software Sednterp. Theratio f ⁄ f0was calculated from the diffusion coefficient,which, in turn, is related to the spreading of the bound-ary, using the software sedfit. Sedimentation equilibriumexperiments were performed at 7280, 10 483 and 14 270 gon enzyme samples at subunit concentrations of 2.5 and25 lm. Data were collected every 3 h at a spacing ofRole of hydrophobic contacts in serine hydroxymethyltransferase R. Florio et al.140 FEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBS0.001 cm with ten averages in a step scan mode. Equilib-rium was checked by comparing scans for up to 24 husing the software winmatch (J. Lary, National Analyti-cal Ultracentrifugation Center, Storrs, CT, USA). Datasets were edited by reedit (J. Lary, National AnalyticalUltracentrifugation Center, Storrs, CT, USA) and ana-lyzed using the software sedphat (National Institutes ofHealth). Data from different speeds were combined forglobal fitting. When a monomer–dimer association modelwas required for data fitting, the monomer molecular masswas fixed at the value determined from the amino acidsequence (45 320 Da).Measurement of the Kdof PLP bindingequilibriumPLP binding equilibria were analyzed taking advantage ofthe protein intrinsic fluorescence quenching observed uponthe binding event [10]. Dissociation constants of bindingequilibria were then calculated from saturation curvesobtained measuring the protein fluorescence emission inten-sity as a function of increasing PLP concentration. Thecofactor (range 1–400 nm) was added to apoenzyme sam-ples (26 nm)at20°Cin50mm NaHepes (pH 7.2), contain-ing 200 lm dithithreitol and 100 lm EDTA. Preliminaryexperiments demonstrated that, with all enzyme forms, thebinding equilibrium was established within the mixing time.Fluorescence emission spectra (300–450 nm; 7 nm emissionslit) were recorded immediately after mixing PLP and apo-enzyme with a LS50B spectrofluorimeter (Perkin-Elmer,Waltham, MA, USA), with excitation wavelength set at280 nm (5 nm excitation slit), at the same temperature andwith a 1 cm path length quartz cell. Data were analyzedaccording to Eqn (2).Activity assaysAll assays were carried out at 20 °Cin50mm NaHepes(pH 7.2), containing 200 lm dithiothreitol and 100 lmEDTA. The serine hydroxymethyltransferase activity wasmeasured with 0.05 lm enzyme samples with l-serine andH4PteGlu as substrates, as previously described [9]. Todetermine the Kmfor l-serine, H4PteGlu was maintained at0.23 mm and the l-serine concentration was varied in therange 0.06–6 mm. For Kmdeterminations of H4PteGlu,l-serine concentrations were held constant at 30 mm andH4PteGlu concentrations varied in the range 3–500 lm. Therate of l-threo-phenylserine cleavage (3 lm enzyme samples)was obtained from spectroscopic measurement of benzalde-hyde production at 279 nm, employing a molar absorptivityvalue of e279= 1400 cm)1Æm)1[12,13]. The Kmfor l-threo-phenylserine was determined by varying the substrateconcentration in the range 4–200 mm.Spectroscopic measurementsFluorescence emission measurements were carried out at20 °C with a LS50B spectrofluorimeter (Perkin-Elmer)using a 1 cm path length quartz cuvette. Fluorescence emis-sion spectra were recorded from 300–450 nm (1 nm sam-pling interval), with the excitation wavelength set at280 nm. Far- (190–250 nm), near-UV (250–310 nm) andvisible (310–500 nm) CD spectra were measured using 0.2and 1 cm path length quartz cuvettes and the resultsobtained were expressed as the mean residue ellipticity (Q),assuming a mean residue molecular mass of 110 per aminoacid residue. UV–visible spectra were recorded with a dou-ble-beam Lambda 16 (Perkin-Elmer). Kinetic measurementsin the activity assays were performed on a HP 8453 diode-array spectrophotometer (Hewlett-Packard, Palo Alto, CA,USA). All spectroscopic measurements were carried out at20 °Cin50mm NaHepes (pH 7.2), containing 200 lm dith-iothreitol and 100 lm EDTA.Data analysisKinetic parameters were determined by nonlinear leastsquares fitting of initial velocity data to the Michaelis–Menten equation using the software prism (GraphPad Soft-ware Inc., San Diego, CA, USA). The concentrations ofmonomeric and dimeric species at equilibrium were calcu-lated from Eqn (1) [29], in which [Deq] and [Et] are theequilibrium concentrations of the dimer and the total con-centration of the enzyme, respectively, expressed as dimerequivalents, and Kdis the dissociation constant of therelated equilibrium.DeqÂü8  Et½þKdÀffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi16  Et½ÂKdþ K2dp8ð1ÞFluorescence data obtained in PLP binding equilibriumexperiments were analyzed according to Eqn (2), in whichFrelis the measured relative fluorescence, F0is fluorescencein the absence of PLP, Finfis fluorescence at infinite PLPconcentration, [E] is the total enzyme subunit concentration,[PLP] is the total cofactor concentration and Kdis the disso-ciation constant of the equilibrium HOLO ¢ APO þ PLP:Frel¼ F0À F0À FinfðÞÂPLP½þE½þKdÀffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiÀ Kdþ PLP½þE½ðÞ2þ4  PLP½þKdðÞÀ4  E½ÂPLP½q2E½ð2ÞR. Florio et al. Role of hydrophobic contacts in serine hydroxymethyltransferaseFEBS Journal 276 (2009) 132–143 ª 2008 The Authors Journal compilation ª 2008 FEBS 141[...]... Scarsdale N, Kazanina G & Schirch V (2000) Role of tyrosine 65 in the mechanism of serine hydroxymethyltransferase Biochemistry 39, 7492–7500 18 Paiardini A, Gianese G, Bossa F & Pascarella S (2003) Structural plasticity of thermophilic serine hydroxymethyltransferases Proteins 50, 122–134 19 Szebenyi DM, Liu X, Kriksunov IA, Stover PJ & Thiel DJ (2000) Structure of a murine cytoplasmic serine hydroxymethyltransferase. .. V (1996) Structural studies on folding intermediates of serine hydroxymethyltransferase using single tryptophan mutants J Biol Chem 271, 2987–2994 25 Cai K & Schirch V (1996) Structural studies on folding intermediates of serine hydroxymethyltransferase using fluorescence resonance energy transfer J Biol Chem 271, 27311–27320 Role of hydrophobic contacts in serine hydroxymethyltransferase 26 Iurescia.. .Role of hydrophobic contacts in serine hydroxymethyltransferase The fraction present in Eqn (1) corresponds to the fraction of subunits that binds PLP at equilibrium  à ( HOLOeq =½EŠ) The concentration of holoenzyme at equilibrium ([HOLOeq]) was derived from the equation for the dissociation constant of the binding equilibrium: À  ÃÁ À  ÃÁ ½EŠ À HOLOeq... Schirch V, Hopkins S, Villar E & Angelaccio S (1985) Serine hydroxymethyltransferase from Escherichia coli: purification and properties J Bacteriol 163, 1–7 10 Cai K, Schirch D & Schirch V (1995) The affinity of pyridoxal 5¢-phosphate for folding intermediates of Escherichia coli serine hydroxymethyltransferase J Biol Chem 270, 19294–19299 11 Schirch V (1998) Mechanism of folate-requiring enzymes in one-carbon... 2008 The Authors Journal compilation ª 2008 FEBS R Florio et al 22 Fu TF, Boja ES, Safo MK & Schirch V (2003) Role of proline residues in the folding of serine hydroxymethyltransferase J Biol Chem 278, 31088–31094 23 Contestabile R, Paiardini A, Pascarella S, di Salvo ML, D’Aguanno S & Bossa F (2001) l-Threonine aldolase, serine hydroxymethyltransferase and fungal alanine racemase A subgroup of strictly... Savithri HS & Subramanya HS (2002) Crystal structure of binary and ternary complexes of serine hydroxymethyltransferase from Bacillus stearothermophilus: insights into the catalytic mechanism J Biol Chem 277, 17161–17169 16 Szebenyi DM, Musayev FN, di Salvo ML, Safo MK & Schirch V (2004) Serine hydroxymethyltransferase: role of glu75 and evidence that serine is cleaved by a retroaldol mechanism Biochemistry... mutagenesis techniques in the study of Escherichia coli serine hydroxymethyltransferase Protein Expr Purif 7, 323–328 27 Schirch V (1997) Purification of folate-dependent enzymes from rabbit liver Methods Enzymol 281, 146– 161 28 Malerba F, Bellelli A, Giorgi A, Bossa F & Contestabile R (2007) The mechanism of addition of pyridoxal 5¢-phosphate to Escherichia coli apo -serine hydroxymethyltransferase. .. Biophys Acta 1248, 81–96 4 Grishin NV, Phillips MA & Goldsmith EJ (1995) Modeling of the spatial structure of eukaryotic ornithine decarboxylases Protein Sci 4, 1291–1304 5 Jansonius JN (1998) Structure, evolution and action of vitamin B6-dependent enzymes Curr Opin Struct Biol 8, 759–769 6 Schneider G, Kack H & Lindqvist Y (2000) The manifold of vitamin B6 dependent enzymes Structure 8, R1–R6 7 Kack H,... hydroxymethyltransferase quinonoid ternary complex: evidence for asymmetric obligate dimers Biochemistry 39, 13313–13323 20 Renwick SB, Snell K & Baumann U (1998) The crystal structure of human cytosolic serine hydroxymethyltransferase: a target for cancer chemotherapy Structure 6, 1105–1116 21 Scarsdale JN, Kazanina G, Radaev S, Schirch V & Wright HT (1999) Crystal structure of rabbit cytosolic serine hydroxymethyltransferase. .. Schneider G & Lindqvist Y (1999) Crystal structure of diaminopelargonic acid synthase: evolutionary relationships between pyridoxal-5¢-phosphate-dependent enzymes J Mol Biol 291, 857–876 8 Paiardini A, Bossa F & Pascarella S (2004) Evolutionarily conserved regions and hydrophobic contacts at the superfamily level: the case of the fold-type I, pyridoxal5¢-phosphate-dependent enzymes Protein Sci 13, 2992– . The role of evolutionarily conserved hydrophobic contacts in the quaternary structure stability of Escherichia coli serine hydroxymethyltransferase Rita. impact of the mutations on the structure of the PLP binding site.Because PLP binding to apo-eSHMT is known toquench the intrinsic fluorescence emission of the enzyme
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Xem thêm: Báo cáo khoa học: The role of evolutionarily conserved hydrophobic contacts in the quaternary structure stability of Escherichia coli serine hydroxymethyltransferase pptx, Báo cáo khoa học: The role of evolutionarily conserved hydrophobic contacts in the quaternary structure stability of Escherichia coli serine hydroxymethyltransferase pptx, Báo cáo khoa học: The role of evolutionarily conserved hydrophobic contacts in the quaternary structure stability of Escherichia coli serine hydroxymethyltransferase pptx