Rat GTP cyclohydrolase I is a homodecameric protein complex containing high-affinity calcium-binding...

J. Mol. Biol. (1998) 279, 189±199

Rat GTP Cyclohydrolase I is a HomodecamericProtein Complex Containing High-affinityCalcium-binding Sites

Michel O. Steinmetz1, Christoph PluÈ ss2, Urs Christen3

Bettina Wolpensinger1, Ariel Lustig4, Ernst R. Werner5

Helmut Wachter5, Andreas Engel1, Ueli Aebi1, Josef Pfeilschifter2,6*and Richard A. Kammerer4

1Maurice E. MuÈ ller Institutefor Microscopy, BiozentrumUniversity of BaselKlingelbergstrasse 70CH-4056 Basel, Switzerland2Department of PharmacologyBiozentrum, University of BaselKlingelbergstrasse 70CH-4056 Basel, Switzerland3Department PDC1F. Hoffmann-LaRoche LtdCH-4070 Basel, Switzerland4Department of BiophysicalChemistry, BiozentrumUniversity of BaselKlingelbergstrasse 70CH-4056 Basel, Switzerland5Institute for MedicalChemistry and BiochemistryUniversity of InnsbruckFritz-Pregl-Strasse 3A-6020 Innsbruck, Austria6Zentrum der PharmakologieKlinikum der Johann WolfgangGoethe-UniversitaÈtTheodor-Stern-Kai 7D-60590 Frankfurt am MainGermany

Correspondence address: Prof. J.Goethe-UniversitaÈ t, Theodor-Stern-

Abbreviations used: ADF, annulresponsive dystonia; EM, electronCH-I, GTP cyclohydrolase I; HDPscanning transmission electron mreaction; [�], mean molar residues20,W, sedimentation coef®cient (wchromatography.

0022±2836/98/210189±11 $25.00/0/mb

Recombinant rat liver GTP cyclohydrolase I has been prepared byheterologous gene expression in Escherichia coli and characterized bybiochemical and biophysical methods. Correlation averaged electronmicrograph images of preferentially oriented enzyme particles revealeda ®vefold rotational symmetry of the doughnut-shaped views with anaverage particle diameter of 10 nm. Analytical ultracentrifugation andquantitative scanning transmission electron microscopy yielded averagemolecular masses of 270 kDa and 275 kDa, respectively. Like theEscherichia coli homolog, these ®ndings suggest that the active enzymeforms a homodecameric protein complex consisting of two ®vefoldsymmetric pentameric rings associated face-to-face. Examination of theamino acid sequence combined with calcium-binding experiments andmutational analysis revealed a high-af®nity, EF-hand-like calcium-bind-ing loop motif in eukaryotic enzyme species, which is absent in bac-teria. Intrinsic ¯uorescence measurements yielded an approximatedissociation constant of 10 nM for calcium and no signi®cant bindingof magnesium. Interestingly, a loss of calcium-binding capacityobserved for two rationally designed mutations within the presumedcalcium-binding loop of the rat GTP cyclohydrolase I yielded a 45%decrease in enzyme activity. This ®nding suggests that failure of cal-cium binding may be the consequence of a mutation recently identi®edin the causative GTP cyclohydrolase I gene of patients suffering fromdopa responsive dystonia.

# 1998 Academic Press Limited

Keywords: digital image processing; dopa responsive dystonia; EF-hand-like calcium-binding loop motif; electron microscopy; homodecamericenzyme complex
*Corresponding author
M. Pfeilschifter, Zentrum der Pharmakologie, Klinicum der Johann WolfgangKai 7, D-60590 Frankfurt am Main, Germany.ar dark ®eld; BH4, tetrahydrobiopterin; CD, circular dichroism; DRD, dopamicroscopy; GFRP, GTP cyclohydrolase I feed-back regulatory protein; GTP-

, hereditary progressive dystonia with marked diurnal ¯uctuation; STEM,icroscopy; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain

ellipticity; rpm, revolutions per minute; AUC, analytical ultracentrifugation;ater, 20�C); NOS, nitric oxide synthase; IMAC, immobilized metal af®nity

981649 # 1998 Academic Press Limited

190 Structural Studies on Rat GTP Cyclohydrolase I

Introduction

GTP cyclohydrolase I (GTP-CH-I) catalyzes the®rst committing step in the multienzyme conver-sion of GTP to folic acid in microorganisms, andto tetrahydrobiopterin (BH4) in higher animals(Nichol et al., 1985). BH4 is involved in a wide var-iety of important physiological functions. Not onlyis it required as an essential co-factor for aromaticamino acid hydroxylases (Kaufman, 1993) and forall nitric oxide synthase (NOS) isoforms (Moncadaet al., 1991), but it has also been suggested to beinvolved in proliferation and growth regulationof erythroid cells (Tanaka et al., 1989) and primedT-cells (Ziegler, 1990).

It has now been well established that abnormal-ities in the metabolism of BH4 are associated withcertain neurological disorders such as Parkinson'ssyndrome (Fujishiro et al., 1990) and Alzheimer'sdisease (Sawada et al., 1987). De®ciency in GTP-CH-I activity causes hyperphenylalaninemia(Niederwieser et al., 1984) and dopa responsivedystonia (DRD), a variant form of Parkinsonism(Ichinose et al., 1994).

The de novo pathway for BH4 biosynthesis hasbeen extensively characterized and is thought to beregulated by the activity of GTP-CH-I (Nichol et al.,1985). To this end, it has been shown that conver-sion of GTP to 7,8-dihydroneopterin triphosphateby GTP-CH-I is the ®rst and rate-limiting step inBH4 biosynthesis (Werner et al., 1996). Changes inGTP-CH-I enzyme activity usually mediate or clo-sely correlate with changes in BH4 levels (Duch &Smith, 1991). GTP-CH-I activity is controlled bothby transcriptional regulation (Werner et al., 1990;PluÈ ss et al., 1996) and by substrate levels(Hatakeyama et al., 1992). For mammalian GTP-CH-I several possible phosphorylation sites havebeen identi®ed (Hatakeyama et al., 1991), and ithas been shown that the enzyme becomes phos-phorylated in vivo (Imazumi et al., 1994). Moreover,the rat liver enzyme is subject to end product feed-back inhibition by BH4 which, in turn, is mediatedby the GTP-CH-I feedback regulatory protein(GFRP; Milstien et al., 1996) previously termed p35(Harada et al., 1993). In contrast, the two otherenzymes implicated in BH4 biosynthesis, 6-pyru-voyl tetrahydropterin synthase and sepiapterinreductase, are both constitutively expressed(Werner et al., 1990).

GTP-CH-I has been puri®ed to homogeneity andcharacterized from different sources includingE. coli (Yim & Brown, 1976), Bacillus subtilis(De Saizieu et al., 1995), Comamonas (Cone et al.,1974), Serratia indica (Kohashi et al., 1980), Drosophi-la melanogaster (Weisberg & O'Donnell, 1986), andhuman (Schoedon et al., 1989), mouse (Cha et al.,1991), and rat (Hatakeyama et al., 1989) liver.Characterization of B. subtilis GTP-CH-I hasrevealed that both calcium and magnesiumions drastically decrease its enzymatic activity (DeSaizieu et al., 1995). A similar effect was alsoobserved for the enzymes puri®ed from rat liver

(Hatakeyama et al., 1989), D. melanogaster(Weisberg & O'Donnell, 1986), S. indica (Kohashiet al., 1980), and E. coli (Yim & Brown, 1976). Incontrast to these data, Schoedon et al. (1992)reported stimulation of the E. coli enzyme by mag-nesium ions. Divalent cations did not affect theactivity of puri®ed GTP-CH-I from Comamonas(Cone et al., 1974), although addition of mag-nesium in the presence of a stimulatory proteinfraction increased its activity. The reasons for thesedifferent, in part contradictory effects of divalentcations of GTP-CH-I activity from different speciesare at present unclear.

E. coli GTP-CH-I has been studied in most detailby a combination of X-ray diffraction analysis andelectron microscopy of freeze-etched specimens(Meining et al., 1995). Its atomic structure has beendetermined by X-ray crystallography revealing a250 kDa homodecameric protein complex consist-ing of two 5-fold symmetric pentameric ringsassociated face-to-face (Nar et al., 1995). As yet, adetailed structural characterization of a mamma-lian GTP-CH-I homolog has remained elusive.Although available sequence information indicatesa high degree of conservation and similaritybetween different GTP-CH-I species (Maier et al.,1995), all homologs characterized to date are largemultimeric complexes, which differ in their exactsize and number of subunits (Yim & Brown, 1976;De Saizieu et al., 1995; Cone et al., 1974; Kohashiet al., 1980; Weisberg & O'Donnell, 1986; Schoedonet al 1989; Cha et al., 1991; Hatakeyama et al., 1989).

To address this important issue and to com-pare its structure with the E. coli homolog, wehave analyzed recombinant rat GTP-CH-I byanalytical ultracentrifugation (AUC), circulardichroism (CD) spectroscopy, and scanning trans-mission electron microscopy (STEM) combinedwith digital image processing. In addition, wehave identi®ed by sequence analysis a high-af®-nity, EF-hand-like calcium-binding loop motif.Calcium binding to GTP-CH-I was investigatedby 45Ca2�-overlay assays and intrinsic ¯uor-escence measurements, and its functional signi®-cance was assayed by mutational analysis andmeasurements of enzyme activity.

Results

Preparation of recombinant wild-type andmutant rat GTP-CH-I

For biophysical and structural characterizationrecombinant rat GTP-CH-I has been prepared byheterologous gene expression in E. coli. Based oncalcium-binding studies (see later), a mutant pro-tein was designed in which aspartate 114 and 116were changed to lysine residues (Figure 4). Thehomogeneity of the af®nity-puri®ed wild-type andmutant polypeptides was assessed by tricine-SDS-PAGE (SchaÈgger & von Jagow, 1987), whichrevealed single bands migrating with mobilities

Structural Studies on Rat GTP Cyclohydrolase I 191

corresponding to their molecular masses of26.6 kDa (i.e. as predicted by their sequences)and with no degradation products beingdetected (Figure 1A). To assess the conformation-al and oligomeric state of recombinant rat GTP-CH-I, CD spectroscopy, AUC, and STEM com-bined with digital image processing were per-formed.

Figure 1. A, Analysis of recombinant wild-type andmutant rat GTP-CH-I after af®nity-puri®cation andthrombin cleavage by tricine-SDS-PAGE (SchaÈgger &von Jagow, 1987) under reducing conditions. Lanes:1, wild-type enzyme; 2, mutant enzyme in which aspar-tate 114 and 116 were replaced by lysine residues. Themigration of marker proteins of known molecular massis indicated to the right and is given in kilodaltons.B, Circular dichroism spectrum of wild-type GTP-CH-I.The spectrum of the mutant enzyme was super-imposable and could not be distinguished from thespectrum of the wild-type protein.

CD spectroscopy, AUC, andquantitative STEM

The CD spectrum recorded from the wild-typeenzyme revealed minima at 208 nm and 222 nm(Figure 1B). A [�]222 value of ÿ12,000 deg. cm2

dmolÿ1 indicated 25 to 30% a-helicity by assumingthat a value of ÿ37,300 deg. cm2 dmolÿ1 corre-sponds to a 100% a-helix content (Chen et al.,1974). These spectroscopic data are consistent withthe recently solved crystal structure of the E. colihomolog yielding an ab conformation for the pro-tein (Nar et al., 1995). Notably, the CD spectrum ofthe mutant GTP-CH-I (data not shown) was indis-tinguishable from that of the wild-type enzyme(Figure 1B), indicating that replacing aspartate 114and 116 by lysine residues did not signi®cantlyinterfere with the enzyme's secondary structure.

The sedimentation coef®cients of wild-type andmutant GTP-CH-I were determined by sedimen-tation velocity centrifugation and their averagemolecular masses by sedimentation equilibriumcentrifugation and quantitative STEM. Sedimen-tation velocity runs revealed s20,w values of 10.7 Sand 11.4 S for the wild-type and mutant enzyme,respectively, values considerably larger than thoseexpected for a monomeric protein with a subunitmass of 26.6 kDa. In both cases, single sharpboundaries were observed, indicating that thesamples were homogeneous. Sedimentation equili-brium measurements yielded average molecularmasses of 283 kDa and 274 kDa for the twoenzyme complexes.

For STEM mass measurements, we recordedelastic dark-®eld images of unstained freeze-driedrecombinant wild-type GTP-CH-I (Figure 2).Enzyme particle images (875) were evaluated andput into a histogram that could be ®tted with asingle Gaussian curve peaking at 266 kDa andwith a half-height width corresponding to a stan-dard deviation of �65 kDa (Figure 2; inset). Cor-recting this value for the measured beam-inducedmass loss (MuÈ ller et al., 1992) yielded a value of275(�67) kDa. The standard deviation of the STEMmass measurements appeared typical and reason-able for particles of this kind and recorded undersimilar conditions (Engel & Colliex, 1993; andreferences therein). The molecular masses obtainedindependently by sedimentation equilibrium andquantitative STEM are consistent with a decamericstructure of both wild-type and mutant enzymecomplexes.

Enzyme morphology

Figure 3 displays an electron micrograph ofnegatively stained recombinant wild-type enzymeparticles recorded by STEM in the annular dark®eld (ADF) mode. The particles appeared fairlyhomogeneous in overall size and shape indicatingadsorption to the support ®lm in a preferred orien-tation. As revealed by the gallery of the contrastreversed high-magni®cation images, almost all

Figure 2. ADF electron micrograph of unstained/freeze-dried rat GTP-CH-I particles recorded by STEM. Lightregions represent high mass density and dark regionslow mass density. Scale bar, 100 nm. Inset: histogram ofmolecular mass measurements of 875 particles ®ttedwith a single Gaussian curve. The displayed valuesrepresent mean and standard deviation.

Figure 3. Contrast reversed ADF electron micrographsof negatively stained recombinant rat GTP-CH-Irecorded by STEM. Scale bars, 40 nm for the low-magni-®cation overview and 10 nm for the high-magni®cationindividual particle images displayed in the gallery onthe right. Bottom image of the gallery: calculated aver-age comprising 21 particles of a particular subclassobtained by multivariate statistical analysis of a set of550 particles with no symmetry imposed. Inset: sameaverage as the bottom image of the gallery but 5-foldrotationally symmetrized and displayed without (left)and with (right) isodensity contours superimposed.Scale bar, 5 nm.


enzyme particles harbored a central stain-®lledcavity. Visual inspection of the images suggests apredominantly ®vefold symmetric particle mor-phology of the preferentially adsorbed enzymemolecules.

A data set comprising 550 doughnut-like projec-tion images of the preferentially adsorbed, wellpreserved, and uniformly stained enzyme particleswas generated. The individual particle imageswere angularly and translationally aligned withrespect to a variety of reference images. For aver-aging, multivariate statistical analysis (van Heel,1984) was used to divide the total data set of 550particle images into 20 subclasses each represent-ing a set of mutually more similar images. Theapparent 5-fold rotational symmetry of the com-plex was about the same in all 20 subclasses (datanot shown). The variation between different classeswas mostly due to differences in the delineation ofthe particle boundary and the central cavity by theheavy metal salt deposition, and by differences inparticle inclination relative to the support ®lm.A high-magni®cation image of the average (withno rotational symmetry imposed) comprizing 21particles of a particular subclass is displayed at thebottom of the gallery in Figure 3. The same aver-age is also displayed further enlarged after 5-foldrotational symmetrization in the inset at the top ofFigure 3. The apparent diameter of the averagedand 5-fold symmetrized doughnut-like views ofthe enzyme particle was approximately 10 nm, andits stain-®lled central cavity revealed an apparent

diameter of about 1 to 3 nm. In Figure 4 we havecompared our calculated particle average obtainedby EM of the recombinant rat GTP-CH-I proteincomplex (Figure 4D; see also Figure 3, inset) withthe atomic structure (Figure 4A) of the E. colienzyme determined by Nar et al. (1995). For thatpurpose, an electron density map was computedand surface rendered from the atomic coordinatesof the E. coli homolog at a resolution of 3 nm(Figure 4B) and projected onto a plane perpendicu-lar to the 5-fold symmetry axis (Figure 4C). Asrevealed from Figure 4, C and D, at this resolutionlevel the resulting doughnut-like projection imageof the E. coli particle appeared indistinguishable inoverall size, shape, and symmetry from our ratparticle average obtained by EM. Correspondingparticle images of the recombinant mutant ratenzyme were indistinguishable in overall size,shape, and symmetry from those of wild-typeGTP-CH-I (data not shown).

Taken together, our morphological data indicatethat both recombinant wild-type and mutant rat

Figure 4. Structural comparison between E. coli (A-C)and recombinant rat (D) GTP-CH-I. A, Ribbon represen-tation of the atomic structure of the E. coli GTP-CH-Idetermined by Nar et al. (1995). B, Same structure as Abut represented as a surface-rendered electron densitymap band-limited to a resolution of 3 nm. C, Calculatedprojection image of B onto a plane perpendicular to the5-fold symmetry axis. D, Calculated average after multi-variate statistical analysis and 5-fold symmetrization ofSTEM ADF projection images of recombinant rat GTP-CH-I particles adsorbed to the specimen support ®lm ina preferred orientation (i.e. with their 5-fold symmetryaxis approximately normal to the support ®lm). Scalebar, 5 nm.

Figure 5. EF-hand-like calcium-binding loop motif inGTP-CH-I. Alignment of known GTP-CH-I sequences(Maier et al., 1995; and references therein) with the con-sensus sequence of an EF-hand loop motif (Kretzingeret al., 1991) and with a calcium-binding 12-residue pep-tide analogue derived from calmodulin (Cal-P12; WoÂ jciket al., 1997). Positions X, Y, Z, and ÿZ in the consensussequence (shown in bold) indicate calcium-coordinatingresidues, which must contain oxygen atom(s) in theirside-chains. The ÿX position is usually occupied by awater molecule, and the residue at ÿ Y complexes cal-cium via its peptide carbonyl group. h, Denotes hydro-phobic residues and stars denote any amino acid.Homology to the calcium-binding loop of the consensusEF-hand sequence is, with the exception of D. discoi-deum, strongly conserved in eukaryotes but absent inbacteria. The two residues that have been mutated inthe rat enzyme are shown as k and k.


GTP-CH-I assume a structure very similar to thatof the E. coli homolog, which was shown to form ahomodecameric complex consisting of two 5-foldsymmetric rings of enzyme subunits associatedface-to-face (Meining et al., 1995; Nar et al., 1995).

Calcium binding to an EF-hand-like loop motifof GTP-CH-I affects its enzymatic activity

Analysis of the GTP-CH-I amino acid sequencerevealed the presence of a distinct sequence motifexhibiting a strong homology to the calcium-binding loop of the consensus EF-hand structuralmotif (Kretzinger et al., 1991). As documented inFigure 5, this 12-residue long sequence motif isstrongly conserved in the various eukaryoticGTP-CH-I homologs, but absent in bacteria. Dot-blot assays were performed to qualitatively assessthe ability of the recombinant rat enzyme to bindcalcium and to compare it to the known calciumbinding of calmodulin. As shown in Figure 6 Aand B, binding of 45Ca2� to the recombinantwild-type enzyme was nearly as effective asbinding of calcium to porcine brain calmodulin.To test whether calcium binding of rat GTP-CH-Idoes in fact involve its EF-hand-like calcium-

binding loop, a mutant enzyme was engineeredby site-directed mutagenesis, in which two of thepossible calcium-coordinating residues, aspartate114 and 116, were changed to lysine residues. Asexpected, this mutant enzyme, which wasdesigned so as to contain the same deviationsfrom the calcium-coordinating amino acid resi-dues as the E. coli GTP-CH-I, yielded an approxi-mately sevenfold reduced calcium bindingcompared to the wild-type enzyme (Figure 6B).The residual calcium binding of the mutantenzyme produced a signal which was onlyslightly stronger than that of bovine serum albu-min used as a negative control (Figure 6A andB).

To assess more quantitatively the calcium-bind-ing behavior of the wild-type and mutant recombi-nant GTP-CH-I, the calcium-dependent change ofthe enzyme's intrinsic ¯uorescence was used as asignal for titration (Figure 6C). Titration of a 20 nMsolution of calcium-depleted wild-type GTP-CH-Iwith calcium yielded simple sigmoidal pro®les of

Figure 6. Calcium binding of recombinant rat GTP-CH-I. Comparison of calcium binding of wild-type andmutant GTP-CH-I with the known binding of calcium tocalmodulin. A, Overlay assay with 45Ca2� B, and corre-sponding densiometric quantitation normalized to cal-cium binding to calmodulin. Equimolar proteinconcentrations of 300 pmol have been used in each case.1, wild-type rat GTP-CH-I; 2, mutant rat GTP-CH-I; 3,porcine brain calmodulin; 4, bovine serum albumin.C, Intrinsic tyrosine (spheres) and tryptophan (squares)¯uorescence of wild-type (®lled symbols) and mutant(open symbols) recombinant rat enzyme as a function ofcalcium concentration. Excitation wavelengths were270 nm for tyrosine and 290 nm for tryptophan resi-dues. Fluorescence emission was monitored at 334 nmfor tyrosine and at 347 nm for tryptophan. The exper-imental points represent mean values of two indepen-dent experiments.

Table 1. Enzymatic activities of wild-type (wt) andmutant (mt) recombinant rat GTP-CH-I with and with-out EDTA or EGTA treatment

Addition GTP-CH-Iactivity

GTP-CH-Iactivity

(% of wt)

wt GTP-CH-I 51.5 � 2.2 100wt GTP-CH-I � 2.5 mM EDTA 45.6 � 0.9 88wt GTP-CH-I � 2.5 mM EGTA 45.7 � 1.6 88mt GTP-CH-I 28.1 � 1.4 54mt GTP-CH-I � 2.5 mM EDTA 28.0 � 1.5 54mt GTP-CH-I � 2.5 mM EGTA 27.8 � 1.0 54

Determination of the GTP-CH-I activity was performed asdescribed in Materials and Methods. Activity of GTP-CH-I isgiven as nmol of neopterin triphosphate per mg of recombina-tion protein per minute. The GTP-CH-I activity of the wild-typeenzyme was set to 100%. The data represent averages plus cor-responding standard deviation from three independent experi-ments.


the corresponding tryptophan (at 347 nm) andtyrosine (at 334 nm) ¯uorescence intensities with amidpoint of transition at a free calcium concen-tration of approximately 10 nM. Saturatingamounts of calcium caused an approximately 10%decrease of intensity for both tryptophan and tyro-sine ¯uorescence (Figure 6C). The best ®t to theexperimental data was obtained by a non-coopera-tive two-state model with a Hill coef®cient of one(Engel & Schalch, 1980) and an apparent equili-brium dissociation constant of 8.4 nM (data notshown). The number of high-af®nity calcium-bind-ing sites per polypeptide can be determined athigh protein concentrations. When the condition

is met that the product of the equilibriumassociation constant and the protein concentrationbecomes much larger than one, the ¯uorescenceintensity pro®le exhibits a sharp bend when the con-centration of bound calcium equals the enzyme con-centration (Engel & Schalch, 1980). Accordingly, at aprotein concentration of 10 mM the number of cal-cium ions bound per GTP-CH-I polypeptide was 1(data not shown). In agreement with the resultsobtained from the 45Ca2�-overlay assays (Figure 6Aand B), no calcium-dependent change of the tyrosineand tryptophan ¯uorescence was observed for themutant enzyme (Figure 6C). Both wild-type andmutant enzymes did not yield any signi®cantchange of their intrinsic ¯uorescence on magnesiumtitration (data not shown), indicating that GTP-CH-Ihad no signi®cant af®nity for magnesium.

To assess the effect of calcium binding on theirenzymatic activity, puri®ed recombinant wild-type and mutant GTP-CH-I were compared inthe absence and presence of calcium in the lowmM range (i.e. with and without EDTA or EGTAtreatment; see Table 1). Whereas the wild-typeprotein yielded a value of 51.5(�2.2) nmolmgÿ1 minÿ1 in the presence of calcium, the corre-sponding activity of the mutant enzyme wasreduced by 45% to a value of 28.1(�1.4) nmolmgÿ1 minÿ1. Moreover, a 12% decrease in wild-type GTP-CH-I activity was observed in the pre-sence of 2.5 mM EDTA (i.e. 45.6(�0.9) nmol mgÿ1

minÿ1) or 2.5 mM EGTA (45.7(�1.6) nmol mgÿ1

minÿ1). As expected, the activity of the mutantenzyme was insensitive to the presence orabsence of calcium: it was 28.1(�1.4) nmol mgÿ1

minÿ1 in the presence of calcium, 28.0(�1.5) nmolmgÿ1 minÿ1 in the presence of 2.5 mM EDTA,and 27.8(�1.0) nmol mgÿ1 minÿ1 in the presenceof 2.5 mM EGTA.

Taken together, our ®ndings indicate that ratGTP-CH-I binds calcium but not magnesium via anEF-hand-like loop motif, and that binding of cal-cium causes an increase of its enzyme activity.


Discussion

Here, we have employed AUC and STEM incombination with digital image analysis and pro-cessing to determine the rat GTP-CH-I to be ahomodecameric enzyme complex consisting of two5-fold symmetric pentameric rings of subunitsassociated face-to-face. Remarkably, the overallmorphology and dimensions of the recombinant ratenzyme revealed a strong similarity to the E. colihomolog (Meining et al., 1995; Nar et al., 1995),although the two species share only 34% identicalamino acid residues. Nar et al. (1995), who recentlysolved the atomic structure of the E. coli enzyme,reported a perfect D5 particle symmetry for the dec-amer and a 2-fold symmetry for the arrangement ofthe two pentameric rings of subunits. Morphologi-cally, the E. coli GTP-CH-I enzyme complex may bedescribed as a torus with an approximate height of65 AÊ and an average diameter of 100 AÊ . OurEM-based data, on the recombinant rat homo-log, suggest that the molecular architectureof mammalian GTP-CH-I, whose amino acidsequences reveal more than 90% identity, are prob-ably all very similar to that of the E. coli enzyme (seeFigure 4).

By examining the amino acid sequence com-bined with calcium-binding experiments and muta-tional analysis, we identi®ed a 12-residue longhigh-af®nity calcium-binding site residing withinthe rat enzyme. This site reveals strong sequencehomology to the calcium-binding loop as it is com-monly found within EF-hand sequence motifs withoxygen atom-containing calcium-coordinating resi-dues at the positions designated X, Y, Z, and ÿZ(Figure 5; Kretzinger et al., 1991). Although the sig-nature of the calcium-binding loop is clearly pre-sent in the eukaryotic, but absent from thebacterial GTP-CH-I homologs (Figure 5), severallines of evidence suggest that this region is not acanonical EF-hand domain. The similarity to a-helices E and F is weak and there is no second EF-hand present to form a pair (Kretzinger et al.,1991). Nevertheless, in the E. coli enzyme, the sitecorresponding to the 12-residue EF-hand-like cal-cium-binding motif forms part of a solvent-exposed loop (Nar et al., 1995). Our ®nding thatthe EF-hand-like loop motif within mammalianGTP-CH-I can bind calcium is further supportedby the observation that synthetic 12-residue cal-cium-binding loop peptides from calmodulin (seeFigure 5) retained their ability to coordinate thedivalent cation (WoÂ jcik et al., 1997).

Known GTP-CH-I sequences cover a wide evol-utionary range and include species that divergedmore than 3000 million years ago (Maier et al.,1995; and references therein). Comparison of theseenzyme sequences reveals that the region contain-ing the EF-hand-like loop motif is strongly con-served among eukaryotes (Figure 5). The onlyexception to this rule represents the Dictyosteliumdiscoideum homolog where, in addition to devi-ations from the DXDXDXXXXXXD pattern, a histi-

dine residue is found at position Z. Accordingly,D. discoideum is phylogenetically the most distantrelative within the eukaryotic kingdom for whichinformation of the corresponding GTP-CH-Isequence is available (Figure 5). This organism isconsidered to have diverged before the diversi®ca-tion of eukaryotes into animals, plants and fungiabout 1000 million years ago. Slight deviations ofthe calcium-binding sequence motif are alsoobserved in Saccharomyces cerevisiae and D. melano-gaster, but in contrast to D. discoideum GTP-CH-I,these differences are conservative in the sense thata calcium-coordinating aspartate has been replacedby an amino acid which also contains oxygenatom(s) within its side-chain. Evidently, the cal-cium-binding motif is not conserved in bacteria(Figure 5). Our results obtained with a recombi-nant mutant rat GTP-CH-I which was designed soas to contain the same deviations from the cal-cium-coordinating sequence motif as the E. colihomolog (KXKXDXXXXXXD), suggest that bothprokaryotic GTP-CH-I species and the D. discoi-deum homolog may not bind calcium at physiologi-cal concentrations.

The effects of divalent cations on the enzymaticactivity of various GTP-CH-I homologs reported inthe literature are contradictory (see Introduction).For example, Hatakeyama et al. (1989) observed a5% and 50% inhibition by calcium and magnesium,respectively, at a concentration of 0.1 mM for therat liver enzyme. In contrast, we found a 12%decrease for recombinant wild-type rat GTP-CH-Ienzyme activity in the absence of calcium. Consist-ent with this latter ®nding, compared with thewild-type rat enzyme, our mutated enzymerevealed a 45% decreased activity, and this activitywas no longer dependent on the presence orabsence of calcium. Moreover, as expected, themutant enzyme exhibited a drastic attenuation ofits calcium-binding af®nity. Due to the strong con-servation of the EF-hand-like loop motif in eukary-otic GTP-CH-I (Figure 5), it is intriguing tospeculate that physiological calcium concentrationsmay provide a possible regulatory mechanism forthe biosynthesis of BH4. Moreover, an importantrole of physiological calcium concentrations for theregulation of eukaryotic GTP-CH-I activity is docu-mented by preliminary results indicating an inter-action of the enzyme complex with calmodulin(U. Christen et al., unpublished data).

The possibility of modulating the enzymeactivity of GTP-CH-I by calcium becomes attractivein the context of a disease known as hereditaryprogressive dystonia with marked diurnal ¯uctu-ation (HPD), also termed dopa responsive dystonia(DRD). HPD/DRD is a progressive autosomalinherited basal ganglia disorder that is caused by ade®ciency of GTP-CH-I activity. Recently, Ichinoseet al. (1994) have identi®ed four independentmutations in the GTP-CH-I gene of patients withHPD/DRD. All cases analyzed were heterozygouswith respect to these mutations, and no additionalmutations were found in the coding region of their


GTP-CH-I gene. One of these mutations representsa single base-pair change in codon 134 leading to anon-conservative amino acid substitution of aspar-tate to valine. Notably, aspartate 134 is the cal-cium-coordinating residue at position ÿZ of theenzyme's EF-hand-like loop motif. Deviations fromthe EF-hand consensus motif commonly result inthe loss of calcium binding (Strynadka & James,1989). Indeed, it has been shown that mutation ofthe calcium-coordinating residue at ÿZ within anEF-hand motif causes a 30 to 100-fold reduction ofits calcium-binding af®nity (Pottgiesser et al., 1994).In contrast, substitutions of the amino acid at theX position only yield a minor decrease in thecalcium-binding af®nity (Pottgiesser et al., 1994).A failure of binding calcium was also observed forour mutant GTP-CH-I, in which the two calcium-coordinating aspartate residues at positions X andY had been changed to lysine, the constituentamino acid at the corresponding sites in the E. colihomolog (Figure 5). Taken together, these ®ndingssuggest that substitution of aspartate 134 to valinemay result in a loss of calcium binding of themutant enzyme which in turn may represent theprimary cause of the observed enzyme de®ciencyand hence lead to the disease HPD/DPD. Furtherdetailed studies will now be necessary to clarifythe exact physiological role of the enzyme's inter-action with calmodulin, and to more systematicallyevaluate the possible involvement of calcium bind-ing to GTP-CH-I in HPD/DRD.

Materials and Methods

Construction of expression plasmids

A rat liver cDNA was kindly provided by Dr K.Hatakeyama, Osaka Medical College, Osaka, Japan. Itwas used as a template for PCR ampli®cation of a DNAfragment coding for residues glycine 12 to serine 241 ofGTP-CH-I (Hatakeyama et al., 1991). Oligonucleotideprimers, p1 (50-CCCGGATCCGGGTTCCCCGAGCGG-GAG-30) and p2 (50-CCCGAATTCTTATTAGCTCCT-GATGAGTGTGAGG-30) were designed to obtain aBamHI site at the 50 end and two TAA translation stopsignals followed by an EcoRI site at the 30 end. Theampli®ed product was ligated into the bacterialexpression vector pET-32a�, a derivative of pET-32a(Novagen) encoding E. coli thioredoxin (trxA), a 6xHistag, and a thrombin cleavage site followed by the mul-tiple cloning site.

The designed mutant enzyme was constructed byPCR as described by Ho et al. (1989) using two sets ofprimers: p1 and p3 (50-TCTCGTCATGTTTCTCTTTAAA-TATAGCATCGTTCAGGACATC-30), and p2 and p4(50-GCTATATTTAAAGAGAAACATGACGAGATGGT-GATTGTG-30).

PCR and DNA manipulations for cloning were per-formed according to standard protocols (Sambrook et al.,1989). Insert sequences of recombinant plasmids wereveri®ed by Sanger dideoxy DNA sequencing.

Synthesis and purification of recombinant proteins

The E. coli JM109(DE3) host strain (Promega) wasused for expression. Production and puri®cation of6xHis-tagged fusion protein by immobilized metalaf®nity chromatography (IMAC) on Ni2�-Sepharose(Novagen) was performed as described in the manu-facturer's instructions.

Separation of recombinant wild-type and mutantGTP-CH-I from the thioredoxin carrier by thrombin clea-vage was carried out as described by Kammerer et al.(1995). Both recombinant proteins contained tenadditional residues GSGAMADIGS which originatedfrom the plasmid and are not part of the rat GTP-CH-Icoding sequence. Identities and concentrations of thepuri®ed proteins were determined by quantitative aminoacid composition analysis. If not stated otherwise, recom-binant proteins were analyzed at room temperature in5 mM sodium phosphate buffer (pH 7.4) supplementedwith 150 mM NaCl.

CD spectroscopy

CD spectra were recorded on a Jasco J 720 spectro-polarimeter. Far-ultraviolet spectra (200-250 nm) weremeasured in a 1 mm path-length quartz cell. Dataanalysis was performed with the Sigma Plot softwarepackage from Jandel Scienti®c.

AUC

Sedimentation equilibrium and sedimentation velocityexperiments of mutant and wild-type GTP-CH-I at con-centrations of 0.1 to 0.5 mg/ml were performed on aBeckman Optima XL-A analytical ultracentrifugeequipped with 12 mm Epon double-sector cells in anAn-60 Ti rotor. Sedimentation velocity runs were per-formed at rotor speeds of 56,000 rpm and 48,000 rpm forthe wild-type and mutant protein, respectively. Sedimen-tation coef®cients were corrected to standard conditions(water, 20�C; Eason, 1986). Sedimentation equilibriumscans were carried out at 6800 rpm (wild-type) and7200 rpm (mutant). Average molecular masses wereevaluated from sedimentation equilibrium runs, usinga ¯oating baseline computer program that adjusts thebaseline absorbance to obtain the best linear ®t ofln(absorbance) versus radial distance square. A partialspeci®c volume of 0.73 ml/g was used for all calcu-lations.

45Ca2�-overlay assays

For 45Ca2�-overlay assays, proteins were dot-blottedonto a nitrocellulose membrane. The membrane wasincubated with 45Ca2�, washed and autoradiographed asdescribed (Maruyama et al., 1984).

Fluorescence measurements

Fluorescence measurements were acquired on a PerkinElmer LS50 spectro¯uorimeter. The measurements werecarried out at room temperature in a 1 cm � 1 cm quartzcell at a protein concentration of 20 nM. Excitation wave-lengths were set at 270 nm and 290 nm for tyrosine andtryptophan, respectively, and ¯uorescence emission wasmonitored at 334 nm and 347 nm, respectively. Spectrawere recorded after successive additions of calciumchloride and corrected for dilutions. Free calcium con-


centrations were determined using a computer programdescribed by Haiech et al. (1979). Data analysis was per-formed with the Sigma Plot software (Jandel Scienti®c).Estimation of the equilibrium dissociation constant forcalcium assuming a simple non-cooperative two-statemodel and determination of the number of binding siteswere achieved as described (Engel & Schalch, 1980).

Determination of enzyme activity

Enzyme activities of puri®ed recombinant GTP-CH-Iwere quanti®ed according to Werner et al. (1990).

EM, and digital image analysis and processing

Sample preparation for EM was performed asdescribed by Bremer and Aebi (1994). The negativelystained specimens were examined using a Vacuum Gen-erator (East Grinstead) STEM HB5 operated at an accel-eration voltage of 100 kV and at a nominal magni®cationof 200,000�. Data recording was performed with anADF detector interfaced to a custom built, digital dataacquisition system (MuÈ ller et al., 1992).

STEM images were analyzed and processed using theIMPSYS (MuÈ ller et al., 1992) and SEMPER 6 (Saxton,1996) image processing software packages run on DECVAX workstations. Brie¯y, a total of 550 images of64 � 64 pixels each containing a single particle withinthem were extracted from larger, 1024 � 1024 pixelimages. Angular and translational alignment with respectto a variety of reference images was performed to bringall particles into register with one another. Subsequently,multivariate statistical analysis (van Heel, 1984) wasapplied for the classi®cation of the aligned particle imagesso as to de®ne homogeneous subsets for averaging.

Mass determination by STEM

Sample preparation for STEM mass measurementswas according to MuÈ ller et al. (1992). The freeze-dried,unstained specimens were examined at room tempera-ture in the STEM in the ADF mode operated at an accel-erating voltage of 80 kV. Digital images comprising512 � 512 pixels (i.e. with each pixel corresponding to0.89 nm at the specimen level) were recorded at a nom-inal magni®cation of 200,000� with an average dose of333(�17) electrons per square nanometer. Processing ofthe STEM data for mass determination was carried outas described previously using the IMPSYS softwarepackage (MuÈ ller et al., 1992).

Acknowledgments

We thank Dr M. Bider for kindly providing the pET-32a expression vector, Prof. J. Engel for generous sup-port, helpful discussions, and invaluable comments onthe manuscript. Dr K. Hatakeyama is acknowledged forthe gift of rat liver GTP-CH-I cDNA, and Ms T.Schulthess for ®gure preparation and critical reading ofthe manuscript. We are grateful to Dr J. B. Heymann, DrS. A. MuÈ ller and Mr L. Hasler for help with digitalimage processing. This work was supported by theDepartment of Education of the Kanton Basel-Stadt, theM. E. MuÈ ller Foundation of Switzerland, the AustrianResearch Funds zur FoÈrderung der wissenschaftlichenForschung (grant P11301 to E.R.W.), the Swiss National

Science Foundation (grant 31-43090.95 to J.P., and grant31-39691.93 U.A.), a grant from the Commission of theEuropean Union (Biomed 2, PL950979 to J.P.), and agrant from the Krebsliga beider Basel to C.P.. M.O.S. andC.P. contributed equally to this work.

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Edited by W. Baumeister

(Received 29 September 1997; received in revised form 2 January 1998; accepted 9 January 1998)

Rat GTP cyclohydrolase I is a homodecameric protein complex containing high-affinity calcium-binding...

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