The structural basis for the regulation of tissue transglutaminase bycalcium ions

Rita Casadio1, Eugenia Polverini1, Paolo Mariani2, Francesco Spinozzi2, Flavio Carsughi2, Angelo Fontana3,

Patrizia Polverino de Laureto3, Gabriella Matteucci4 and Carlo M. Bergamini4

1Interdipartimental Biotechnology Research Center (CIRB), University of Bologna, Italy; 2Institute of Physical Sciences, INFM, University of Ancona,

Italy; 3CRIBI Biotechnology Center, University of Padova, Italy; 4Department of Biochemistry and Molecular Biology, University of Ferrara, Italy

The role of calcium ions in the regulation of tissue transglutaminase is investigated by experimental approaches and

computer modeling. A three-dimensional model of the transglutaminase is computed by homology building on

crystallized human factor XIII and is used to interpret structural and functional results. The molecule is a prolate

ellipsoid (6.2 � 4.2 � 11 nm) and comprises four domains, assembled pairwise into N-terminal and C-terminal

regions. The active site is hidden in a cleft between these regions and is inaccessible to macromolecular substrates in

the calcium-free form. Protein dynamics simulation indicates that these regions move apart upon addition of calcium

ions, revealing the active site for catalysis.

The protein dimensions are consistent with results obtained with small-angle neutron and X-ray scattering. The

gyration radius of the protein (3 nm) increases in the presence of calcium ions (3.9 nm), but it is virtually unaffected

in the presence of GTP, suggesting that only calcium ions can promote major structural changes in the native protein.

Proteolysis of an exposed loop connecting the N-terminal and C-terminal regions is linearly correlated with enzyme

inactivation and prevents the calcium-induced conformational changes.

Keywords: calcium conformational changes; ligand effect; protein modeling; small-angle scattering;

transglutaminase.

Transglutaminases catalyze the post-translational modificationof proteins at glutamine residues, with formation of isopeptidebonds [1,2]. Multiple isoenzymic forms are found in extra-cellular fluids and in animal cells. Within the intracellularcompartment, the predominant isoenzyme is the soluble type-IItissue transglutaminase. This protein is involved in programmedcell death [3], as its activation by calcium leads to cross-linkageof membrane and cytoskeletal proteins, marking irreversiblealterations of cellular homeostasis [4]. In erythrocytes as well asother cells, the enzyme is latent during the normal cell lifespanand is subjected to fine regulation, involving GTP and calcium.GTP binding by transglutaminase inhibits calcium binding andtransglutaminase activity, whereas calcium binding inhibits GTPbinding [5]. Additional metabolites (e.g. sphingosylphosphocho-line, a breakdown product of membrane phospholipids) may beinvolved in this regulatory mechanism [6]. In situ the enzyme isactivated only after a decrease in GTP and increase in calcium inthe cytosol [7,8]. Recent evidence suggests that, in its inactivestate, the enzyme acts as a G-protein in signal-transductionpathways, disclosing an unexpected bifunctional role [9,10],which needs clarification.

A major effort of our laboratories has been directed towardscharacterization of this isoenzyme from red blood cells [11±13]

with respect to its protein-modifying activity. The effects ofmodulators are related to putative conformational changes [11],which need to be characterized in the three-dimensionalstructure of the protein. However, of the different transglutami-nases, only the structure of the human A2 homodimer of factorXIII has been elucidated at high resolution [14,15]. Multiplesequence alignments of the members of the transglutaminasefamily indicate homology at the core domains and majordifferences at the N-terminus and C-terminus, which areresponsible for the overall low level of sequence identity.Nevertheless, conservation of amino acid functional propertiesindicates conservation of tertiary structure within the family andthis is the basis of homology building of the type-IItransglutaminase on the factor XIII crystal structure [10,16].Using these models and a domain-deletion approach, it waspossible to demonstrate that constructs containing domains 1and 2 are the minimal essential structures required fortransamidating activity, while a 47-residue region at the startof the core domain of the type-II transglutaminase is endowedwith GTPase and ATPase activity [16], and that domain 4 isinvolved in stabilization of the active conformation of GTPase[10]. However, the role of calcium ions in promoting theconformational changes leading to cross-linking of the activeconformation remains to be elucidated.

To clarify this point we have simulated calcium±proteininteraction, by the use of protein dynamics, on a three-dimensional model of the enzyme. Results obtained by acombination of proteolytic, electrophoretic and small-angle-scattering (SAS) techniques support a model in which theprotein core domain, containing the catalytic site, becomesaccessible to the solvent as a consequence of calciumbinding.

Eur. J. Biochem. 262, 672±679 (1999) q FEBS 1999

Correspondence to C. M. Bergamini, Department of Biochemistry and

Molecular Biology, University of Ferrara, Via L. Borsari 46, 44100 Ferrara,

Italy. Fax: +39 532 202723, Tel.: +39 532 291425

Abbreviations: SAS, small-angle scattering; SANS, small-angle neutron

scattering; SAXS, small-angle X-ray scattering.

(Received 21 December 1998; revised 10 March 1999; accepted

16 March 1999)

q FEBS 1999 Structure and activation of transglutaminase (Eur. J. Biochem. 262) 673

MATERIALS AND METHODS

Preparation of erythrocyte transglutaminase

Preparation of the enzyme was performed using a purificationscheme that proceeds through chromatography on DEAE-cellulose and DEAE-Sepharose, fractionation with poly(ethy-lene glycol) and affinity chromatography on immobilizedheparin [12]. The purified transglutaminase was homogeneous,as determined by SDS/PAGE [17], although non-enzymaticfragmentation took place slowly during storage, as previouslydescribed [12]. The molecular mass was calculated as77 124 Da from the amino acid composition of tissuetransglutaminase. Analytical ultracentrifugation by the standardrate zonal sedimentation procedure was carried out to checkpossible changes in the aggregation state of transglutaminaseinduced by ligands.

Proteolysis

Transglutaminase was subjected to proteolysis by incubationwith individual proteinases (trypsin, chymotrypsin or elastase) at

80 : 1 weight ratio in 50 mm Tris/HCl/1 mm 2-mercaptoethanol,pH 7.5, for 60±90 min, with the eventual addition of 2 mmCaCl2 or 0.5 mm GTP. Proteolysis was quenched with 1 mmphenylmethanesulfonyl fluoride; the mixture was analyzed forresidual activity by a standard assay of amine incorporation intodimethylcasein [18]. The extent of proteolysis was detected bySDS/PAGE, and the remaining intact protein was determined bydensitometry of the stained gel. Peptides released duringproteolysis were subjected to N-terminal sequence deter-mination after blotting on to poly(vinylidene difluoride)membranes by automated Edman degradation in a model 70Applied Biosystems Sequenator. On other occasions, trans-glutaminase, after complete digestion with chymotrypsin in thepresence of GTP, was employed for SAS studies (see below).

Computer modeling

The model of endothelial transglutaminase was built following amethod based on sequence homology using the tools available inthe what if program [19]. The sequence of endothelialtransglutaminase [20] and that of subunit A of factor XIIIA(as found in the SwissProt database) were aligned using the

Fig. 1. Sequence alignment of tissue (i.e.

human endothelial) transglutaminase with

factor XIII. Inserted regions are shown by dashes

in either sequence. Identical residues are printed

on a dark background.

674 R. Casadio et al. (Eur. J. Biochem. 262) q FEBS 1999

clustalw Multiple Sequence Alignment tool [21]. The crystalstructure of factor XIIIA was obtained from the BrookhavenProtein Data Bank [14]. To construct the model, backbone atomswere generated by copying directly from the model in regionswith identical sequence length. In regions differing by a fewamino acids (producing gaps in the alignment), backbone atomswere generated via a loop search and the fill gap tool present inwhat if. Energy minimization of these loops was performedwith the GROMOS force field [22], using a steepest-descentmethod to attain convergence. Side-chain conformations weregenerated using the rotamer library included in the sameprogram. Bumps were resolved with small rotations around side-chain torsion angles and the structure was regularized for correctbond length and angles. Owing to the large number of atoms inthe whole protein (. 10 000), the refined structure wasminimized with the CHARMM force field [23] implementedin the TINKER code [24]. Molecular dynamic simulation ofwhole proteins in the absence or presence of three calciumions bound to their putative binding sites (see below) wasalso performed with the CHARMM force field [23]. Adistance-dependent dielectric constant value of 1 was used.After energy minimization, molecular dynamic simulation of thewhole protein was run without constraints (after heating for5 ps) for about 50 ps, at 300 K. Models were visualized usingmolscript [25].

SAS experiments

SAS experiments using neutron scattering (SANS) wereperformed on V4 or KWS II instruments, at the Hahn-MeitnerInstitut (HMI) in Berlin and at Forschungszentrum Julich (FZJ)in Julich, Germany, respectively. In these experiments different

ranges of the scattering vector Q were explored by employingdifferent combinations of sample-to-detector distance andwavelength of the focused neutron beam. The actual Q rangesobserved were 0.13±1.1 nm21 at HMI and 0.07±0.8 nm21 atFZJ. The protein sample was dissolved in buffer prepared withheavy water and analyzed at room temperature. Experimentalintensities were corrected for background, buffer contributionand detector inhomogeneities, and converted into absolutescattering cross-sections by calibration. Some SAS experimentswere repeated with a small-angle X-ray-scattering (SAXS)pin-hole camera, installed on a Rigaku±Denki rotatinganode, working at 8 kW. The radius of gyration (Rg) ofthe particles in solution was estimated by the Guinierapproximation [26]:

dS

dV�Q� � dS

dV�O� exp�2Q2R2

g=3� �1�Here dS/dV(O) is the cross-section at Q = 0 given by

dS

dV�O� � N�Sb 2 rsV�2 �2�

where N is the number of scattering particles per unit volume,Sb the sum of scattering length of all the nuclei of wholescattering particles, rs the scattering length density of thesolvent and V the scattering particle volume [26].

In the fitting procedure of experimental SAS data to Eqn (1)in the so-called Guinier region, we applied a large cut off, as it isknown that the criterion QRg , 2 is appropriate for estimatingscattering curves obtained for proteins [27]. The distance-distribution function P(r), the frequency of vectors connectingsmall volumes within the entire volume of the scatteringparticle, was calculated as described previously [27], using the

Fig. 2. Model of the calcium-free form of tissue type-II transglutaminase obtained by homology building on the structure of factor XIIIA [12] as

described in the text. Domains 1, 2, 3 and 4 are colored, respectively, in red, yellow, green and blue. The figure was generated with molscript [22].

q FEBS 1999 Structure and activation of transglutaminase (Eur. J. Biochem. 262) 675

indirect Fourier transform of Glatter and coworkers and theappropriate ITR program [28,29].

RESULTS

Computer modeling of transglutaminase structure

The model of the transglutaminase structure was built on thesequence of the human endothelial enzyme based on thealignment with the crystal structure of factor XIII (Fig. 1), asdetailed in the Materials and methods section. Results obtainedon the ligand-free inactive enzyme (the T state) are presented inFig. 2. They indicate that the protein is a wide prolate ellipsoidwith a flat base. The length of the three main axes of the proteinare 11, 6.2 and 4.2 nm, which would give rise to a theoreticalgyration radius of 2.84 nm for the protein in solution. In themodel, at least four domains resembling those of factor XIII areeasily identified [14,30]. The N-terminal domain contains thefirst 139 amino acids and is characterized by a large content

of b structures. The second domain extends from amino acid140 to 454 and contains virtually all the a-helical regions of theprotein. It harbors three putative calcium-binding regions [31],as well as the active site, comprising Cys277, His335 andAsp358 very close to each other, forming the catalytic triad [32].The active center itself is shielded from contact with solventbecause it is buried within a narrow cleft with walls formed bythis domain and by the following C-terminal domains 3 and 4(Fig. 3). The latter domains, spanning amino acids 479±585 and586±687, contain b structures arranged in a barrel-likeconformation. The connection between domain 2 and 3 isrepresented by a flexible solvent-exposed loop, the structure ofwhich is modified in the presence of calcium (see below).

The GTP-binding site can be located by considering our dataon affinity labeling [12] and proteolysis (indicating the locationof the GTP-binding site in the N-terminal fragment), the knownprotein sequence and the report from Greenberg's laboratorythat region 1±180 is required for the Mg-associated GTPaseactivity of transglutaminase [33]. Four lysine residues arepresent in positions 30, 74, 173 and 176 and are shown in thethree-dimensional model. These lysines are positioned in pairs,

Fig. 3. Location of the active site comprising the catalytic triad Cys277

(colored in purple), His335 and Asp358 (colored in violet). The four

lysines among the first 180 protein residues (Lys30, Lys74, Lys173 and

Lys176) are colored in red; one of them (Lys173 or Lys176) is involved in

GTP binding [13]. The putative calcium-binding regions (comprising

residues 146±162, 229±234 and 434±452 [28]) are highlighted in green in

the transglutaminase model.

Fig. 4. Overlapping of the calcium-free transglutaminase model (in

gray) with that obtained after molecular dynamics simulation in the

presence of three calcium ions (domains 1 and 2 are colored in green and

domains 3 and 4 are colored in red) as described in the text. The

unwinding of the a helix spanning residues 451±459 is marked by the arrow.

676 R. Casadio et al. (Eur. J. Biochem. 262) q FEBS 1999

exposed to the solvent and close to both extremities of thecatalytic domain (Fig. 3). Recent studies have shown that eitherLys173 or Lys176 is potentially involved in GTP binding. Threehighly conserved acidic regions (comprising residues 146±162,229±234 and 434±452), which are believed to representcalcium-binding sites [31], are also shown in the model.

Protein dynamic simulation in the presence of calcium ions

The effects of calcium ions on the conformation of trans-glutaminase are simulated by considering the interaction of thebare ions with the three main putative binding sites, which arepresumably occupied by calcium under saturating concen-trations. In the model, these regions display considerablestructural organization consisting of a turn±helix±turn motif,then a short turn, and then a loop±strand±turn motif, followedby a helix close to the loop connecting domains 2 and 3 (Fig. 3).After energy minimization and dynamics, the calcium ions arewithin 0.35 nm of oxygen atoms in the side-chains of Asp151(site 1), Asp232 and Asp234 (site 2) and Ser449, Glu451,Glu452 and the carbonyl oxygen of Pro446 (site 3). Moleculardynamic simulation for up to 50 ps does not lead to significantdistortion of protein secondary-structure motifs, as shown bysuperimposition of the structure of the calcium-free trans-glutaminase on that of the calcium-bound enzyme (Fig. 4).However, it is evident that the a helix comprising Glu451 toAla459, near the surface-exposed loop, unwinds and loses itssecondary structure. As a consequence, domain 3 and 4 move

apart from each other: a large gap is initiated, which points tothe active-site region, without significant interference withhydrogen-bonding among the catalytic triad residues.

Limited proteolysis

When the transglutaminase is subjected to limited proteolysis bya number of proteinases of distinct specificity, the enzyme chainis always cleaved, in the absence of ligands, to generate twopeptides of apparent molecular mass 56 and 31 kDa, asdetermined by SDS/PAGE (Fig. 5). With each proteinase tested,the addition of calcium ions promotes further degradation ofthe large peptide into smaller fragments while in thepresence of GTP the yield of peptides is increased,indicating a more selective cleavage with preservation ofthe larger fragment containing the GTP-binding site. Duringproteolysis, the enzyme is also inactivated, with a linearcorrelation between the amount of cleaved protein and theextent of inactivation (Fig. 6).

N-Terminal sequencing of the peptides demonstrated thatthe 56-kDa fragment has a blocked N-terminus (thuscorresponding to the N-terminal portion of the intact protein)while the N-terminal sequences of the 31-kDa fragments, foreach proteinase, are Thr-Arg-Ala-Asn-His-Leu-Asn-Lys-Leu(chymotrypsin), Leu-Ala-Glu-X-Glu-Glu (trypsin) and Ala-Glu-X-Glu and Asn-His-Leu-Asn (elastase). In the primarystructure of human endothelial transglutaminase these sequencesare present between residues 453 and 475, the region of theexposed loop between domains 2 and 3, defined above [31].When proteolysis was carried out on the enzyme that hadbeen subjected to affinity labeling at the nucleotide site withradioactive GTP derivative [12], radioactivity migratedexclusively with the large N-terminal fragment (data notshown). Attempts to separate the fragments by size-exclusionchromatography were unsuccessful, appreciable separationbeing achieved only by ion-exchange chromatography inthe presence of high concentrations of neutral salts. CDspectra of the isolated fragments are indicative of appreciablehelical structure in the larger one and the exclusive presenceof b structures in the smaller one (data not shown), as expectedfrom the modeling predictions.

SAS experiments on native transglutaminase

Samples of native transglutaminase dissolved in 2H2O buffer, inthe presence and absence of GTP and calcium, were analyzed bySANS. The scattering cross-sections, obtained at HMI, are

Fig. 5. Limited proteolysis of tissue transglutaminase by proteinases of

different specificity. The enzyme was incubated for 60 min at 25 8C with

the indicated proteinases at 100 : 1 weight ratio before SDS/PAGE. Lane 1,

native transglutaminase; lanes 2, 5, 8, proteolysis without ligands; lanes 3, 6,

9, proteolysis in the presence of 1.8 mm calcium; lanes 4, 7, 10, proteolysis

in the presence of 0.4 mm GTP. Lane 11, molecular mass standards.

Fig. 6. Correlation between percentage of

proteolysis and inactivation of the enzyme

during proteolysis by trypsin, elastase and

chymotrypsin in the presence and absence of

ligands. Accumulated data for increasing periods

of treatment. (W) In the absence of ligands; (X) in

the presence of 2.5 mm calcium; (K) in the

presence of 0.5 mm GTP.

q FEBS 1999 Structure and activation of transglutaminase (Eur. J. Biochem. 262) 677

reported in Fig. 7, in the form of the Guinier plot, vs. Q2. Thedata fitted with a similar slope in the absence of ligands and inthe presence of GTP, while significant differences wereobserved for the enzyme incubated in the presence of 2±5 mmcalcium. The low Q region appeared to be linear; the gyrationradii and the values obtained by fitting the experimental curvesusing Eqn (1) are reported in Table 1. Aspects that should benoted are (a) the similarity, within experimental variation, of thegyration radius in the absence of ligands to that calculated fromthe theoretical model; (b) the increase in the gyration radius ofthe scattering particle from 3.15 to 3.8 nm in the presence ofcalcium; (c) the small, but significant, decrease in gyrationradius induced by saturation with GTP. The results from theexperiments performed at the FZJ, which are also reported inTable 1 together with values measured by SAXS, move in the

same direction, but also demonstrate that the contemporaneouspresence of GTP and calcium yields values for the gyrationradius that correspond to those calculated in the presence ofcalcium alone. A similar conclusion on the prevalent role ofcalcium in dictating the final conformational state of the proteinwas reached in previous kinetic studies [11]. Data were alsoanalyzed by the distance-distribution function, the P(r) plot,which demonstrates a bimodal distribution in the absence ofligands as well as in the presence of GTP. A first peak, centeredaround 3.0 nm, thus yielded a value close to the gyration radiuscalculated from the Guinier plot, while the curve extrapolated toa maximal value close to 15 nm corresponding to the maximalaxis of the protein, calculated from the model (Fig. 8). Additionof calcium converted the curve into a three-modal one,extrapolating to a maximal value of 14±15 nm, suggestingagain a broadening of the protein on calcium binding.

SAS on proteolyzed and denatured transglutaminase

Guinier plots relative to proteolyzed transglutaminase revealedbiphasic behavior, as indicated by a change in slope in the small-angle region of the Guinier plots; this may be related tomoderate aggregation of the proteolyzed protein in 2H2O [34].After measurement, small amounts of precipitated material wereeffectively recovered from the cell. However, as effects of largeaggregates are negligible in Guinier analysis at relatively largeangles, which more closely correspond to features of themonomers [35], significant data were collected that should helpto shed light on the effects of ligands. As reported in Table 1, thelarge increase in gyration radius induced by calcium in thenative enzyme was no longer detected, so that cleavage ofthe peptide chain at the exposed loop prevents theoccurrence of the calcium-dependent widening in proteolyzedtransglutaminase.

Additional experiments were carried out on intact trans-glutaminase in the presence of a range of concentrations ofguanidine, to identify the possible occurrence of distinctscattering centers related to the domains themselves, but wedid not detect resolvable signals. In contrast, a distinct gyrationradius of 1.9 nm was measured as the only resolvable entity, as

Fig. 7. Guinier plots of neutron-scattering data obtained at HMI for the

native transglutaminase dissolved in 2H2O buffer in the absence of

ligands (W), in the presence of 1.8 mm calcium (X) and in the presence of

0.4 mm GTP (P). The curves are arbitrarily displaced on the cross-section

axis for clarity. The solid lines correspond to the best fit through the

experimental points for which the Guinier law is satisfied: the arrows show

the higher cut-off values of the Q2 range used for the Guinier analysis

(QRg = 1.5).

Fig. 8. Distance-distribution functions, r2P(r), for the native transglu-

taminase dissolved in 2H2O buffer in the absence of ligands (W), in the

presence of 0.4 mm GTP (P) and in the presence of 1.8 mm calcium (X).

The intensity of the r2P(r) functions were normalized to the same total area.

Table 1. SAS experimental results. Gyration radii (Rg) were determined

by Guiner approximation from SAXS and SANS curves. The forward cross-

sections dSdV�0� are reported only for SANS experiments and were calculated

by Guinier approximation. nTGase, pTGase and gnd indicate, respectively,

native transglutaminase, the proteolyzed enzyme and guanidinium chloride

at different concentrations. (a) Neutron-scattering experiments performed at

HMI; (b) neutron-scattering experiments performed at FZJ; (c) X-ray-

scattering experiments. The nominal protein concentrations (number of

proteins per unit of volume, cm23) were 3.9 � 1016 in HMI and 3.0 � 1016

in FZJ experiments.

Rg �nm� dSdV�0� (cm21) N �cm23�

nTGase (a) 3.14 �̂ 0.05 0.279 �^ 0.004 2�.8 � 1016

nTGase (b) 3.19 �̂ 0.14 0.108 �^ 0.008 1�.4 � 1016

nTGase (c) 3.14 �̂ 0.42 ± ±

nTGase/Ca2+ (a) 3.83 �^ 1.13 0.375 �^ 0.013 3�.7 � 1016

nTGase/GTP (a) 2.96 �̂ 0.05 0.259 �^ 0.004 2�.6 � 1016

nTGase/GTP/Ca2+ (b) 3.80 �^ 0.08 0.192 �^ 0.006 1�.9 � 1016

pTGase (b) 2.98 �̂ 0.26 0.029 �^ 0.019 2�.5 � 1016

pTGase/Ca2+ (b) 3.07 �^ 0.48 0.021 �^ 0.101 2�.1 � 1016

pTGase/gnd 0�.5 m (b) 2.98 �^ 0.31 0.013 �^ 0.002 1�.4 � 1016

pTGase/gnd 2�.6 m (b) ± ± ±

pTGase/gnd 1�.0 m (c) 1.90 �^ 0.56 ± ±

678 R. Casadio et al. (Eur. J. Biochem. 262) q FEBS 1999

the guanidine experiments were carried out on proteolyzedtransglutaminase. This value is consistent with that expected foreither the isolated N-terminal or C-terminal region. It is likelythat this signal arises from the C-terminal fragment, which ismore resistant than the N-terminal moiety to guanidinedenaturation, as judged by CD signals (C. M. Bergaminiet al., unpublished results).

DISCUSSION

The increasing interest in transglutaminases because of theirmedical relevance [36] led us to undertake a structural study ofthe protein to clarify the basis of its regulation. There isaccumulating evidence that the enzyme can act as a bifunctionalprotein in pathways regulating cell cycling [37] and proteincross-linking, at the time of cell death [3]. In this perspective itmust be recalled that the cross-linking activity is latent incycling cells, through the combined action of two effectors,calcium and GTP, which, respectively, activate and inhibit thetransamidating activity [11]. This is achieved through confor-mational changes, which have been amply demonstrated by anumber of techniques, although their molecular details need tobe elucidated. Quite recently, data have been collected thatcontribute to our understanding of the GTP-stabilized confor-mation [10]. The aim of the present report was to clarify themolecular basis of the activation of the transamidating activityby calcium ions.

The study was carried out by computing the plausible modelof the protein to simulate relevant structural changes and tocheck them experimentally by limited proteolysis and SASscattering, which is an emerging and particularly usefultechnique for determining protein size and shape in solution[38]. The model was generated on the primary structure of atypical transglutaminase (human endothelial transglutaminase)by sequence alignment and homology with the three-dimen-sional structure of factor XIII. It is generally known that proteinsin the same family display similar three-dimensional structureseven if they share low sequence homology. According to thissimulation, transglutaminase has a cake-like shape consisting oftwo masses, with the active and regulatory sites present in thelarger N-terminal region. On visual inspection our model issimilar to those presented in [10] and [16].

In the ligand-free protein, the active site is buried in a cleftbetween the N-terminal and C-terminal regions, hidden fromcontact with the solvent or macromolecular substrates. Thisfeature was also noted in crystals of factor XIII [14,15]. Ourscattering data indicate that GTP makes the contact between theN-terminal and C-terminal region closer than in the ligand-freeprotein, as suggested from the small decrease in the radius ofgyration detected in the absence of ligands. The most strikingresult is, however, observed in the presence of calcium, whichpromotes a massive broadening of the protein structure, asindicated by the 0.8-nm increase in gyration radius.

A crucial structure in the protein is a flexible loop on theenzyme surface between domains 2 and 3, at the border betweenthe N-terminal and C-terminal regions. The loop, which is by farthe preferred site of cleavage by proteinases, probably acts like ahinge to allow movement of these regions. This type ofregulatory mechanism is frequent in multidomain proteins [39]and involves widening of the clefts to make active sites availableto substrates. Dynamic simulations of calcium binding to theprotein confirm that this loop is heavily distorted and unwound,with an alteration in the shape of the protein and the relativepositions of domains 3 and 4. Domain 3 is claimed to beprincipally involved in the inhibition of the transamidating

activity in the absence of calcium [10]. It is noteworthy that thestructural changes induced by calcium are observed aftermolecular dynamic simulation for only a very short time (i.e.50 ps). This and the low stoichiometry used in the simulation (3calcium ions per molecule, instead of 6 as previously reported inequilibrium dialysis experiments [11]) could explain thedifference in the extent of the conformational changes detectedby molecular dynamic simulation as compared with thoseobserved in SAS experiments. On comparing our data with thoseavailable for the crystalline factor XIII, we noted that, at the endof the dynamics simulation, we did not detect any flipping of thecis peptide bonds such as was found previously by Weiss andassociates for factor XIII [15]. This point needs to be addressedin future studies.

The SAS data obtained using erythrocyte transglutaminase asa representative and well-characterized member of the tissue-type transglutaminase family are consistent with the observa-tions obtained by molecular dynamics simulation. In particular,we noted that (a) the loop is the only region cleaved in the nativeprotein and that GTP and calcium influence the yield of theproteolytic fragments (these fragments carry the expectedbinding sites and display the secondary structure predicted bythe model); (b) the values of the gyration radii determinedexperimentally correspond closely to those predicted by themodel; (c) the addition of calcium widens the whole protein, andthe major axis increases from 11 nm (measured in the absenceof the cation) to 15 nm; (d) the change in conformation isobserved in the intact but not the cleaved protein. This supportsthe notion that physical interruption of the peptide chaininterferes with the hinge movement mediated by the crucial loopconnecting the N-terminal and C-terminal regions, making theenzyme fully inactive. In contrast, C-terminal-deletion experi-ments demonstrated that the N-terminal region alone is active inthe absence of the C-terminal moiety [16]. This indicatesonce more that the physical separation of the N-terminal andC-terminal regions across the connecting loop is crucial forthe regulation of the transglutaminase transamidating activity.Yee and associates [40] tried to detect the calcium-dependentconformational changes in factor XIII during activation bystudying X-ray-diffraction patterns of single crystals in theabsence and presence of cations, but they failed to detect them,possibly because of constraints in the crystal structure.

ACKNOWLEDGEMENTS

Analytical ultracentrifugation by the standard rate zonal sedimentation

procedure was kindly carried out by Professors E. Chiancone and P. Vecchini

of the Department of Biochemistry of the University `La Sapienza' in Rome.

C. M. B. was supported by grants from the Italian MURST and C.N.R.,

and R. C. was supported partially by a grant for a target project in

Biotechnology from C.N.R. and by a grant to the project `Biocatalisi e

Bioconversioni' from the Italian MURST.

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