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Originally published In Press as doi:10.1074/jbc.M403481200 on April 14, 2004

J. Biol. Chem., Vol. 279, Issue 25, 26716-26725, June 18, 2004
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Crystal Structure of Transglutaminase 3 in Complex with GMP

STRUCTURAL BASIS FOR NUCLEOTIDE SPECIFICITY*

Bijan Ahvazi{ddagger}§, Karen M. Boeshans{ddagger}, and Peter M. Steinert¶{dagger}

From the {ddagger}X-ray Crystallography Facility, Office of Science and Technology and Laboratory of Skin Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892-8023

Received for publication, March 29, 2004 , and in revised form, April 13, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Epidermal-type Transglutaminase 3 (TGase 3) is a Ca2+-dependent enzyme involved in the cross-linking of structural proteins required in the assembly of the cell envelope. We have recently shown that calcium-activated TGase 3, like TGase 2, can bind, hydrolyze, and is inhibited by GTP despite lacking structural homology with other GTP-binding proteins. Here we report the crystal structure determined at 2.0 Å resolution of TGase 3 in complex with GMP to elucidate the structural features required for nucleotide recognition. Binding affinities for various nucleotides were found by fluorescence displacement to be as follows: guanosine 5'-3-O-(thio)triphosphate (GTP{gamma}S) (0.4 µM), GTP (0.6 µM), GDP (1.0 µM), GMP (0.4 µM), and ATP (28.0 µM). Furthermore, we found that GMP binds as a reversible, noncompetitive inhibitor of TGase 3 transamidation activity, similar to GTP{gamma}S and GDP. A genetic algorithm similarity program (GASP) approach (virtual ligand screening) identified three compounds from the Lead QuestTM data base (Tripos Inc.) based on superimposition of GTP{gamma}S, GDP, and GMP guanine nucleotides from our crystal structures to generate the minimum align flexible fragment. These three were nucleotide analogs without a phosphate group containing the minimal binding motif for TGase 3 that includes a nucleoside recognition groove. Binding affinities were measured as follows: TP349915 (Kd = 4.1 µM), TP395289 (Kd = 38.5 µM), TP394305 (Kd = 1.0 mM). Remarkably, these compounds do not inhibit but instead activate TGase 3 transamidation by about 10-fold. These results suggest that the nucleotide binding pocket in TGase 3 may be exploited to either enhance or inhibit the enzymatic activity as required for different therapeutic approaches.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transglutaminases (TGases)1 comprise a family of Ca2+-dependent enzymes that catalyze the transfer of the {gamma}-carboxamide group from protein-bound glutamine residue to the {epsilon}-amino group of protein-bound lysine residues or other primary amines. This reaction results in the formation of N{epsilon}-({gamma}-glutamyl)lysine isopeptide cross-links between (and within) polypeptide substrates, giving rise to stable protein polymers (1-4). TGase activity is important in both intra- and extracellular reactions involved in apoptosis, cell morphology, cell adhesion, tissue repair, bone ossification, tumor growth, and metastasis (3, 6-11). Six of the nine known human TGase enzymes are expressed in terminally differentiating epithelia such as the epidermis and involved in the assembly of the cell envelope. At least two TGases are required for effective barrier formation (4, 12), including the TGase 1, which is usually bound to plasma membrane (13, 14) and the cytosolic TGase 3 (15, 16).

TGase 3 is expressed in the epidermis in the last stage of terminal differentiation (17, 18), in the inner root sheath, and in the medullary layers in hair follicles (19). Although far less TGase 3 is expressed than TGase 1, TGase 3 accounts for the majority of the total activity in the epidermis (15, 21, 22). Both enzymes are expressed in differentiated epidermal cells but seem to have different roles. Cross-linking of major structural proteins such as trichohyalin and keratin intermediate filaments is preformed preferentially by TGase3 enzyme in hair follicles (22), whereas TGase 3 cannot compensate for cell envelope formation in patients with lamellar ichthyosis involving mutations in the TGase1 gene (21).

We recently presented biochemical and crystallographic evidence for the binding of GTP{gamma}S and GDP to active TGase 3 and showed that TGase 3 catalyzes GTP hydrolysis to GDP (23). These studies revealed the reciprocal effect of Ca2+ ions and GTP with respect to TGase 3 activity. GTP binding inhibits TGase transamidation activity, whereas calcium binding inhibits GTP binding and activates the enzyme (23, 24). In this report we further explore whether GMP can also bind and have inhibitory effects similar to those demonstrated for GTP{gamma}S and GDP (23).

To gain more insight into the structural basis for nucleotide binding and specificity, we determined the crystal structure of the active TGase 3 enzyme in complex with GMP at 2.0-Å resolution and measured the binding affinity for different guanine nucleotides by fluorescence titration. Three nucleotide analogs were identified from a virtual ligand screening experiment using the GMP crystal structure. In contrast to guanine nucleotide inhibitors, these compounds are shown to activate TGase 3 transamidation. These results reveal that the nucleotide binding pocket in TGase 3 may be exploited to either enhance or inhibit the enzymatic activity as required for different therapeutic approaches.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Protein Expression and Purification—Recombinant human TGase 3 as a zymogen was expressed in the baculovirus system and purified as described (26). The zymogen (in a 10-ml solution at a concentration of 15 mg/ml in 20 mM Tris-HCl (pH 8.0) buffer containing 1 mM EDTA and 125 mM NaCl) was proteolyzed with 3 mg of dispase (Roche Applied Science) at 37 °C for 30 min. The protein was purified by anion exchange chromatography using a Mono QTM column eluting in a 500 mM NaCl gradient at 150 mM NaCl. The protein was extensively dialyzed against 25 mM Tris-HCl (pH 8.0) containing 1 mM EDTA and 125 mM NaCl.

Crystallization and Data Collection—Crystallization trials of TGase 3·GMP complex were performed using the hanging-drop vapor diffusion method. Stock solutions of active enzyme (10 mg/ml, previously dialyzed against 25 mM Tris-HCl (pH 8.0) containing 1 mM EDTA and 125 mM NaCl) were supplemented with 0.1 mM {beta}-octyl glucoside, 0.575 mM CaCl2, 1 mM MgCl2, and 1 mM GMP. Equal volumes (2 µl each) of protein and reservoir/precipitant solution were mixed and equilibrated at 21 °C over well solutions containing 100 mM sodium-Hepes (pH 7.0) and a concentration gradient of 4-10% (w/v) in polyethylene glycol 6000.

Thin, plate-like crystals appeared after 2 days and were harvested into reservoir solution containing 20% 2-methyl-2, 4-pentanediol cryoprotectant and flash-frozen into liquid nitrogen with a rayon mounting loop (Oxford Cryosystems, Oxford, UK) before data collection. All data were collected at the National Synchrotron Light Source, Brookhaven National Laboratories using beamline X9B with an ADSC Quantum 4 detector with a wavelength of 0.92 Å, crystal distance-to-detector of 180 mm, and oscillation range of 1.0° and exposure for 30 s. The diffraction data from sets of crystal were indexed, processed, scaled, and merged using the HKL2000 suite of program (27). Data collection statistics of crystals are summarized in Table I.


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  		TABLE I
Crystallographic parameters and data processing Statistics

 
Structure Solution and Refinement—The crystal structure of TGase 3·GMP was solved by molecular replacement using AmoRe (28) using Protein Data Bank entry 1RLE [PDB] (23) as the search model (excluding the GTP{gamma}S model). Resolution ranges of 10 to 3.5 Å and 10 to 3.0 Å were used for the rotation and translation searches, respectively. Two solutions to both the rotation and translation searches permitted the positioning of two molecules within the asymmetric unit.

The initial R-factor from the molecular replacement solutions was 30.5% for all the data between 10 and 3.0 Å of resolution. Rigid-body refinement using CNS (29) followed by simulated annealing with the strict application of 2-fold non-crystallographic symmetry (30) improved the solutions, yielding a final R factor of 28.5%. Electron density for GMP was clear in a 2 Fo - Fc electron density map, permitting an unambiguous fitting of a GMP model. Initial phases were further improved using solvent-flattening (31) with 2-fold non-crystallographic symmetry molecular averaging (32). For final refinement, the non-crystallographic symmetry restraints were released. Six cycles of refinement and manual model rebuilding into SIGMAA-weighted 3 Fo - 2 Fc, Fo - Fc, and composite simulated-annealing omit-map electron density maps (33) resulted in an R-factor of 20.6% at 2.0 Å (using all data with no {sigma} cutoff and including low resolution data from 20-Å resolution with bulk solvent correction). Each step of the rebuilding procedure was monitored using the free R-factor (24.1%) and a residue real space correlation coefficient as a guide (34). Water molecules were included in the model based on hydrogen bonding and the presence of reproducible peaks in Fo - Fc the electron density map. The stereochemistry of the structure was analyzed with the programs PRO-CHECK (35) and CNS (29). Figures were generated with Molscript (36), Raster3D (37), and Sybyl (25) programs.

Microcalorimetric Titration Studies of Nucleotide Binding of TGase 3—The thermodynamic properties of nucleotide binding to the activated TGase 3 were measured by isothermal titration calorimetry using a MicroCal VP-ITC calorimeter at 30 °C. Before measurements, the activated TGase 3 (6.8 µM), dialyzed three times against 20 mM Tris-HCl (pH 8.0) and 1 mM standard CaCl2 solution (Fisher), was placed in a 1.38-ml sample cell. The activated TGase 3 was titrated with GMP nucleotide (0.19 mM) in a series of automatic injections starting with 5 µl each into the protein solution and following with 10- and 20-µl aliquots using a 250-µl syringe. After each injection, a 5-min pause was allowed to achieve a stable base line. Heat produced due to dilution was measured by injecting the nucleotide solution into sample cells from which TGase 3 protein was omitted. For each titration step, the heat of dilution was subtracted. Data were fitted to appropriate binding models, and thermodynamic parameters were determined from nonlinear least-squares fits using ORIGINTM software.

TGase 3 Activity Assay—TGase activity was measured by incorporation of [14C]putrescine into casein (38). Assays were done at 37 °C in 0.5-ml portions of 0.1 M Tris-HCl (pH 7.5) containing 1% N,N-dimethylated casein (Sigma), 0.5 µCi of putrescine (118 mCi/mmol), 1 mM dithiothreitol, 5 mM CaCl2, and 1 mM EDTA. Reaction blanks contained no added calcium. Reactions were stopped with the addition of ice-cold 7% (w/v) trichloroacetic acid, and incorporated radioactivity was collected by filtration through GF/A glass fiber filters (Whatman) wet with ice-cold 5% (w/v) trichloroacetic acid and successively washed 3 times with 5 ml of ice-cold 5% (w/v) trichloroacetic acid. Filters were dried and solubilized in 5 ml of CytoScint (ICN). Inhibition assays contained 0-500 µM GMP at 3 different CaCl2 concentrations.

TGase 3 Quantitative Metal Analyses—All plastic and glassware were incubated in 5 N HCl for period of 36 h and rinsed with double-distilled water that had been passed over a Chelex 100 column. For buffer and transition metal solutions, Chelex 100-treated water was used throughout. Before adjustment of the pH, Chelex 100 resin was added (0.5 g/100 ml), and the solution was stirred for 45 min; after adjustment of the pH, the solution was passed through a 0.2-µm filter and stored in metal-free plastic vials at -20 °C. Before measurements, TGase 3 zymogen (10 mg/ml) was treated with dispase (Roche Applied Science) at 37 °C for 30 min and dialyzed 5 times against 20 mM Tris-HCl (pH 8.0) and 5 mM EGTA. The EGTA solution was removed by dialysis against 20 mM Tris-HCl (pH 8.0). Bound metal ions were detected using a PerkinElmer Life Sciences ELAN 6100 inductively coupled plasma-mass spectrometry dynamic reaction cell apparatus.

TGase 3 Fluorescence Titration and Displacement—Titration experiments were performed at 25 °C using a PerkinElmer LS50 fluorescence spectrometer. The fluorescence of GTP{gamma}S trisodium salt BODIPY FL thioester (BODIPY FL GTP{gamma}S, Molecular Probes, Eugene, OR) and TNP-GTP were measured at an excitation wavelength of 475 nm (spectral bandpass, 2.5 nm) and emission wavelength of 513 nm (bandpass 2.5 nm), corresponding to the maximum in the fluorescence difference spectrum between free and TGase 3-bound BODIPY FL GTP{gamma}S. Either BODIPY FL GTP{gamma}S was added in small aliquots (2-10 µl) to a given solution of TGase 3 (40-200 nM) or TGase 3 from a concentrated stock solution was added in aliquots to a given concentration of BODIPY FL GTP{gamma}S (32 nM). The total change of the sample volume was less than 10% in every case. The buffer used for experiments with activated TGase was 20 mM Tris-HCl (pH 8.0), 1 mM CaCl2 and for the inactivated form was 20 mM Tris-HCl (pH 8.0), 1 mM MgCl2. The following equation was used for non-linear least-square fitting of the data: F = F0 + {Delta}F ((nE + L + KD - ((nE + L + KD)2-4LE)1/2)/2nE), where F is the measured signal, F0 is the signal without binding, {Delta}F is the maximal change of the signal at saturation, E and L are the total concentrations of titrated substance and ligand, respectively, and n and KD are the number of binding sites and the dissociation constant. For the fluorescence displacement experiments different nucleotides from stock solutions were added in small aliquots to a mixture of BODIPY FL GTP{gamma}S and TGase 3 (0.48 and 0.43 µM, respectively). The data fitting for non-linear least square was obtained from a previously derived formula (39).

Virtual Ligand Screening and Modeling of the Nucleotide Binding Pocket of TGase3—The GASP program was used to superimpose GTP{gamma}S, GDP, and GMP guanine nucleotides from our crystal structures to generate the "minimum align flexible fragment." This fragment signature query was used to scan a Tripos fragment data base (Lead QuestTM, 84,509 molecules as of 10 Dec, 2003). The SYBYL UNITY tools were used as a source of fragment molecules for the creation of a signature data base with two-dimensional fragment similarity. The starting data base was screened for all compounds with molecular mass less than 600 Da, yielding 117 molecules. The two-dimensional output molecules were used to generate three-dimensional coordinate molecules from the CONCORD module tool in SYBYL program.

The FlexS module implemented in SYBYL was then used to predict conformations and orientations of the nucleotide analogs relative to the pharmacophore pattern template (map of properties common to all active conformations of a set of ligands). The best 10 compound scores from FlexS, which are heavily based on shared volume and influenced by similar groups in space, were subsequently used for flexible docking method in FlexX. The orientations of three compounds with the best conformational scores (TP349915, TP395289, TP394305) were visually inspected.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Overall Architecture of TGase 3·GMP Complex—The crystal structure of TGase 3·GMP complex at 2.0 Å of resolution was solved. Except for the substitution of GMP nucleotide, the crystallization conditions used here were identical to those previously used to crystallize this enzyme in the presence of GTP{gamma}S including 0.575 mM CaCl2 and 1 mM MgCl2 (23). The final 2 Fo - Fc exp(iac) electron density map in the region of the catalytic triad is shown in Fig. 1A. Fig. 1B shows the overall architecture and binding site for GMP. The TGase 3·GMP complex crystal contains two independent protomers (692 residues each, subunit A and B) within the asymmetric unit. The overall conformation of the two subunits are essentially identical, similar to that reported earlier for the activated TGase 3 either in the absence or the presence of guanine nucleotide (23, 40, 41). The root mean square deviation between the 673 C{alpha} carbon atoms of the A and B subunits is 0.43 Å in the complex, indicating that overall conformations are similar. The two subunits in each of the structures lack electron density for a flexible loop between residues 462-478 in subunit A and 460-472 in subunit B. This flexible hinge region harbors Ser469-Ser470, the cleavage site used for proteolytic activation of the TGase 3 zymogen. Because the protein is known to be a monomer in solution (15), the presence of a dimer as the crystallographic asymmetric unit is likely of no functional relevance.



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  		FIG. 1.
A, view of the final portion of a 2.0-Å resolution (2 Fo - Fc) exp(i{alpha}c) composite omit electron density map contoured at 1.0-{sigma} level around the catalytic triad of TGase 3·GMP. B, ribbon image of the structure of human TGase 3·GMP complex. The four domains are the {beta}-sandwich (red), the catalytic core (blue), the {beta}-barrel 1 (magenta), and {beta}-barrel 2 (orange). The Ca2+ ions are shown in yellow, and the Mg2+ ion is in green. In B a single molecule of GMP nucleotide in ball-and- stick is visible in the monomer.

 
The TGase 3 architecture consists of four domains ({beta}-sandwich domain, catalytic core domain, {beta}-barrel 1, and {beta}-barrel 2 domains), which we described previously (23, 40, 41). The core domain harbors the active site residues Cys272, His330, and Asp353. This catalytic triad is shielded from contact with the solvent because it is buried within a narrow cleft with walls formed by two {beta}-sheet strands of the catalytic core domain and the carboxyl terminus of {beta}-barrel 1 domain. In addition, the core domain possesses two functional Ca2+ and one Mg2+ metal-binding site indicated in Fig. 1B. The TGase 3·GMP structure has two non-proline cis peptide bonds (Arg268-Tyr269 in the {beta} strand before the loop of the {alpha}-helix containing the active site Cys272 residue and Asn382-Phe383 in a loop adjoining two {alpha}-helices of the core domain) and cis-peptide bond at Gly367-Pro368.

The GMP Nucleotide Binding Pocket—The electron density unequivocally indicates the presence of a bound GMP molecule in the crystal structure (Fig. 2A, left panel). The GMP nucleotide binds to the same site as GTP{gamma}S and GDP, a deep cleft located between the core domain and the {beta}-barrel 1 domain (Fig. 2A, right panel). This cleft consists of residues Asn168 and Arg169 from the core domain and Lys485, Lys487, Val488, Met491, Leu492 (first {beta}-strand), Arg587, Asp588, Ile590, Leu591, and Asp592 (last {beta}-strand) from {beta}-barrel 1. Fig. 2B shows how the guanine ring is recognized in the structure. The specificity for binding by guanine nucleotides versus adenine nucleotides is conferred by the pattern of hydrogen bond donors and acceptor interactions with the major-groove face of the guanine moiety. Ile590 uses its backbone nitrogen and oxygen to form direct hydrogen bonds with the O-6, N-1, and N-2 atoms of the guanine base. The side chain of Arg587 forms a hydrogen bond with the N-7 group of the guanine ring and {alpha}-phosphate oxygen of GMP nucleotide and stabilizes a water-mediated interaction with O-6 through N{epsilon} of Arg587. An additional hydrogen bond to the phosphate oxygen atom is made with the backbone nitrogen of Val488. The GMP nucleotide binding cleft is defined on one side by the Arg169 side chain stacking with the guanine ring and the side chains from Val488, Leu492, and Ile589 sandwiching from the other side. This binding cleft shows no similarity to those of traditional large and small G proteins, which rely on a (N/T)KXD consensus motif for hydrogen-bonding interactions with the guanine ring (42-44).



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  		FIG. 2.
A, left panels, stereo view of the 2.0-Å resolution 3 Fo - 2 Fc electron density map around the GMP contoured at 1.0-{sigma} level in the. Right panel, view of cleft surface (yellow) and the surrounding residues within 6.5 Å of the GMP nucleotide located at the interface of the core domain (top) and the {beta}-barrel 1 domain (bottom). B, a view of the guanine nucleotide-binding site pocket in TGase 3·GMP complex. The hydrogen bonds and ion pair interactions are shown as dashed lines. All the important residues in the guanine nucleotide-binding site pocket and the GMP are shown in ball-and-stick. C, identification of key residues involved in the coordination with metal ions in sites 1, 2, and 3 in TGase 3 complex. In site 1, the Ca2+ ion in the TGase 3·GMP forms direct contacts with the main chain carbonyl oxygen atoms of Ala221, Asn224, Asn226, the carboxyl side-chain oxygen of Asn224, Asp 228, and to a water molecule. Site 2 exists near the end of catalytic core domain, next to the {alpha}-helical segment (residues Asn430-Glu448), leading to the loop connecting the {beta}-barrel 1 domain. The Ca2+ ion forms contacts with the carboxyl side-chain atoms of Asn393, Glu443, and Glu448, the main chain carbonyl oxygen atom of Ser415, and two water molecules. Mg2+ in site 3 binds in the vicinity of the loop segment, leading to the catalytic His330 at the active site. This Mg2+ ion forms contacts with the carboxyl side-chain oxygen atoms of Asp301 and Asp303, the side-chain atoms of Asn305, the main chain carbonyl oxygen atoms of Ser307, and a water molecule. The loop bearing residues 320DKGSDS325 has moved from its position in the native enzyme, closing the channel.

 
The Effect of GMP Nucleotide on Metal Ion Binding—We have shown previously that active TGase 3 can bind three Ca2+ ions. That in site 1 is constitutively bound, whereas those in sites 2 and 3 are acquired during activation and are required for activity. In particular, binding at site 3 opens a channel through the enzyme and exposes two tryptophan residues near the active site that are thought to be important for enzyme reaction (45). In the TGase 3·GMP structure we find that the three divalent cation-binding sites are occupied, with site 3 showing significant differences (Fig. 2C) from TGase 3 structure determined in the absence of nucleotides (23). A Ca2+ ion with pentagonal bipyramidal coordination occupies site one identically to that previously reported in the GTP{gamma}S-bound and -unbound nucleotide structure (23, 40, 41). The second metal site is occupied by a Ca2+ ion but with a change in coordination compared with the activated TGase 3 (40, 41). In the absence of guanine nucleotide, the coordination involves a bidentate interaction with the side-chain oxygen atoms of Glu443. However, in the TGase 3·GMP complex, Glu443 provides monodentate coordination. This may be attributable to a shift in the position of the nearby His416 side chain, providing a hydrogen bond the to Glu443 side chain. This shift in the coordination of Ca2+ at site 2 appears to derive from a more dramatic change in metal occupancy at the third divalent cation-binding site.

When no nucleotide is present, site 3 is bound by a Ca2+ ion with a similar hepta coordination as in the activated TGase 3 form (40). In the GMP-bound structure the third site is occupied by an octahedrally coordinated Mg2+ ion. The identification of a Mg2+ ion is based on a number of criteria such as coordination and bonding distances. The observed octahedral geometry is more consistent with Mg2+ ligation, since Ca2+ ion favors pentagonal bipyramid heptacoordination. The short distance to nearby oxygen atoms (2.0-2.1 Å) also supports the identification of the Mg2+ ion. The clarity of the simulated-annealing Fo - Fc omit electron density map strongly supports the assignment of Mg2+ at this site. Bond distances to the metal-oxygen ligands typically range from 2.0 to 2.1 Å for Mg2+ and 2.1 to 2.8 Å for Ca2+. Moreover, bond-valence calculation parameters (46) further support this assignment as a Mg2+ site. Finally, an inductively coupled plasma-mass spectrometry dynamic reaction cell-based analysis of these crystals indicated the existence of 1.0 Mg2+ ion/mol, 2.0 Ca2+ ions/mol, and the absence of any other metals. The occupation of site 3 by Mg2+ and the associated 9-Å movement of C{alpha} atoms of the flanking 320DKGSDS325 loop are clearly observed when compared with the TGase 3 structure solved in the absence of guanine nucleotide (40, 41).

TGase 3 Binding and Regulation by GMP Nucleotide—We directly measured the interaction of recombinant, purified active TGase 3 with GMP nucleotide by isothermal calorimetry as shown in Fig. 3A. In this case the GMP bound to TGase 3 at a molar ratio of nearly 1:1, similar to other previously reported guanine nucleotides (23). The dissociation constants and heat of binding were determined to be Kd = 1.6 ± 0.2 µM ({Delta}H = -10.3 ± 0.2 kcal/mol), which is similar to those of GTP{gamma}S. Transamidation assays indicate that GMP is also a potent inhibitor of TGase 3 enzyme activity (Fig. 3B). In the presence of a lower concentration of Ca2+ ion, TGase 3 is completely inhibited by micromolar concentrations of GMP nucleotide. An increase in the concentration of Ca2+ in the assay mixture progressively suppresses the inhibition by GMP nucleotides.



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  		FIG. 3.
A, isothermal calorimetric titration of nucleotide binding to TGase 3. To solutions of the activated forms were added a series of injections of 5, 10, and 20 µl of GMP, and the heat absorbed or liberated was recorded (upper panel). The lower panel records the net heat changes. The heat of dilution was determined from different injections of a solution without protein subtracted to determine heat changes due solely to nucleotide binding. The data shown are representative of one of three experiments, each performed in triplicate; the S.D. of replicates was <5%. B, inhibition of TGase 3 by increasing concentrations of GMP for four TGase 3 concentrations ({diamondsuit}, 1.0 mM; {blacksquare}, 0.50 mM; , 0.25 mM; and {blacktriangledown}, 0.10 mM). Increasing concentrations of GMP were preincubated for 30 min with recombinant activated TGase 3. C, activities of TGase 3 in the presence and absence of nucleotide were measured by incorporation of [14C]putrescine into casein. The - and + indicate the absence or presence of 0.575 mM Ca2+ and 1 mM Mg2+ in the standard assay reaction.

 
The TGase 3·GMP complex activity was investigated under conditions similar to those utilized in crystallization (1 mM MgCl2 and 0.575 mM CaCl2). These divalent cation concentrations were shown to be optimal for crystallization. In the presence of 0.575 mM CaCl2 alone enzyme activity was less than 10%. An addition of 1 mM MgCl2 increased the transamidation activity to 15% that of active TGase 3. The observed activity was less than 1% in the presence of 1 mM MgCl2 alone as shown in Fig. 3C. The transamidation activity of TGase 3·GMP complex begins to increase when it is titrated with CaCl2/MgCl2, with a molar ratio greater than 0.6 (data not shown), consistent with previously reported results (41). These results indicate that the enzyme bound with GMP nucleotide has very low activity when there are Ca2+ ions at sites 1 and 2 and when one Mg2+ is at site 3. Therefore the Mg2+ ion plays an important role in nucleotide hydrolysis and inhibition of TGase 3 activity. Furthermore, these results suggest that at physiological intracellular and extracellular concentrations of these factors, TGase 3 can display a distinct spectrum of activities. To determine whether hydrolysis of GTP was required for the inhibition of TGase 3 activity, active TGase 3 was preincubated with GTP in the presence of 1 mM MgCl2 and 0.575 mM CaCl2 at 37 °C for 60 min. Preincubation did not increase the potency of inhibition, indicating that GTP hydrolysis was not required for inhibition of TGase activity.

We additionally investigated the binding of BODIPY FL GTP{gamma}S-labeled guanine nucleotides (Fig. 4A) by fluorescence titration and fluorescence displacement for GTP{gamma}S, GTP, GMP, and ATP. Fig. 4B shows the titration of four different concentrations (43, 72, 173, and 180 nM) of active TGase 3 enzyme with different concentrations of labeled BODIPY FL GTP{gamma}S up to 1 µM. The observed dissociation constant was 60 nM. The fluorescence titration range of 0-1 µM active TGase 3 enzyme in the presence of 0.575 mM Ca2+ alone is identical to fluorescence changes observed with active TGase 3 containing no Ca2+ ion and 1 mM Mg2+ (data not shown). To determine the binding affinity of unlabeled GTP{gamma}S, GTP, and GMP using fluorescence displacement, a mixture of labeled BODIPY FL GTP{gamma}S (0.48 µM) and TGase 3 enzyme (0.43 µM) has been titrated with unlabeled guanine nucleotides. Fig. 4C represents the release of BODIPY FL GTP{gamma}S bound to TGase 3 after the addition of successive amounts of GTP{gamma}S, GTP, and GMP. This indicates that GTP{gamma}S and GMP rather than GTP releases BODIPY FL GTP{gamma}S more efficiently from TGase 3. Bound GTP{gamma}S, GTP, and GMP proved to be a better competitor than ATP (see "Discussion"). The dissociation constants are summarized in Table II. Kinetics measured from a mixture of labeled BODIPY FL GTP{gamma}S with TGase 3 do not show a significant change in signal over 20 min, indicating that GTPase activity of TGase 3 under these conditions is exceedingly slow and there is virtually no reaction over the time course of the experiments. These results further support our crystallographic observation that the GTP{gamma}S, GTP, and GMP molecules are recognized through the "DNA major groove face" of the purine.



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  		FIG. 4.
A, fluorescence of BODIPY FL GTP{gamma}S (right) and TNP-GTP (left) used in the experiment. B, the titration of 4 different concentrations (, 43 nM; {blacktriangleup}, 72 nM; {blacksquare}, 173 nM; {diamondsuit}, 180 nM) of active TGase 3 enzyme with a 0-1000 nM concentration of labeled BODIPY FL GTP{gamma}S. C, chase of BODIPY FL GTP{gamma}S nucleotides bound to TGase 3 by unmodified nucleotides. Upon excitation wavelength of 475 nm and an emission wavelength of 513 nm the BODIPY FL GTP{gamma}S nucleotide binding to TGase 3 was specifically monitored by the increase in fluorescence. Unmodified nucleotides used as competitors were GMP ({blacktriangleup}, 0.4 µM), GTP ({blacksquare}, 0.6 µM), and GTP{gamma}S (, 0.4 µM).

 


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  		TABLE II
Binding of the active TGase 3 for different nucleotides

 
Nucleotide Analogs That Stimulate TGase Activity—To gain insight into nucleotide recognition by TGase 3 and to facilitate the design of TGase 3 nucleotide analogs, we applied a genetic algorithm similarity program approach to identify the structural basis for alteration of nucleotide analog specificity. In this study we generated a minimum of three pharmacophore features, (i) an aromatic ring that maps 5- and 6-member rings, (ii) a hydrogen bond acceptor, and (iii) a hydrophobic (aromatic) feature from the superposition of guanine nucleotides. The phosphate atom was eliminated from the subset of nucleotide analogs in the search. This provides an opportunity for variance in the search to contribute to the correlation.

The three nucleotide analog representative compounds (TP349915, TP395289, TP394305) from the Lead QuestTM data base were proposed by FlexX, as shown in Fig. 5. The radius of 6.5 Å around the guanine nucleotide was chosen to include residues Ser166-Arg169, Lys485-Ala493, Tyr421, and Arg587-Asp592 in the calculation. The results were qualitatively evaluated by visual inspection of pharmacophore models (Fig. 6A). The binding of these three nucleotide analog compounds to TGase 3 enzyme was determined by the effect on transamidation by incorporation of [14C]putrescine into casein (Fig. 6, B and C). The binding affinities were TP349915 (Kd = 4.1 µM), TP395289 (Kd = 38.5 µM), and TP394305 (Kd = 1.0 mM). These specific nucleotide analogs do not inhibit but instead fully activate the TGase 3 transamidation activity in vitro (Fig. 6D). The three molecules occupy 369.4, 361.1, and 280.0 Å3 of the GMP cleft volume, respectively. The structure-activity relationship of nucleotide analogs suggests that the TGase 3 nucleotide-binding site is highly tolerant of small changes in the parent compound. For instance, the nucleotide analogs lacking the 2-amino group of the purine moiety still bind to TGase 3 as determined by transamidation assay (Fig. 6B).



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  		FIG. 5.
Stereo view representation of compounds TP349915 (upper), TP395289 (middle), TP394305 (lower) with predicted docked conformations and residues within 6.5 Å. Compounds bind in a cleft defined by residues Asn168 and Arg169 from the core domain and Lys485, Lys487, Val488, Met491, Leu492 (first {beta}-strand), Arg587, Asp588, Ile590, Leu591, and Asp592 (last {beta}-strand) from {beta}-barrel 1. The three molecules occupy 369.4, 361.1, and 280.0 Å3 of the total cleft volume, respectively.

 



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  		FIG. 6.
A, superposition of compounds TP349915, TP395289, and TP394305 and the mapped surface properties of residues within a 6.5-Å radius cutoff; brown, lipophilic residues; blue, hydrophilic residues. B, the binding of three nucleotide analog compounds to TGase 3 enzyme as a function of incorporation of [14C]putrescine into casein with the binding affinity of TP349915 (Kd = 4.1 µM), TP395289 (Kd = 38.5 µM), and TP394305 (Kd = 1.0 mM). C, differential loading of TGase 3 with nucleotide analog compounds with Ca2+ and Mg2+ ions results in stark changes in enzyme activity. The assays of TGase 3 activity were determined in the presence of 0.575 mM CaCl2 and/or 1 mM MgCl2 and were measured by the incorporation of [14C]putrescine into casein. Shown are TGase 3 transamidation activities of the activated form of TGase 3 enzyme in the presence of GTP and nucleotide analogs (TP349915, TP395289, TP394305) from Lead QuestTM data base compounds. D, chase of BODIPY FL GTP{gamma}S-nucleotides bound to TGase 3 by unmodified nucleotide analog compounds. Unmodified nucleotide analogs used as competitors were TP349915 ({diamondsuit}), TP395289 ({blacktriangleup}), and TP394305 (). The binding affinity for ATP ({blacksquare}) was determined to be Kd = 28 µM by fluorescence displacement.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We previously provided direct evidence that TGase3 enzyme, like TGase 2, binds, hydrolyzes, and is inhibited by GTP (23). This report shows that GMP also acts as a reversible, noncompetitive inhibitor for TGase 3 enzyme activity. Isothermal calorimetry, fluorescence titration, and fluorescence displacement were used to demonstrate guanine nucleotide binding to TGase 3 (Figs. 3 and 4). Moreover, although calcium ions activate the enzyme, GMP nucleotide binding leads to the inhibition of the enzyme activity in a concentration-dependent manner (Fig. 3, B and C). An increase in Mg2+ ion concentration is associated with loss of the enzyme transamidation activity. The biochemical data in Fig. 3, B and C, suggests that Ca2+ ion and GMP/Mg2+ reciprocally regulate the enzyme in a fashion similar to GTP{gamma}S and GTP nucleotide (23, 24).

Earlier comparisons of the native TGase 3 structure with TGase 3 bound to GTP{gamma}S and GDP (23) identified three regions involved in conformational changes (switch regions). These included the binding site of guanine nucleotides (switch-I), the change in coordination of Ca2+ ion in site 2 (switch-II), and replacement of a Ca2+ by Mg2+ at site 3 and the associated movement of the 320DKGSDS325 sequence motif (switch-III) (23, 24). These subtle rearrangements in the TGase 3·GMP complex crystal structure led to a similar closing of the substrate entry channel due to GMP binding. Examination of surface representation of the TGase 3·GMP complex revealed that access to the central channel of the enzyme active site is closed. The loss of transamidation activity accompanying GMP binding may be explained on this basis (Fig. 3C).

Examination of the TGase 3·GMP structure reveals that a Mg2+ ion replaces Ca2+ at site 3 (23, 40, 41). Moreover, we have shown that an increase in Mg2+ ion concentrations alone even without GMP nucleotide can inhibit the enzyme, raising the possibility that GMP binding and Mg2+ ion substitution at site 3 represent a coordinated event in the inhibition of TGase 3 (23, 41). A combination of changes in the enzyme structure stemming from the binding of GMP and Mg2+ ion causes the closing of the channel required for substrate access to the active site (24, 45). The guanine nucleotide-binding site is far from the active site (22.9 Å from C{alpha} atom of Cys272). The GMP molecule imparts the inactive state through a set of local structural rearrangements and not through its direct occlusion of the active site. In the course of our fluorescence titration study, we utilized two fluorescent probes BODIPY FL GTP{gamma}S and TNPGTP (Fig. 4A); however, only BODIPY FL GTP{gamma}S bound. The fluorescence of the BODIPY FL GTP{gamma}S probe can be used in competition experiments as a reliable reporter of the binding of the BODIPY FL GTP{gamma}S derivative to the protein since the signal of the bound derivative should not be modified by the presence of the derivatized nucleotides. This is consistent with our structural findings that the ribose moiety of GMP forms many contacts with surrounding protein side chains; the TNP substituent attached to the ribose moiety would prevent binding of TNP-GTP, whereas the fluorescent substituent of BODIPY FL GTP{gamma}S could be solvent-exposed.

To understand the molecular basis for nucleotide selectivity by TGase 3 and to guide further nucleotide analog design, we used the automated computational approach GASP to detect pharmacophore features from the superimposed GTP{gamma}S, GDP, and GMP nucleotides in a search for appropriate nucleotide analogs (Fig. 5). Three specific nucleotide analog compounds (TP349915, TP395289, TP394305) from the Lead Quest TM data base were found to bind to TGase 3·GTP, displacing GTP and increasing the transamidation of [14C]putrescine into casein (Fig. 6, B and C). Fluorescence displacement experiments determined the binding affinities as follows: TP349915, Kd = 4.1 µM; TP395289, Kd = 38.5 µM; TP394305, Kd = 1.0 mM. Notably, these compounds do not inhibit but, instead, activate the TGase 3 transamidation activity in vitro (Fig. 6D). The nucleotide groove seems to be the main determinant for target affinity. The poorer binding of ATP with only micromolar affinity can be rationalized from the lack of the 2-amino group of the purine. Interestingly, the nucleotide analogs that increase transamidation activity all lack phosphate groups, as shown in Fig. 6C. Our results suggest that novel compounds targeting the GTP-binding site may be designed to either inhibit or activate TGase 3.

In summary it is clear that the enzymatic activities of TGase 3 are controlled by local concentrations of Ca2+, Mg2+, and nucleotides. Under physiological conditions, intracellular GTP levels between 50 and 150 µM, free Ca2+ ion levels around 100 nM, and free Mg2+ concentrations in the micromolar range are sufficient to keep TGase 3 in a latent state (20). TGase 3 would be expected to be largely in the GDP-bound state with Mg2+ ion at site 3. Because the Mg2+-ATP is more abundant and is a strong inhibitor of TGase 3 (23, 41), one would expect TGase 3 to display minimal hydrolysis activity intracellularly. The activity would be recovered upon Ca2+ ion entry, since higher Ca2+ ion concentrations reverse the inhibition by GTP nucleotide (23). It has been reported that local concentrations of GTP and Ca2+ ion regulate intracellular TGase 3 activity (5). TGase 3 enzyme acts in epidermal keratinocytes during its late differentiation and apoptosis stages, where GTP might be depleted and free Ca2+ ion might increase. The work described here suggests that the nucleotide binding pocket in TGase3 may be exploited to either enhance or inhibit the enzymatic activity as needed for different therapeutic approaches.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1SGX [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{dagger} Deceased April 7, 2003. This paper is dedicated to Dr. Peter M. Steinert on the anniversary of his death. Back

§ To whom correspondence should be addressed: X-ray Crystallography Facility, NIAMS, 50 South Dr., Bldg. 50, Rm. 1345, Bethesda, MD 20892-8023. Tel.: 301-402-4086; Fax: 301-480-0742; E-mail: ahvazib{at}mail.nih.gov.

1 The abbreviations used are: TGase, transglutaminase; GTP{gamma}S, guanosine 5'-O-(thiotriphosphate); TNP-GTP, 2'- (or 3'-)-O-(trinitrophenyl) guanosine 5'-triphosphate, trisodium salt; BODIPY FL GTP{gamma}S, GTP{gamma}S trisodium salt BODIPY FL thioester. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Zbigniew Dauter at Brookhaven National Laboratory NSLS for assistance with the data collection at X9B Beamline. We thank Dr. Ulrich Baxa for assistance with the VP-isothermal calorimetry and fluorescence titration experiment. We thank Dr. Craig Hyde for numerous insightful comments.



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G. E. Begg, L. Carrington, P. H. Stokes, J. M. Matthews, M. A. Wouters, A. Husain, L. Lorand, S. E. Iismaa, and R. M. Graham
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