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Proc Natl Acad Sci U S A. Mar 5, 2002; 99(5): 2743–2747.
Published online Feb 26, 2002. doi:  10.1073/pnas.042454899
PMCID: PMC122418
Biochemistry

Structural basis for the guanine nucleotide-binding activity of tissue transglutaminase and its regulation of transamidation activity

Abstract

Tissue transglutaminase (TG) is a Ca2+-dependent acyltransferase with roles in cellular differentiation, apoptosis, and other biological functions. In addition to being a transamidase, TG undergoes a GTP-binding/GTPase cycle even though it lacks any obvious sequence similarity with canonical GTP-binding (G) proteins. Guanine nucleotide binding and Ca2+ concentration reciprocally regulate TG's transamidation activity, with nucleotide binding being the negative regulator. Here we report the x-ray structure determined to 2.8-Å resolution of human TG complexed with GDP. Although the transamidation active site is similar to those of other known transglutaminases, the guanine nucleotide-binding site of TG differs markedly from other G proteins. The structure suggests a structural basis for the negative regulation of transamidation activity by bound nucleotide, and the positive regulation of transamidation by Ca2+.

Tissue transglutaminase (TG, also called type II transglutaminase) catalyzes the Ca2+-dependent formation of a new amide bond between the γ-carboxamide of glutamine and the epsilon-amino group of lysine or another primary amine (1) (see scheme below).

TG activity, which is found in the cytosol, plasma membrane, and nucleus of cells, has been implicated in a variety of physiological activities and pathological processes, including neuronal growth and regeneration (24), bone development (56), angiogenesis (7), wound healing (7), cellular differentiation, and apoptosis (810). During apoptosis, for example, TG-catalyzed crosslinking of proteins results in the irreversible formation of scaffolds that could prevent the leakage of harmful intracellular components (11). Retinoic acid (RA)-stimulated increases in TG expression and activation accompany RA-induced cellular differentiation (8, 12). This increased TG expression, coupled with the finding that two of the primary targets for TG, the eukaryotic initiation factor eIF-5A and the retinoblastoma gene product (13, 14), are essential for cell viability has led to the suggestion that TG activity is necessary for ensuring cell survival under conditions of differentiation or cellular stress. It has also been proposed that the dysregulation of TG activity may be associated with neurodegenerative conditions such as Alzheimer's disease and Huntington's disease (1517).

TG's ability to bind and hydrolyze GTP with affinity and rates like those of traditional G proteins distinguishes it from other transglutaminases and suggests that TG, like other G proteins, participates in signaling pathways (1821). Among the studies implicating TG as a signal transducer in biological response pathways, the best documented is its role in α1-adrenergic receptor-mediated stimulation of phospholipase C-δ activity (2123). It was originally reported that an ≈70- to 80-kDa GTP-binding protein (named Gh) was responsible for coupling α1-adrenergic agonists to the stimulation of phosphoinositide lipid metabolism (24), and it was subsequently demonstrated that Gh was identical to TG (21). The GTP-binding/GTPase cycle of TG is closely linked to its transamidation activity, with guanine nucleotide binding having a negative regulatory effect that can be overcome by high concentrations of Ca2+ (18, 19).

Because of the lack of sequence similarity between TG and either the large or small G proteins, the structural basis for TG's ability to bind guanine nucleotides with high affinity and hydrolyze GTP was not understood. The structural mechanism by which guanine nucleotide binding exerts such marked regulatory effects on transamidation activity is also unknown. To address these questions, we overexpressed and purified full-length human TG and determined its three-dimensional structure to 2.8-Å resolution by x-ray crystallography.

Materials and Methods

Expression and Purification of Human TG.

TG was amplified with primers that introduced a XhoI site before the initial ATG codon and an EcoRI site after the stop codon for the ORF of TG, by using a pGEX-MCS-HTG plasmid as a template (25), and then subcloned into pET-28a vector (Novagen) to create TG with an N-terminal His6 tag. Overnight cultures from colonies of Escherichia coli Bl21 (DE3) cells (Novagen) transformed with the expression vector were grown at 37°C. These were used to inoculate 1 liter of TP medium [2% bacto-tryptone/1.5% yeast extract/0.2% Na2HPO4/0.1% KH2PO4/0.8% NaCl/0.2% glucose (all wt/vol)]. The bacterial cells were then grown at 25°C until the cell density reached an OD600 reading of 0.6, at which point the temperature was reduced from 25°C to 18°C before induction with 1 μM isopropyl β-d-thiogalactoside (IPTG). The cultures were grown overnight at 18°C and then the cells were harvested by centrifugation at 4°C.

All protein purification steps were performed on ice. Cell pellets from 4 liters of culture were lysed by sonication in 150 ml of lysis buffer (50 mM Na2HPO4, pH 7.5/400 mM NaCl/5 mM benzamidine/5 mM 2-mercaptoethanol) with 50 μM GTP, 50 μM ATP, and 50 μg/ml PMSF. Both GTP and ATP were included in the lysis buffer as possible stabilizing agents as ATP as well as GTP has been suggested to bind to TG (25). After sonication, Triton X-100 was added to a final concentration of 0.5% (vol/vol). Cell debris was removed by high-speed centrifugation and the supernatant was loaded onto a column containing 5 ml of Talon metal-affinity resins (CLONTECH). The column was washed with 150 ml of lysis buffer containing 20 μM GDP, and then further washed with 150 ml of 50 mM Hepes (pH 7.0)/150 mM NaCl/5 mM 2-mercaptoethanol/20 μM GDP/5 mM imidazole. The TG fusion protein was eluted with 50 mM Hepes (pH 7.0)/50 mM NaCl/5 mM 2-mercaptoethanol/20 μM GDP/160 mM imidazole. The eluted protein was loaded onto a MonoQ anion exchange column (Pharmacia Biotech) equilibrated with 50 mM Mes (pH 6.5)/50 mM NaCl/10% (vol/vol) glycerol/1 mM EDTA/5 mM DTT. After washing with the equilibration buffer, human TG was eluted by using a gradient of 150 mM to 450 mM NaCl in the same buffer. The fractions containing TG were pooled and concentrated to 2 ml by using UltraPrep filtration (Millipore, molecular weight cutoff = 30,000), and then loaded onto a HiLoad 26/60 Superdex S-200 gel filtration column (Pharmacia Biotech) and eluted with 50 mM Hepes (pH 7.0)/100 mM NaCl/10% (vol/vol) glycerol/1 mM EDTA/5 mM DTT at 0.5 ml/min. Fractions containing TG were pooled and concentrated. Purity of TG was confirmed by SDS/PAGE, and its transamidation activity was assayed by the hydroxylamine method (26). The specific activity of TG is ≈1.0 μmol/min per mg, comparable to the value reported for guinea pig liver tissue TG (27). The guanine nucleotide-binding activity of purified TG was confirmed by photoaffinity labeling with [α-32P]GTP (19) and by monitoring guanine nucleotide-mediated inhibition of transamidation.

Crystallization of Human TG.

Crystals of TG were obtained by the sitting-drop vapor diffusion method, by mixing 20 mg/ml TG in 20 mM Hepes (pH 7.0)/1 mM EDTA/EGTA/5 mM DTT/20% (vol/vol) glycerol with equivalent amounts of precipitation solution containing 50 mM Mes (pH 6.6), 200 mM NaCl, 50 mM MgCl2, 6–8% PEG 3350, and 5 mM DTT. Drops were set against 1 ml of precipitation solution plus 20% (vol/vol) glycerol at 4°C. Crystals usually appeared within a day and reached the full size of 0.4 mm × 0.2 mm × 0.1 mm in 2–3 days. The crystals belong to space group P212121, with unit cell constants of a = 136.478 Å, b = 168.797 Å, and c = 236.568 Å. After soaking in 50 mM Hepes (pH 7.0)/200 mM NaCl/5 mM MgCl2/30% (vol/vol) glycerol/20% (wt/vol) PEG 3350 for about 2 weeks, a 2.8-Å data set was collected at the Advanced Photon Source (Chicago) Beamline BioCAT 12C at 100 K. Reflection data were processed by using the program suite denzo/xdisplay/scalepack (Table (Table1;1; ref. 28).

Table 1
Data collection and refinement statistics

Structural Analysis.

The TG structure was solved by the molecular replacement method using the program molrep (29) with the crystal structure of the human factor XIIIa as a search model (PDB ID code: 1GGU; ref. 30). Six independent molecules were found in the asymmetric unit, with orientations consistent with the observed pseudo-622 symmetry seen in the self-rotation function search and locations consistent with the pseudo-B centering observed in the low resolution Patterson map. The amino acid residues of factor XIIIa were replaced with the corresponding residues of TG. After rigid body refinement with the program cns (31), the model was subjected to successive cycles of simulated annealing refinement using cns and manual model building by using the program o (32). Noncrystallographic symmetry (NCS) between the six independent molecules was used in electron density averaging and during early refinement. In the last stages of refinement, side-chain atoms of several residues involved in different crystal packing environments were released from NCS restraints. The final model was refined to a final R factor of 0.232 and a final Rfree of 0.272 (Table (Table1)1) and has been deposited in the Protein Data Bank (PDB ID code: 1KV3).

Results and Discussion

Structure of TG.

The x-ray crystallographic model has six independent TG molecules in the asymmetric unit of a P212121 unit cell, and they are organized as three dimers to give an approximate P6122 space group. The overall structure of a TG dimer is shown in Figure Figure1.1. Each monomer has four distinct domains: the amino-terminal β-sandwich domain (shown in green) consisting of residues Met-1 to Phe-139, the transamidation catalytic core domain (red) consisting of Ala-147 to Asn-460 (marked by the essential Cys-277 in ball-and-stick), and two carboxy-terminal β-barrel domains (the first in blue and the second in yellow), which include Gly-472 to Tyr-583, and Ile-591 to Ala-687, respectively. The general domain structure for TG is similar to that for factor XIIIa (30). Dimerization buries 2,783 Å2 of surface area (i.e., the sum of the surface buried by each monomer), with each monomer contributing the tip of the first β-barrel domain to the interface. The remainder of the dimerization interface consists of the second β-barrel domain from one monomer, and the β-sandwich domain, the catalytic domain, and the second β-barrel domain from the other monomer.

Figure 1
Overall structure of a human tissue transglutaminase (TG) dimer with bound GDP. TG is shown in ribbon drawing with the β-sandwich domain, the catalytic core domain, and the first and second β-barrel domain shown in green, red, cyan, and ...

The guanine nucleotide-binding site is located in a cleft between the catalytic core and the first β-barrel domain (Fig. (Fig.1),1), close to the dimerization interface. The electron density unambiguously shows one GDP molecule bound to each of the six TG monomers within the asymmetric unit (Fig. (Fig.2).2). Finding bound GDP was unexpected because no GDP was present in the final purification or crystallization steps, and its presence testifies to both its tight binding and slow exchange. The majority of the residues contacting GDP come from the end of the first β-strand of the first β-barrel domain and the loop that connects it to the second β-strand, as well as from the last β-strand of the β-barrel domain (Fig. (Fig.1).1). The catalytic domain contains two residues interacting with the guanine base.

Figure 2
Stereoview of an electron density map (2FoFc, 1.2σ, GDP omitted, 2.8-Å resolution) of the GDP-binding pocket. An atomic model of the final structure is embedded in the electron density. Drawing prepared from MOLSCRIPT (44) and ...

Guanine Nucleotide-Binding Site of TG.

Overall, the architecture for the guanine nucleotide-binding site on TG differs markedly from the nucleotide-binding domain conserved among the α subunits of large heterotrimeric G proteins and small Ras-related G proteins, which have five helices surrounding a six-stranded β-sheet (33). In heterotrimeric G proteins, the Gα subunits also contain a helical domain adjacent to the nucleotide-binding site. This helical domain probably enables the Gα subunits to bind guanine nucleotides with high affinity in the absence of Mg2+. Mg2+ is essential for the high-affinity binding of guanine nucleotides to Ras-related small G proteins, and guanine nucleotide exchange factors work by weakening the binding of Mg2+ (34). Like Gα, TG can bind GDP with high affinity in the absence of Mg2+ (35), and the TG structure does not reveal any bound Mg2+. Although both large and small G proteins have serine and threonine residues that bind to the β- and γ-phosphates of the guanine nucleotide and participate in Mg2+ ion coordination (33, 36), TG lacks amino acids with either hydroxyl or carboxyl side-chain moieties in the vicinity of the nucleotide phosphate groups. Several positively charged side chains surround the phosphate moieties of the bound GDP on TG (Figure (Figure33 Left). Arg-580 forms two ion pairs with the α- and β-phosphates, with the β-phosphate being positioned near the main chains of Arg-478 and Val-479 and forming a hydrogen bond with the nitrogen of Val-479 (Fig. (Fig.33 Left). Arg-478, Val-479, and Arg-580 are all conserved in tissue transglutaminases but not in other transglutaminases (Fig. (Fig.4).4).

Figure 3
Comparisons between the atomic interactions of GDP with TG (Left) and Ras (Right). Hydrogen bonds and ion pair interactions are shown in dashed lines. The GDP molecule is shown in ball-and-stick. TG and Ras residues are shown in thin sticks. Drawing prepared ...
Figure 4
Sequence alignment of different members of the human transglutaminase family with TG numbering on the bottom. F13A represents factor XIIIa and B4.2 represents the erythrocyte band 4.2 protein. Conserved residues are in pink; TG catalytic triad residues ...

There are a number of other interesting points of comparison between the guanine nucleotide-binding pocket of TG and the pocket for the traditional large and small G proteins. Of particular interest is the binding site for the guanine ring moiety. In both heterotrimeric large G proteins and small G proteins, the highly conserved NKXD motif plays an essential role in binding the guanine ring. The x-ray crystallographic structures of the Gα subunits of retinal transducin and the Gi1 protein (33, 37, 38), as well as Ras (36), show that the asparagine residue of the NKXD sequence forms a hydrogen bond with the N7 atom of the guanine moiety, whereas the aspartic acid (Asp-119 of Ras in Fig. Fig.33 Right) forms hydrogen bonds with the N1 and N2 atoms. The NKXD motif is not present in TG. Rather, a main-chain oxygen from Tyr-583 forms hydrogen bonds with the N1 and N2 atoms of the guanine base, and Ser-482 Oγ forms an additional hydrogen bond with N2 (Fig. (Fig.33 Left). In addition, O6 of the base forms a hydrogen bond with the main chain nitrogen of Tyr-583, a conserved residue in tissue transglutaminases.

In the TG structure, the guanine base sits in a hydrophobic pocket formed by the side chains from Phe-174, Val-479, Met-483, Leu-582, and Tyr-583 (Fig. (Fig.33 Left). The conserved phenylalanine residue might stabilize one side of the guanine ring through aromatic stacking interactions. There is no such corresponding phenylalanine in heterotrimeric G proteins. Both the Gα subunits of retinal transducin and the Gi1 protein use the methylene carbons of a second lysine residue within the signature NKXD motif (where X is the second lysine) to fulfill a similar function by making van der Waals contacts with one side of the guanine ring. However, it is worth noting that in Ras and other related small G proteins, a conserved phenylalanine (Phe-28 of Ras in Fig. Fig.33 Right) approaches one side of the guanine ring at an approximately 90° angle (36) and has been suggested to participate in π–π stacking interactions. Mutation of this phenylalanine to leucine in the small G proteins Ras and Cdc42 yields constitutive GTP–GDP exchange activity, and in both cases gives rise to malignant transformation (39, 40). In TG, the opposite side of the guanine ring is in contact with the conserved residues Val-479 and Met-483, such that these residues together with Phe-174 sandwich the guanine moiety (Fig. (Fig.33 Left). This arrangement is not observed in Ras or other small G proteins, whereas in the Gα subunits of transducin and Gi1, a conserved threonine residue within the carboxyl-terminal domain of the Gα subunits serves a function similar to that of Val-479 and Met-483 in TG (33).

In both Gα subunits and Ras-related small G proteins, a conserved glutamine is essential for GTP hydrolysis. Although TG has no such glutamine, it is capable of hydrolyzing GTP with a turnover number (≈1 mol of 32Pi released per min per mol of TG) similar to the intrinsic rates of GTP hydrolysis measured for Gα subunits, and the GTPase-activating protein (GAP)-catalyzed hydrolytic rates of small G proteins (41). Given that the β-phosphate of the guanine nucleotide is pointed toward the Arg-478–Val-479 dipeptide (Fig. (Fig.33 Left), the γ-phosphate would need to rotate around the β-phosphate–O3′ bond to avoid clashing with the side chains of these amino acids. This rotation would bring the γ-phosphate into the vicinity of the positively charged side chains of Lys-173 and Arg-476. A plausible mechanism for TG-catalyzed GTP hydrolysis may involve a water hydrogen bonded to either the side chain of Lys-173 or Arg-476 as the nucleophilic attacking group. The positive charges of Lys-173, Arg-476, and Arg-478 would likely help orient the γ-phosphate group as well as stabilize the negative charges that develop on the γ-phosphate group during hydrolysis. Mutations of Lys-173 significantly impair GTP hydrolysis, which is consistent with this proposal (35). In this mechanistic formulation, either Arg-476 or Arg-478 could serve as the “arginine finger,” which has been shown to be essential for stabilizing the transition states for GTP hydrolysis by both large and small G proteins (42). Lys-173, Arg-476, and Arg-478 are conserved or conservatively substituted (Lys–Arg) in tissue transglutaminases.

Studies have shown that among the transglutaminase family only TG (TG2 in Fig. Fig.4)4) can bind and use guanine nucleotides to regulate transamidation. Indeed, multiple sequence alignments of different human transglutaminases show that the amino acid residues involved in GDP binding in TG are not remotely conserved (see the blue dots in Fig. Fig.4,4, which are placed below the residues essential for GTP-binding to TG2). For example, Phe-174 is replaced by aspartic acid in the factor XIIIa sequence. On the other hand, the sequences of all TGs known to bind guanine nucleotides are highly conserved, including those residues that form the nucleotide-binding site.

Regulation of Transamidation Activity.

The TG structure also provides clues regarding the regulation of its enzymatic transamidation activity. It has been well established that Cys-277 is the essential nucleophile for transamidation (30). In the TG structure, Cys-277 is located in the middle of a groove within the catalytic domain (Fig. (Fig.1)1) and participates in a catalytic triad, Cys-277–His-335–Asp-358 (Fig. (Fig.5),5), similar to what has been reported for factor XIIIa (30). These three catalytic residues are conserved in all members of the transglutaminase family (Fig. (Fig.4).4). In the GDP-bound form of TG, access to the transamidation active site is blocked by a loop connecting the third and fourth β-strands, as well as by a loop connecting the fifth and sixth β-strands of the first β-barrel domain (the loops are shown in blue in Fig. Fig.5).5). Tyr-516, which is conserved in TGs and located in the first loop, forms a hydrogen bond with Cys-277 (Fig. (Fig.5).5). Transamidation activity requires an accessible Cys-277, and Tyr-516 with its associated loop from the first β-barrel domain must move to make the active site accessible to substrates. The GDP molecule engages both the first and last β-strands of the first β-barrel domain, which should maintain the inactive state by stabilizing the loops that block access to the catalytic domain. This observation would likely account for the observations that guanine nucleotide binding inhibits transamidation activity (18, 25).

Figure 5
Transamidation active site of TG. A close-up view of the juxtaposition of the catalytic triad consisting of Cys-277–His-335–Asp-358 and Tyr-516 relative to the guanine nucleotide-binding site. Cys-277, His-335, Asp-358, Tyr-516, and GDP ...

Calcium ions exert an activating signal for transamidation (18, 19). The structures for factor XIIIa complexed to calcium, strontium, and ytterbium show that a major Ca2+-binding site is formed by the side chains of the conserved Asn-436, Asp-438, Glu-485, and Glu-490, and by the main chain oxygen of Ala-457 (ref. 43; also see Fig. Fig.6).6). The putative Ca2+-binding site on TG is located near the end of the loop that connects the catalytic transamidation domain to the first β-barrel domain. Unlike the case for factor XIIIa, this site is distorted in TG, with the largest difference occurring in the vicinity of Ser-419 (equivalent to Ala-457 in factor XIIIa, Fig. Fig.6).6). In TG, peptide Ile-416–Ser-419 forms a β-strand antiparallel with peptide Leu-577–Glu-579. Apparently these hydrogen bonds involved in β-sheet formation can support the first β-barrel domain and further stabilize the nucleotide-binding site. Calcium binding, by altering the position of the Ile-416–Ser-419 peptide, would eliminate these stabilizing effects and could thereby weaken nucleotide binding, as has been observed experimentally (18). In factor XIIIa, the equivalent peptide, Asn-454–Ala-457, forms an antiparallel β-strand with Asp-458–Tyr-441, which stabilizes calcium binding. Glutamic acid residues 447 and 452 may also undergo Ca2+-induced conformational changes that would further impact the nucleotide site and weaken nucleotide binding. In an apoptotic cell, falling nucleotide levels and increasing Ca2+ levels would activate TG's transamidation activity.

Figure 6
Comparison of the calcium-binding sites of TG (green) and factor XIIIa (red). In factor XIIIa, the loop involved in calcium binding is oriented toward the Ca2+-binding site, whereas in TG-GDP, the same loop is oriented toward GDP. Figure prepared ...

Subtle differences in the conformations induced by GTP versus GDP could explain some reports that have shown differences in the extent of transamidation activity measured for the two nucleotide states of TG (18, 19). The ability of TG to undergo a GTP-binding/GTPase cycle that is conformationally coupled to its enzymatic transamidation activity potentially offers an interesting example of a G protein that has a “built-in” effector enzyme activity, and perhaps underlies the unique architecture of its guanine nucleotide-binding site. It also raises the likelihood that distinct types of extracellular stimuli (e.g., retinoic acid; ref. 27) and as yet undescribed types of regulatory proteins will be involved in the regulation of the GTP-binding and GTP-hydrolytic activities of TG, relative to those that have been reported for the more traditional large and small G proteins.

Acknowledgments

We thank J. Widom for expert advice on protein production and purification, C. Westmiller for expert secretarial assistance, and the BioCAD staff at the Advanced Photon Source. This work was supported by National Institutes of Health Grants CA59021 (to J.C.) and GM61762 (to R.A.C.).

Abbreviation

TG
transglutaminase

Footnotes

This paper was submitted directly (Track II) to the PNAS office.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.rcsb.org (PDB ID code 1KV3).

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