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

Abstract

Tissue transglutaminase (TG) is a Ca 2؉ -dependent acyltransferase with roles in cellular differentiation, apoptosis, and other biological functions. In addition to being a transamidase, TG undergoes a GTP-binding͞GTPase cycle even though it lacks any obvious sequence similarity with canonical GTP-binding (G) proteins. Guanine nucleotide binding and Ca 2؉ 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 Ca 2؉ .T issue transglutaminase (TG, also called type II transglutaminase) catalyzes the Ca 2ϩ -dependent formation of a new amide bond between the ␥-carboxamide of glutamine and the -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 (2-4), bone development (5-6), angiogenesis (7), wound healing (7), cellular differentiation, and apoptosis (8-10). 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 (15-17).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 (18)(19)(20)(21). Among the studies implicating TG as a signal transducer in biological response pathways, the best documented is its role in ␣1-adrenergic receptor-mediated stimulation of phospholipase C-␦ activity (21-23). It was originally reported that an Ϸ70-to 80-kDa GTP-binding protein (named Gh) was responsible for coupling ␣1-adrenergic agonists to the stimulation of phosphoinositide lipid metabolism (24), and it was subsequently demonstrated that Gh was identical to TG (21)....

Isopeptidase activity of human transglutaminase 2: disconnection from transamidation and characterization by kinetic parameters

“…It was a surprise that TG2-I presented functional nucleotide sensitivity similar to the wild type TG2 despite its weak GTP binding while TG2-T presented slightly higher nucleotide binding affinity and accordingly higher GTP sensitivity compared to the wild type. One possibility is that the interaction of BODIPY-GTPγS with non canonical nucleotide binding sites of TG2 does not lead to the increases of its fluorescent intensity (Kiraly et al 2009;Liu et al 2002) or the slight difference between the natural and labelled nucleotides could be responsible for these deviances. It cannot be excluded either that the replacement of Trp 332 , similarly to Cys 277 , may break the integrity and balance of the highly conserved interface between the 159-173 nucleotide binding loop and the amino acids surrounding the active site (Murthy et al 2002) modifying the affinity of nucleotide binding while still keeping its regulatory function.…”

Transglutaminase 5 is regulated by guanine–adenine nucleotides

“…These residues have been identified following a mutagenesis approach (indicated by the asterisk in Figure 2A) [20] and by X-ray studies of TGase2 complexed with GDP (indicated by the circles in Figure 2A) [13,14]. We observed that the key residues interacting with GTP in TGase2 (Ser-171, Phe-174 and Arg-476/478/578) are either identical or conservatively replaced in TGase5 (Ser-172, Trp-175 and Lys-510/512/613).…”

“…The inactive conformation of TGase2 shows the nucleophilic Cys-277 hydrogen-bonded to Tyr-516, and the same interaction may well exist between Cys-278 and His-550 in TGase5. Liu et al [13] have suggested that when Ca 2+ binds to TGase2, the β-barrel 1 domain moves away from the catalytic core breaking this hydrogen bond and opening up the transamidation catalytic pocket. A similar mechanism may explain why Ca 2+ binding in TGase5 down-regulates GTP hydrolysis.…”

“…TGase2 is multifunctional protein and a bifunctional enzyme with both protein-cross-linking and GTP-hydrolysing activities [2]. Moreover, TGase2 is also a signal-transduction GTPase protein (G h ), which is coupled with the α 1 -adrenergic receptor and mediates the activation of phospholipase C. The X-ray structure of human TGase2 bound with GDP has been solved, and the main GDP-binding residues have been identified [13].…”

A model for the reaction mechanism of the transglutaminase 3 enzyme

“…human factor XIIIa (hfXIIIa) , TGase 2 (Liu et al, 2002), TGase 3 enzymes (Ahvazi et al, 2002;2003), and a fish enzyme (fTG, equivalent to mammalian TGase 2, Noguchi et al, 2001). All consist of four domains that are similar in organization and fold ( Figure 1a, b for TGase 3): the amino terminal β-sandwich domain; the catalytic core domain which contains the conserved active site triad of Cys272, His330 and Asp353 (using TGase 3 residue numbers); the β-barrel 1 domain; and the β-barrel 2 domain at the carboxy terminus.…”