Mechanism of allosteric regulation of transglutaminase 2 by GTP

Abstract

Allosteric regulation is a fundamental mechanism of biological control. Here, we investigated the allosteric mechanism by which GTP inhibits cross-linking activity of transglutaminase 2 (TG2), a multifunctional protein, with postulated roles in receptor signaling, extracellular matrix assembly, and apoptosis. Our findings indicate that at least two components are involved in functionally coupling the allosteric site and active center of TG2, namely (i) GTP binding to mask a conformationally destabilizing switch residue, Arg-579, and to facilitate interdomain interactions that promote adoption of a compact, catalytically inactive conformation and (ii) stabilization of the inactive conformation by an uncommon H bond between a cysteine (Cys-277, an active center residue) and a tyrosine (Tyr-516, a residue located on a loop of the ␤-barrel 1 domain that harbors the GTP-binding site). Although not essential for GTP-mediated inhibition of cross-linking, this H bond enhances the rate of formation of the inactive conformer.protein conformation ͉ GTP inhibition ͉ transamidase activity A llosteric regulation of enzymes by conformational change is an important means of biological control, involving residues that functionally couple ligand binding at the allosteric site to modification of the catalytic site. Transglutaminase type 2 (TG2), also known as tissue TG or G h (high molecular weight GTP-binding protein), is a multifunctional protein that is allosterically regulated by calcium and GTP (1). TG2 catalyzes calcium-dependent transamidation reactions, resulting in posttranslational amine modification of proteins or cross-linking of interchain glutamine and lysine residues to form N (␥-glutamyl)lysine isopeptide bonds, which confer rigidity and protease resistance on protein complexes (2). TG2 is also a GTPase (3) and mediates intracellular signaling by various G protein-coupled receptors (4-6).GDP-bound human TG2 (7) is comprised of four domains: an N-terminal ␤-sandwich, a core domain in which the transamidase active site catalytic triad (Cys-277, His-335, and Asp-358) and transition-state stabilizing residue (Trp-241) (8) are buried and inaccessible to substrate, and two ␤-barrels. Nucleotide binds mainly to residues from the first and last strands (amino acids 476-482 and 580-583) of ␤-barrel 1 and to two core domain residues (Lys-173 and Phe-174) that protrude on a loop to meet ␤-barrel 1 (7, 9, 10). This is postulated to stabilize two ␤-barrel 1 loops that block access to the catalytic site (7). One of these loops protrudes into the core domain localizing Tyr-516 within hydrogen-bonding distance of Cys-277 (7). This is postulated to prevent Cys-277 interaction with the substrate (7,11,12). Calcium-activated TG2 has unique conformational epitopes (13) and is less compact (14-16) and less resistant to protease digestion (1, 10, 14) than GTP-bound TG2. Allosteric mechanisms governing the conformational switch between transamidase and GTPase functions have yet to be elucidated.Mutation of Arg-579 in rat TG2 (Arg-580 in ...

Structural and Phylogenetic Analyses of the GP42 Transglutaminase from Phytophthora sojae Reveal an Evolutionary Relationship between Oomycetes and Marine Vibrio Bacteria

“…Intramolecular disulfide bridges between Cys-256 and Cys-271 and between Cys-262 and Cys-282 stabilize the body domain, whereas a third bridge links Cys-305 with Cys-527 in the tail domain. The presence of disulfide bridges is quite unusual because they can interfere with conformational changes upon activation in mammalian TGases (1,20). The seventh cysteine, Cys-290, is the active-site nucleophile for TGase activity.…”

Opposing Effects of Two Tissue Transglutaminase Protein Isoforms in Neuroblastoma Cell Differentiation

“…This finding has important implications for studies of the role of TG2 expression in human cancer and for TG2 as an anticancer treatment target. Although the transamidase catalytic triad and transition statestabilizing residue are present in both TG2-L and TG2-S proteins, TG2-S lacks the Arg-580 residue that is critical for GTP binding and inhibition of transamidase activity (13)(14)(15). Our current data show that TG2-L with an Ala mutation of the Arg-580 residue induces, whereas wild-type TG2-L blocks, neuroblastoma cell differentiation and that inhibition of transamidase activity with a chemical inhibitor blocks the cell differentiation induced by both TG2-S and the R580A mutant TG2-L. Taken together, our data indicate that TG2-L blocks neuroblastoma cell differentiation through Arg-580-mediated GTP binding and inhibition of transamidation and that TG2-S induces cell differentiation through its transamidase activity not being inhibited due to the lack of Arg-580-mediated GTP binding.…”

“…Residue-Because TG2-L contains, but TG2-S lacks, the GTP-binding Arg-580 residue that is required for maintaining the intracellular transamidase activity of TG2 latent (13,14), we tested whether the Arg-580 residue is critical for the opposite effects of TG2-L and TG2-S on neuroblastoma cell differentiation. Neuroblastoma BE(2)-C cells were transfected with constructs encoding empty vector, TG2-S, TG2-L, transamidase-deficient mutant TG2-L (C277S), GTP-binding-deficient mutant TG2-L (R580A), or mocktransfected.…”

“…The biological function of TG2 is, therefore, dependent on whether transamidation or GTP binding is switched on, in a mutually exclusive way. Mutation of the critical GTP-binding residue Arg-580 to Ala increases the sensitivity of TG2 to calcium activation and results in disinhibition of intracellular transamidase activity (14,15).Two isoforms of TG2 mRNA and protein have been characterized (16,17 2 The abbreviations used are: TG2, tissue transglutaminase; TG2-L, TG2 long isoform; TG2-S, TG2 short isoform; TG2-T, both TG2 long and short isoforms; atRA, all-trans-retinoic acid; siRNA, small interfering RNA; VIP, vasoactive intestinal peptide; RT, reverse transcription. …”

“…The biological function of TG2 is, therefore, dependent on whether transamidation or GTP binding is switched on, in a mutually exclusive way. Mutation of the critical GTP-binding residue Arg-580 to Ala increases the sensitivity of TG2 to calcium activation and results in disinhibition of intracellular transamidase activity (14,15).…”