Transglutaminase 2 (TG2) is a ubiquitously expressed member of an enzyme family catalyzing Ca(2+)-dependent transamidation of proteins. It is a multifunctional protein having several well-defined enzymatic (GTP binding and hydrolysis, protein disulfide isomerase, and protein kinase activities) and non-enzymatic (multiple interactions in protein scaffolds) functions. Unlike its enzymatic interactions, the significance of TG2's non-enzymatic regulation of its activities has recently gained importance. In this review, we summarize all the partners that directly interact with TG2 in a non-enzymatic manner and analyze how these interactions could modulate the crosslinking activity and cellular functions of TG2 in different cell compartments. We have found that TG2 mostly acts as a scaffold to bridge various proteins, leading to different functional outcomes. We have also studied how specific structural features, such as intrinsically disordered regions and embedded short linear motifs contribute to multifunctionality of TG2. Conformational diversity of intrinsically disordered regions enables them to interact with multiple partners, which can result in different biological outcomes. Indeed, ID regions in TG2 were identified in functionally relevant locations, indicating that they could facilitate conformational transitions towards the catalytically competent form. We reason that these structural features contribute to modulating the physiological and pathological functions of TG2 and could provide a new direction for detecting unique regulatory partners. Additionally, we have assembled all known anti-TG2 antibodies and have discussed their significance as a toolbox for identifying and confirming novel TG2 regulatory functions.

The Escherichia coli chemoreceptors for serine (Tsr) and aspartate (Tar) and several bacterial class III adenylyl cyclases (ACs) share a common molecular architecture; that is, a membrane anchor that is linked via a cytoplasmic HAMP domain to a C-terminal signal output unit. Functionality of both proteins requires homodimerization. The chemotaxis receptors are well characterized, whereas the typical hexahelical membrane anchor (6TM) of class III ACs, suggested to operate as a channel or transporter, has no known function beyond a membrane anchor. We joined the intramolecular networks of Tsr or Tar and two bacterial ACs, Rv3645 from Mycobacterium tuberculosis and CyaG from Arthrospira platensis, across their signal transmission sites, connecting the chemotaxis receptors via different HAMP domains to the catalytic AC domains. AC activity in the chimeras was inhibited by micromolar concentrations of L-serine or L-aspartate in vitro and in vivo. Single point mutations known to abolish ligand binding in Tar (R69E or T154I) or Tsr (R69E or T156K) abrogated AC regulation. Co-expression of mutant pairs, which functionally complement each other, restored regulation in vitro and in vivo. Taken together, these studies demonstrate chemotaxis receptor-mediated regulation of chimeric bacterial ACs and connect chemical sensing and AC regulation.The canonical chemotaxis receptors Tsr for serine and Tar for aspartate of Escherichia coli have been studied extensively as model signaling systems that regulate bacterial swimming behavior (1-5). Ligand binding to the periplasmic domain of the receptor initiates a biochemically defined cascade of events that finally results in reversal of the flagellar beat (4, 6). The chemotaxis receptors are tripartite proteins with a periplasmic receptor anchored in the cytoplasmic membrane by two transmembrane spans followed by a HAMP domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins of chemotaxis, and protein phosphatases (7)) which adjoins the second transmembrane helix and serves as a signal converter followed by an output domain as C terminus (3). Homodimerization of Tsr or Tar chemoreceptors is required for function, and the ligands bind in the periplasmic interface between the subunits (8 -11). The ubiquitous HAMP domains are signal converters of 55-60 amino acids with a homodimeric fourhelical parallel coiled-coil conformation (12). In the chemotaxis receptors the HAMP domain operates as a C-terminal histidine kinase control unit that has no known intrinsic enzymatic activity. As this molecular architecture is predominant in the 30 known sensor kinases of E. coli, a variety of chimeric sensor proteins has been generated successfully in the past that consist of modules derived from different E. coli sensors (see examples in Refs. 13-15).A similar tripartite domain architecture as in chemotaxis receptors exists in several class III AC 2 isoforms in eubacteria. Class III AC isoforms outnumber all others, are prevalent in bacteria, and include all eukaryotic ACs (16 -17). ...

TG2 (transglutaminase 2) is a calcium-dependent protein cross-linking enzyme which is involved in a variety of cellular processes. The threshold level of calcium needed for endogenous and recombinant TG2 activity has been controversial, the former being more sensitive to calcium than the latter. In the present study we address this question by identifying a single amino acid change from conserved valine to glycine at position 224 in recombinant TG2 compared with the endogenous sequence present in the available genomic databases. Substituting a valine residue for Gly224 in the recombinant TG2 increased its calcium-binding affinity and transamidation activity 10-fold and isopeptidase activity severalfold, explaining the inactivity of widely used recombinant TG2 at physiological calcium concentrations. ITC (isothermal titration calorimetry) measurements showed 7-fold higher calcium-binding affinities for TG2 valine residues which could be activated inside cells. The two forms had comparable substrate- and GTP-binding affinities and also bound fibronectin similarly, but coeliac antibodies had a higher affinity for TG2 valine residues. Structural analysis indicated a higher stability for TG2 valine residues and a decrease in flexibility of the calcium-binding loop resulting in improved metal-binding affinity. The results of the present study suggest that Val224 increases TG2 activity by modulating its calcium-binding affinity enabling transamidation reactions inside cells.