Opening up about Tissue Transglutaminase: When Conformation Matters More than Enzymatic Activity

Abstract

Tissue transglutaminase (tTG), also referred to as type 2 transglutaminase or Gα_h, can bind and hydrolyze GTP, as well as function as a protein crosslinking enzyme. tTG is widely expressed and can be detected both inside cells and in the extracellular space. In contrast to many enzymes, the active and inactive conformations of tTG are markedly different. The catalytically inactive form of tTG adopts a compact "closed-state" conformation, while the catalytically active form of the protein adopts an elongated "open-state" conformation. tTG has long been appreciated as an important player in numerous diseases, including celiac disease, neuronal degenerative diseases, and cancer, and its roles in these diseases often depend as much upon its conformation as its catalytic activity. While its ability to promote these diseases has been traditionally thought to be dependent on its protein crosslinking activity, more recent findings suggest that the conformational state tTG adopts is also important for mediating its effects. In particular, we and others have shown that the closed-state of tTG is important for promoting cell growth and survival, while maintaining tTG in the open-state is cytotoxic. In this review, we examine the two unique conformations of tTG and how they contribute to distinct biological processes. We will also describe how this information can be used to generate novel therapies to treat diseases, with a special focus on cancer.

Keywords: GTP-binding; cancer; cell death; conformational changes; protein crosslinking; tissue transglutaminase; transamidation.

Fig. 1

Structure and Function of tTG. (A) tTG catalyzes several different reactions: the most important of these is transamidation, or crosslinking of glutamine and lysine residues, which is shown schematically. (B) Crystal structures of tTG reveal two different conformational states. On the left, tTG is shown in the closed-state conformation (PDB code 231KV3). As the cartoon shows, the crosslinking substrate binding site (open wedge in blue crosslinking domain) is occluded. Addition of Ca2+ shifts the protein to the openstate conformation, shown on the right (PDB code 2Q3Z), which reveals this substrate binding site. An excess of GDP or GTP returns the protein to the closed-state. For either crystal structure, the crosslinking catalysis domain is indicated with an orange arrow, while the area where nucleotide would bind is circled and indicated with a blue arrow.

Fig. 2

The nucleotide binding pocket of tTG. (A) Key residues in the nucleotide binding site of tTG (PDB code 1KV3). Arg 580 hydrogen bonds to several parts of the bound GDP nucleotide, while Phe 174 makes a π-stacking interaction with the guanine ring system. Ser 171 does not interact directly with the bound GDP, but forms hydrogen bonds to Phe 174 which may be important to stabilize its π-stacking interaction with the nucleotide. (B) Alignment of the nucleotide binding residues from the crosslinking domain of tTG across different species, and between different TG family members. T162, Q163, Q164, F166, Q169, and K173 are all highly conserved among tTG variants, but poorly conserved among TG family members. Figure panel adapted from ^[71].

Fig. 3

Important bonds which stabilize the conformations of tTG. (A) One of three calcium binding sites identified for TG3. In TG3 (cyan, PDB code 1NUD), calcium is bound by Asp 301 and Asn 305. Upon binding these residues, it pulls on Ser 323 to shift the nearby loop and provide access to the substrate binding site. In blue, tTG (PDB code 1KV3) is overlayed. tTG has Asp and Asn residues very close to those in TG3. (B) Key hydrogen bonds which stabilize the closed-state of tTG. Tyr 516 hydrogen bonds to Cys 277 (left), while on the other face of the protein, Asp 434 and Asn 681 form one hydrogen bond, and Trp 254 and Lys 677 form another. Of these latter four bonds, only Trp 254 makes the bond via backbone atoms.

Fig. 4

The structure of tTG does not change upon binding GDP or GTP. (A) Overlayed crystal structures of GDP-bound tTG (PDB code 1KV3, blue, yellow, and green) and GTP-bound tTG (PDB code 4PYG, colored by temperature factor, redder shades suggest more uncertainty in residue position). Gray spheres show the position of an open-state bound peptide sequence overlayed from open-state tTG (PDB code 2Q3Z). There is almost no change in the structure of tTG when GTP is bound rather than GDP. The only visibly changed loop (black arrow) also has very high temperature factors in the GTP-bound structure. (B) Overlayed crystal structures of GDP-bound tTG [same as in (A)] and ATP-bound tTG (PDB code 3LY6, colored by temperature factor). The same trends are present as in (A), save that the minimally varied loop (black arrow) has lower temperature in the ATP-bound structure, and perfectly overlays with the GDPbound structure.

Fig. 5

tTG behaves in some ways like a classical G-protein. (A) A peptide matching the orange sequence at the C-terminus of tTG is able to block binding between tTG and PLC, which it binds following activation by the α1-adrenergic receptor. Peptides matching regions shown in green do not block the interaction. The sequence in orange is inaccessible to solvent, and presumably binding partners, while tTG is in the closed-state. (B) Proposed mechanism by which tTG signals. For a classical G-protein, such as Gαq, the protein normally exists in a GDP (red pentagon) bound state. Receptors bind and stimulate GDP dissociation. The protein then rapidly binds GTP (green hexagon), which is in excess relative to GDP in cells. This causes a structural change (which is not seen in tTG crystal structures), and allows binding and activation of downstream signaling partners (“Sig. Par.”, shown in purple). We suggest a possible alternate mechanism in which the nucleotide bound forms of tTG are all structurally similar, and have effectively identical signaling ability. Receptors which bind to the open-state of tTG cause the dissociation of both GDP and GTP, and reveal the inner-face of the C-terminus of the protein to solvent, which then specifically binds downstream signaling partners.

Fig. 6

Regions of tTG associated with binding partners. Fibronectin binds to tTG via it’s N-terminal region (colored red), while the blue colored region is a BH3 domain, and binds proteins such as Bax. In orange is the region also shown in Fig. 5, from which a peptidomimetic can be made that binds PLC.

Fig. 7

Inhibitors of tTG stabilize the protein in the open-state. Ac-P-DON-L-P-F-NH2, Z-Don, Zed754, and a peptidomimetic similar to Zed754 (right side) were all demonstrated to stabilize the open-state of tTG via X-Ray crystallography ^{[35,107,108].} VA4, VA5, NC9 ^[109], MDC ^[67], and TTGM 5826 ^[110] were each demonstrated to stabilize the open-state of tTG through assorted biochemical assays. Notably, very large amounts of MDC were needed to stabilize the tTG open-state. CP4d has been reported to modestly stabilize the closed-state of tTG ^[73] or the open-state of tTG ^[109] depending upon the experimental system, and its true effects on tTG structure are at best inconclusive currently.

Fig. 8

X-Ray crystal structures of close homologues to tTG. X-Ray crystal structures have been solved of (A) inactive TG3 (PDB code 1NUG), (B) active TG3 (PDB code 1NUD), (C) inactive Factor XIIIA (PDB code 1FIE), and (D) active Factor XIIIA (PDB code 1EVU). Each protein has four domains, substantially similar to tTG, but neither has the radical conformational shift of tTG upon binding calcium to adopt an activated state.