Crystal structure of transglutaminase 2 with GTP complex and amino acid sequence evidence of evolution of GTP binding site

Abstract

Transglutaminase2 (TG2) is a multi-functional protein involved in various cellular processes, including apoptosis, differentiation, wound healing, and angiogenesis. The malfunction of TG2 causes many human disease including inflammatory disease, celiac disease, neurodegenerative diseases, tissue fibrosis, and cancers. Protein cross-linking activity, which is representative of TG2, is activated by calcium ions and suppressed by GTP. Here, we elucidated the structure of TG2 in complex with its endogenous inhibitor, GTP. Our structure showed why GTP is the optimal nucleotide for interacting with and inhibiting TG2. In addition, sequence comparison provided information describing the evolutionary scenario of GTP usage for controlling the activity of TG2.

Figure 1. Crystal structure of TG2 in complex with GTP.

A. Domain boundary of TG2. Domains are gold and the amino acid residue numbers corresponding to each domain are indicated. B. Ribbon diagram of the structure of TG2 in complex with GTP. Three molecules in the asymmetric unit are shown (Chain A, Chain B and Chain C). C. Symmetric dimer formed by Chain B and Chain C is shown. D. Superposition of the structure of the TG2/GTP complex with known structures. Structurally different regions are indicated by red-dot circles.

Figure 2. Stoichiometry of TG2 in solution.

A. Gel-filtration chromatogram of TG2. A profile obtained from TG2 in buffer containing 20 mM Tris-HCl at pH 8.0 and 150 mM NaCl is shown. Protein size markers are indicated. B. Multi-angle light scattering (MALS) measurement of TG2 in complex with GTP. The x-axis and y-axis indicate the elution volume and molecular mass, respectively. C. Monomeric structure of TG2 in complex with GTP. The active site is shown by a red circle. The GTP binding site is magnified for better visualization.

Figure 3. Environment of the GTP binding site in TG2.

An omit density map contoured at the 1-σ level around GTP. The residues involved in the GTP interaction are indicated. H-bonds are shown as black-dashed lines. The whole GTP binding site is shown in the left panel. Sequence alignment between TG2, TG3, and Factor XIII for sequence comparison of the GTP binding site is shown in the right panel.

Figure 4. Comparison of GTP binding sites.

A. Environment of the GTP binding site of the TG2/GTP complex. Critical residues for the formation of the GTP binding site are indicated. B. Environment of the GTP binding site of the TG2/GDP complex. C. Environment of the GTP binding site of the TG2/ATP complex. D. Superposition of the GTP binding site of the TG2/GTP complex with the TG2/ATP complex and the TG2/GDP complex.

Figure 5. The condition of cysteines at the redox sensor and active site.

A. Cys230 and Cys370 at the redox sensor in TG2 formed a disulfide bond, even under highly reduced conditions. B. Cys277 and Cys336 at the active site in TG2 did not form a disulfide bond.

Figure 6. Sequence alignment between inter-isotype TG family members.

Amino acid residues conserved at the GTP binding site are shown in red. Amino acid residues perfectly and partially conserved across the isotype are highlighted with green or blue, respectively.

Figure 7. Sequence alignment between inter-species TG2 family members.

Conserved GTP-binding amino acid residues are highlighted in red. Amino acid residues perfectly or partially conserved across the species are highlighted ingreen orblue, respectively.