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. 2007 Dec;5(12):e327.
doi: 10.1371/journal.pbio.0050327.

Transglutaminase 2 undergoes a large conformational change upon activation

Daniel M Pinkas  1 , Pavel Strop, Axel T Brunger, Chaitan Khosla
Affiliations

Transglutaminase 2 undergoes a large conformational change upon activation

Daniel M Pinkas et al. PLoS Biol. 2007 Dec.

Abstract

Human transglutaminase 2 (TG2), a member of a large family of enzymes that catalyze protein crosslinking, plays an important role in the extracellular matrix biology of many tissues and is implicated in the gluten-induced pathogenesis of celiac sprue. Although vertebrate transglutaminases have been studied extensively, thus far all structurally characterized members of this family have been crystallized in conformations with inaccessible active sites. We have trapped human TG2 in complex with an inhibitor that mimics inflammatory gluten peptide substrates and have solved, at 2-A resolution, its x-ray crystal structure. The inhibitor stabilizes TG2 in an extended conformation that is dramatically different from earlier transglutaminase structures. The active site is exposed, revealing that catalysis takes place in a tunnel, bridged by two tryptophan residues that separate acyl-donor from acyl-acceptor and stabilize the tetrahedral reaction intermediates. Site-directed mutagenesis was used to investigate the acyl-acceptor side of the tunnel, yielding mutants with a marked increase in preference for hydrolysis over transamidation. By providing the ability to visualize this activated conformer, our results create a foundation for understanding the catalytic as well as the non-catalytic roles of TG2 in biology, and for dissecting the process by which the autoantibody response to TG2 is induced in celiac sprue patients.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Reactions Catalyzed by TG2
TG2 can catalyze the transamidation of Gln to a suitable amine or the deamidation of Gln to Glu.
Figure 2
Figure 2. Inactivation of TG2 by a Reactive Gluten Peptide Mimic
(A) In the pathogenesis of celiac sprue, TG2 deamidates specific Gln residues in gluten peptides to Glu. (B) The inhibitor Ac-P(DON)LPF-NH2 mimics a gluten peptide sequence that has high affinity for TG2. DON is the electrophilic amino acid 6-diazo-5-oxo norleucine. (C) The active-site Cys residue of TG2 nucleophilically attacks DON, resulting in a stable thioether adduct.
Figure 3
Figure 3. Overall Structures of GDP-Bound and Inhibitor-Bound TG2
The crystal structures are shown as ribbons, and simplified cartoons are included for clarity. (A and B) The N-terminal β-sandwich is shown in blue (N), the catalytic domain (Core) in green, and the C-terminal β-barrels (β1 and β2) in yellow and red, respectively. (A) GDP-bound TG2 [16]. (B) TG2 inhibited with the active-site inhibitor Ac-P(DON)LPF-NH2. (C) The N-terminal β-sandwich and catalytic domains of the two structures are superimposed, highlighting the conformational change. The GDP-bound structure is shown in blue and the inhibitor-bound structure in gold.
Figure 4
Figure 4. The Active Site of TG2 and Enzyme–Inhibitor Interactions
(A) Electrostatic potential surface of TG2 (red indicates negative charge; blue, positive; contoured at −15 kBT to +15 kBT) in the vicinity of the peptide inhibitor. (Carbon is indicated by cyan; nitrogen by blue; and oxygen by red.) (B) Surface representation of the active-site tryptophan bridge. W332, W241, and inhibitor are shown in green, red, and cyan, respectively. The proposed acyl-acceptor approach site is indicated. (C) Stereo representation of the active site of TG2. The backbone of TG2 is shown as ribbons. The bridge tryptophans and a T360 that resides in front of the proposed acyl-acceptor entrance are shown as sticks with semitransparent surfaces. It can be seen that the bridging tryptophan residues reside on separate loops above the catalytic Cys (sulfur is indicated by yellow). The thioether attachment of the inhibitor (cyan indicates inhibitor carbons, and gray indicates TG2 carbons) is also evident. (D) Hydrogen-bonding interactions between TG2 and the peptide are shown as dashed lines. (E) Schematic diagram of hydrophobic interactions between TG2 and the inhibitor.
Figure 5
Figure 5. σA Weighted Electron Density Maps (2Fo-Fc) Contoured at 1σ in the Vicinity of Cys-370 and Cys-371
(A) In the GDP-bound structure [16], Cys-370 and Cys-371 are reduced. (B) In the inhibitor-bound structure, the cysteine residues form a vicinal disulfide bond, causing the intervening peptide bind to take on a cis configuration. (Carbon is indicated by grey, nitrogen by blue, oxygen by red, and sulfur by yellow.)
Figure 6
Figure 6. Nondenaturing PAGE Conformation Study of Recombinant Human TG2
TG2 was incubated with effectors, then subjected to electrophoresis. Lane 1: TG2 incubated without effectors. As purified, this sample assumes two conformational states. Extended dialysis of purified TG2 leads to virtual disappearance of the faster-migrating species, suggesting that in the absence of both GTP and calcium, the protein adopts an open-like conformation. Lane 2: incubation of TG2 with GTP and MgCl2, allosteric inhibitors of the enzyme, increases the relative abundance of the conformation with higher electrophoretic mobility. Lane 3: incubation of TG2 with CaCl2, the enzyme activator, reduces the relative abundance of the higher mobility conformer. The lower overall intensity of this lane can be explained by oligomerization, as evidenced by multiple bands of lower electrophoretic mobility (unpublished data). Lane 4: active-site–inhibited TG2 incubated without effectors assumes a conformation with the lower electrophoretic mobility. Lane 5: incubation of active-site–inhibited TG2 with GTP and MgCl2 does not cause a shift in mobility as it does for uninhibited TG2.
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