Fig. 1.

Mutations of residues involved in GTP-binding or the Cys-277–Tyr-516 H bond affect TG2 conformation. (A) Model of GDP-bound human TG2 showing the transamidase active center (Cys-277, His-335, and Asp-358) and other key residues [(Protein Data Bank ID code 1KV3) (7); core domain backbone, pale pink; β-barrel 1 domain, green]. Carbon atoms are gray, except those of active site residues (cyan), and GDP (orange). Other atoms include oxygen, red; nitrogen, deep blue; sulfur, yellow; phosphorous, bright pink. Arg-580 in human TG2 is equivalent to Arg-579 in rat TG2. (B and C) Samples (3 μg) of WT and mutant TG2 were incubated (3 h at 25°C) in buffer (50 mM Tris·HCl, pH 7.5/50 mM NaCl/0.5 mM EDTA/1.0 mM MgCl₂/5 mM DTT/GTP as indicated). nPAGE was performed with 0.5 mM MgCl₂, and the indicated GTP concentrations were included in gel solutions and running buffer. Data are representative of two to eight experiments. (D) Sedimentation coefficient distributions, c_M(s), for 13 μM WT (solid line), Y516F (dashed line), and R579A (dotted line) in 10 mM Tris·HCl pH 7.5/100 mM NaCl/0.5 mM EDTA/1 mM DTT/500 μM GTP at 4°C. Molar mass was fixed at 78.0 kDa for 2.5–3.8 S species. Data are representative of three to four experiments. (E) Disulfide trapping of Cys-277 with a cysteine substituted for Tyr-516. Samples (3 μg) of WT and mutant TG2 were oxidized (2 h at 25°C) in buffer (50 mM Tris·HCl, pH 7.5/50 mM NaCl/200:800 μM Cu²⁺:phenanthroline/50 μM GTP), then incubated (1 h at 25°C) in 0.5 mM MgCl₂, without or with 20 mM DTT, as indicated. nPAGE was performed as in B. Data are representative of two experiments.