Opposite effects of Ca2+ and GTP binding on tissue transglutaminase tertiary structure

Tissue transglutaminase (tTG) belongs to a class of enzymes that catalyze a cross-linking reaction between proteins or peptides, The protein activity is known to be finely tuned by Ca2+ and GTP binding. In this study we repor t the effects of these ligands on the enzyme structure, as revealed by circ ular dichroism, and steady-state and dynamic fluorescence measurements, We have found that calcium and GTP induced opposite conformational changes at the level of the protein tertiary structure, In particular the metal ions w ere responsible for a small widening of the protein molecule, as indicated by anisotropy decay measurements and by the binding of a hydrophobic probe such as 1-anilino-8-naphthalenesulfonic acid (ANS), Unlike Ca2+, the nucleo tide binding increased the protein dynamics, reducing its rotational correl ation lifetime from 32 to 25 ns, preventing also the binding of ANS into th e protein matrix. Unfolding of tTG by guanidinium hydrochloride yielded a t hree-state denaturation mechanism, involving an intermediate species with t he characteristics of the so-called "molten globule" state. The effect of G TP binding (but not that of Ca2+) had an important consequence on the stabi lity of tissue transglutaminase, increasing the free energy change from the native to the intermediate species by at least approximate to 0.7 kcal/mol . Also a greater stability of tTG to high hydrostatic pressure was obtained in presence of GTP, These findings suggest that the molecular mechanism by which tTG activity is inhibited by GTP is essentially due to a protein con formational change which, decreasing the accessibility of the protein matri x to the solvent, renders more difficult the exposure of the active site.