Calcium binding of transglutaminases: A Ca-43 NMR study combined with surface polarity analysis

Transglutaminases (TGases) form cross-links between glutamine and lysine si de-chains of polypeptides in a Ca2+-dependent reaction. The structural basi s of the Ca2+-effeet is poorly defined. Ca-43 NMR. surface polarity analysi s combined with multiple sequence alignment and the construction of a new h omology model of human tissue transglutaminase (tTGase) were used to obtain structural information about Ca2+ binding properties of factor XIII-A(2), tTGase and TGase 3 (each of human origin). 43Ca NMR provided higher average dissociation constants titrating on a wide Ca2+-concentration scale than p revious studies with equilibrium dialysis performed in shorter ranges. Thes e results suggest the existence of low affinity Ca2+ binding sites on both FXIII-A and tTGase in addition to high affinity ones in accordance with our surface polarity analysis identifying high numbers of negatively charged c lusters. Upon increasing the salt concentration or activating with thrombin , FXIII-A(2) partially lost its original Ca2+ affinity; the NMR data sugges ted different mechanisms for the two activation processes. The NMR provided structural evidence of GTP-induced conformational changes on the tTGase mo lecule diminishing all of its Ca2+ binding sites. NMR data on the Ca2+ bind ing properties of the TGase 3 are presented here it binds Ca2+ the most tig htly, which is weakened after its proteolytic activation. The investigated TGases seem to have very symmetric Ca2+ binding sites and no EF-hand motifs .