Calcium binding of transglutaminases: a 43Ca NMR study combined with surface polarity analysis.

Transglutaminases (TGases) form cross-links between glutamine and lysine side-chains of polypeptides in a Ca2+-dependent reaction. The structural basis of the Ca2+-effect is poorly defined. 43Ca NMR, surface polarity analysis combined with multiple sequence alignment and the construction of a new homology model of human tissue transglutaminase (tTGase) were used to obtain structural information about Ca2+ binding properties of factor XIII-A2, tTGase and TGase 3 (each of human origin). 43Ca NMR provided higher average dissociation constants titrating on a wide Ca2+-concentration scale than previous studies with equilibrium dialysis performed in shorter ranges. These 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 clusters. Upon increasing the salt concentration or activating with thrombin, FXIII-A2 partially lost its original Ca2+ affinity; the NMR data suggested different mechanisms for the two activation processes. The NMR provided structural evidence of GTP-induced conformational changes on the tTGase molecule diminishing all of its Ca2+ binding sites. NMR data on the Ca2+ binding properties of the TGase 3 are presented here; it binds Ca2+ the most tightly, which is weakened after its proteolytic activation. The investigated TGases seem to have very symmetric Ca2+ binding sites and no EF-hand motifs.

Synthesis, pH-dependent structural characterization, and solution behavior of aqueous aluminum and gallium citrate complexes.

Reactions of Al(III) and Ga(III) with citric acid in aqueous solutions, yielded the complexes (NH(4))(5)[M(C(6)H(4)O(7))(2)].2H(2)O (M(III) = Al (1), Ga (2)) at alkaline pH, and the complexes (Cat)(4)[M(C(6)H(5)O(7))(C(6)H(4)O(7))].nH(2)O (M(III) = Al (3), Ga (4), Cat. = NH(4)(+), n = 3; M(III) = Al (5), Ga (6), Cat. = K(+), n = 4) at acidic pH. All compounds were characterized by spectroscopic (FT-IR, (1)H, (13)C, and (27)Al NMR, (13)C-MAS NMR) and X-ray techniques. Complex 1 crystallizes in space group P1, with a = 9.638(5) A, b = 9.715(5) A, c = 7.237(4) A, alpha = 90.96(1) degrees, beta = 105.72(1) degrees, gamma = 119.74(1) degrees, V = 557.1(3) A(3), and Z = 1. Complex 2 crystallizes in space group P1, with a = 9.659(6) A, b = 9.762(7) A, c = 7.258(5) A, alpha = 90.95(2) degrees, beta = 105.86(2) degrees, gamma = 119.28(1) degrees, V = 564.9(7) A(3), and Z = 1. Complex 3 crystallizes in space group I2/a, with a = 19.347(3) A, b = 9.857(1) A, c = 23.412(4) A, beta = 100.549(5) degrees, V = 4389(1) A(3), and Z = 8. Complex 4 crystallizes in space group I2/a, with a = 19.275(1) A, b = 9.9697(6) A, c = 23.476(1) A, beta = 100.694(2) degrees, V = 4432.8(5) A(3), and Z = 8. Complex 5 crystallizes in space group P1, with a = 7.316(1) A, b = 9.454(2) A, c = 9.569(2) A, alpha = 64.218(4) degrees, beta = 69.872(3) degrees, gamma = 69.985(4) degrees, V = 544.9(2) A(3), and Z = 1. Complex 6 crystallizes in space group P1, with a = 7.3242(2) A, b = 9.4363(5) A, c = 9.6435(5) A, alpha = 63.751(2) degrees, beta = 70.091(2) degrees, gamma = 69.941(2) degrees, V = 547.22(4) A(3), and Z = 1. The crystal structures of 1-6 reveal mononuclear octahedral complexes of Al(III) (or Ga(III)) bound to two citrates. Solution NMR, on both 4- and 5- species, reveals rapid intramolecular exchange of the bound and unbound terminal carboxylates. Upon dissolution in water, the complexes, through a complicated reaction cascade, transform to oligonuclear 1:1 species that, in agreement with previous studies, represent the thermodynamically stable state in solution. The data provide, for the first time, structural details of low MW, mononuclear complexes of Al(III) (or Ga(III)) with citrate that are dictated, among other factors, by pH. The properties of 1-6 may provide clues relevant to their biological association with humans.