Ambrus A, Bányai I, Weiss M S, Hilgenfeld R, Keresztessy Z, Muszbek L, Fésüs L
University of Debrecen, Department of Biochemistry and Molecular Biology, Hungary.
J Biomol Struct Dyn. 2001 Aug;19(1):59-74. doi: 10.1080/07391102.2001.10506720.
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.
转谷氨酰胺酶（TGases）在依赖Ca2+的反应中，使多肽的谷氨酰胺和赖氨酸侧链之间形成交联。Ca2+效应的结构基础目前还不清楚。利用43Ca核磁共振、表面极性分析结合多序列比对以及构建人组织转谷氨酰胺酶（tTGase）的新同源模型，来获取有关凝血因子XIII - A2、tTGase和TGase 3（均源自人类）的Ca2+结合特性的结构信息。与之前在较短Ca2+浓度范围内进行的平衡透析研究相比，43Ca核磁共振在较宽的Ca2+浓度范围内滴定得到了更高的平均解离常数。这些结果表明，除了高亲和力的Ca2+结合位点外，凝血因子XIII - A和tTGase上还存在低亲和力的Ca2+结合位点，这与我们通过表面极性分析鉴定出大量带负电荷的簇的结果一致。增加盐浓度或用凝血酶激活后，凝血因子XIII - A2部分失去其原有的Ca2+亲和力；核磁共振数据表明这两个激活过程的机制不同。核磁共振提供了GTP诱导tTGase分子构象变化从而减少其所有Ca2+结合位点的结构证据。本文展示了TGase 3的Ca2+结合特性的核磁共振数据；它与Ca2+结合最紧密，在蛋白水解激活后这种结合会减弱。所研究的转谷氨酰胺酶似乎具有非常对称的Ca2+结合位点，且没有EF手基序。
