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.
Ambrus
A
A
University of Debrecen, Department of Biochemistry and Molecular Biology, Hungary.
Bányai
I
I
Weiss
M S
MS
Hilgenfeld
R
R
Keresztessy
Z
Z
Muszbek
L
L
Fésüs
L
L
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005176:Factor XIII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D011503:Transglutaminases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D011994:Recombinant Proteins; D005176:Factor XIII; C498712:TGM3 protein, human; D011503:Transglutaminases; D002118:Calcium