Structural Investigation of Transglutaminases

Ambrus, Attila
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The structural basis of the Ca2+ effect on transglutaminases is poorly defined. Therefore, it was our intention to initiate 43Ca NMR, surface polarity analysis combined with multiple sequence alignment studies to obtain structural information about the Ca2+ binding properties of the Factor XIIIA2, TGase 2 and TGase 3 (each of human origin). Since Ca2+ is also implicated in the folding of TGase 2, refolding studies were also performed to elucidate the nature of these effects. We have constructed a new homology model of human TGase 2 on the basis of the highest resolution (2.1Å) X-ray structure of cellular FXIII zymogen available. Our structure is more accurate than all the previously published homology models of human TGase 2. We have performed surface polarity analysis on the high resolution structure of cellular FXIII zymogen and on our homology model of TGase 2. Results are combined with that of the multiple sequence alignment of TGases and in this manner we could identify probably all the potential Ca2+ binding sites on these molecules; their numbers are more than reported earlier. From the previously published three putative sites of TGase 2, two possess negativity on the surface but the third one does not. Interestingly, in the recently determined high affinity binding pocket of FXIII, only one amino acid shows considerable negative potential on the molecular surface. The potential Ca2+ binding sites of all TGases can be predicted using the results of this analysis. In accordance with the high number of negatively charged clusters in our surface polarity analysis, 43Ca NMR provided higher (but still millimolar) average dissociation constants titrating on a wide Ca2+ concentration scale than previous studies did by equilibrium dialysis in shorter ranges. These results suggest the existence of low affinity Ca2+ binding sites on both FXIII-A and TGase 2 in addition to the well-known high affinity ones. Increasing the salt concentration or activating with thrombin, FXIII-A2 partially lost its original Ca2+ affinity in contrast to previous results, which showed no effects. The NMR data suggests different mechanisms for the proteolytic and salt activation processes of FXIII. The NMR provided structural evidence for the GTP induced conformational changes of TGase 2 molecule diminishing all of its Ca2+ binding sites. The NMR data on the Ca2+ binding properties of the first analyzed TGase 3 are presented here; TGase 3 binds Ca2+ the most tightly which weakens after proteolytic activation. Structural investigation of TGases 42 The investigated TGases have very symmetric Ca2+ binding sites and no EF-hand motifs. The refolding of the recombinant TGase 2 molecule to its catalytically active form from inclusion bodies essentially needs the presence of a helper material with higher molecular mass, but only in the initiation phase. In natural conditions it is probably a chaperon, while in vitro, it could be e.g. PEG. Ca2+ and nucleotides are ascribed as effector molecules in the early phase of the tructural reconstitution of TGase 2; the most efficient condition if they are together in the refolding buffer. Two optimal concentrations of PEG (probably two different folding pathways) and a relatively long time scale (with two activity maxima) for the evolution of the final structure of TGase 2 were identified during its refolding. An optimized refolding procedure of the non-fused recombinant TGase 2 is reported.