Thermodynamics of binding of regulatory ligands to tissue transglutaminase

Carlo M. Bergamini, Alessia Dondi, Vincenzo Lanzara, Monica Squerzanti, Carlo Cervellati, Katy Montin, Carlo Mischiati, Gianluca Tasco, Russell Collighan, Martin Griffin, Rita Casadio

Research output: Contribution to journalArticle

Abstract

The transamidating activity of tissue transglutaminase is regulated by the ligands calcium and GTP, via conformational changes which facilitate or interfere with interaction with the peptidyl-glutamine substrate. We have analysed binding of these ligands by calorimetric and computational approaches. In the case of GTP we have detected a single high affinity site (K (D) approximately 1 muM), with moderate thermal effects suggestive that binding GTP involves replacement of GDP, normally bound to the protein. On line with this possibility no significant binding was observed during titration with GDP and computational studies support this view. Titration with calcium at a high cation molar excess yielded a complex binding isotherm with a number of "apparent binding sites" in large excess over those detectable by equilibrium dialysis (6 sites). This binding pattern is ascribed to occurrence of additional thermal contributions, beyond those of binding, due to the occurrence of conformational changes and to catalysis itself (with protein self-crosslinking). In contrast only one site for binding calcium with high affinity (K (D) approximately 0.15 muM) is observed with samples of enzyme inactivated by alkylation at the active site (to prevent enzyme crosslinkage and thermal effects of catalysis). These results indicate an intrinsic ability of tissue transglutaminase to bind calcium with high affinity and the necessity of careful reassessment of the enzyme regulatory pattern in relation to the concentrations of ligands in living cells, taking also in account effects of ligands on protein subcellular compartimentation.
LanguageEnglish
Pages297-304
Number of pages8
JournalAmino Acids
Volume39
Issue number1
DOIs
Publication statusPublished - 22 Dec 2009

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Thermodynamics
Guanosine Triphosphate
Ligands
Calcium
Hot Temperature
Catalysis
Titration
Thermal effects
Enzymes
Binding Sites
Proteins
Dialysis
Alkylation
Glutamine
Crosslinking
Isotherms
Cations
Catalytic Domain
Cells
transglutaminase 2

Bibliographical note

Published online before printing

Keywords

  • transglutaminase
  • binding of ligands
  • calcium
  • GTP
  • isothermal titration calorimetry

Cite this

Bergamini, C. M., Dondi, A., Lanzara, V., Squerzanti, M., Cervellati, C., Montin, K., ... Casadio, R. (2009). Thermodynamics of binding of regulatory ligands to tissue transglutaminase. Amino Acids, 39(1), 297-304. https://doi.org/10.1007/s00726-009-0442-5
Bergamini, Carlo M. ; Dondi, Alessia ; Lanzara, Vincenzo ; Squerzanti, Monica ; Cervellati, Carlo ; Montin, Katy ; Mischiati, Carlo ; Tasco, Gianluca ; Collighan, Russell ; Griffin, Martin ; Casadio, Rita. / Thermodynamics of binding of regulatory ligands to tissue transglutaminase. In: Amino Acids. 2009 ; Vol. 39, No. 1. pp. 297-304.
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Bergamini, CM, Dondi, A, Lanzara, V, Squerzanti, M, Cervellati, C, Montin, K, Mischiati, C, Tasco, G, Collighan, R, Griffin, M & Casadio, R 2009, 'Thermodynamics of binding of regulatory ligands to tissue transglutaminase' Amino Acids, vol. 39, no. 1, pp. 297-304. https://doi.org/10.1007/s00726-009-0442-5

Thermodynamics of binding of regulatory ligands to tissue transglutaminase. / Bergamini, Carlo M.; Dondi, Alessia; Lanzara, Vincenzo; Squerzanti, Monica; Cervellati, Carlo; Montin, Katy; Mischiati, Carlo; Tasco, Gianluca; Collighan, Russell; Griffin, Martin; Casadio, Rita.

In: Amino Acids, Vol. 39, No. 1, 22.12.2009, p. 297-304.

Research output: Contribution to journalArticle

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T1 - Thermodynamics of binding of regulatory ligands to tissue transglutaminase

AU - Bergamini, Carlo M.

AU - Dondi, Alessia

AU - Lanzara, Vincenzo

AU - Squerzanti, Monica

AU - Cervellati, Carlo

AU - Montin, Katy

AU - Mischiati, Carlo

AU - Tasco, Gianluca

AU - Collighan, Russell

AU - Griffin, Martin

AU - Casadio, Rita

N1 - Published online before printing

PY - 2009/12/22

Y1 - 2009/12/22

N2 - The transamidating activity of tissue transglutaminase is regulated by the ligands calcium and GTP, via conformational changes which facilitate or interfere with interaction with the peptidyl-glutamine substrate. We have analysed binding of these ligands by calorimetric and computational approaches. In the case of GTP we have detected a single high affinity site (K (D) approximately 1 muM), with moderate thermal effects suggestive that binding GTP involves replacement of GDP, normally bound to the protein. On line with this possibility no significant binding was observed during titration with GDP and computational studies support this view. Titration with calcium at a high cation molar excess yielded a complex binding isotherm with a number of "apparent binding sites" in large excess over those detectable by equilibrium dialysis (6 sites). This binding pattern is ascribed to occurrence of additional thermal contributions, beyond those of binding, due to the occurrence of conformational changes and to catalysis itself (with protein self-crosslinking). In contrast only one site for binding calcium with high affinity (K (D) approximately 0.15 muM) is observed with samples of enzyme inactivated by alkylation at the active site (to prevent enzyme crosslinkage and thermal effects of catalysis). These results indicate an intrinsic ability of tissue transglutaminase to bind calcium with high affinity and the necessity of careful reassessment of the enzyme regulatory pattern in relation to the concentrations of ligands in living cells, taking also in account effects of ligands on protein subcellular compartimentation.

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KW - transglutaminase

KW - binding of ligands

KW - calcium

KW - GTP

KW - isothermal titration calorimetry

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Bergamini CM, Dondi A, Lanzara V, Squerzanti M, Cervellati C, Montin K et al. Thermodynamics of binding of regulatory ligands to tissue transglutaminase. Amino Acids. 2009 Dec 22;39(1):297-304. https://doi.org/10.1007/s00726-009-0442-5