Publikationsrepositorium - Helmholtz-Zentrum Dresden-Rossendorf

Tissue transglutaminase (TGase 2) is a multifunctional enzyme that catalyzes the formation of covalent crosslinks between protein-bound glutamine and primary amine substrates (transamidase activity) but also functions as a GTP-binding protein (Gh protein). These two functions are associated with an “open” and a “closed” conformation, respectively, being tightly regulated by Ca²⁺ and GDP/GTP levels. In recent years, several assays for the transamidase activity have been published [1], leaving the GTP-binding function virtually untouched.
Here, we report a novel assay to quantify the GTP-binding activity of human TGase 2, which follows the increase in fluorescence anisotropy of an optimized fluorescein-labeled GTP probe upon binding to the protein. Validity of the assay was ensured by means of the (endogenous) ligands GTP, GTPγS and GDP showing inhibitory potencies (IC₅₀) for displacement of the new probe comparable to reported values [2]. ATP, commonly not considered as being an inhibitor of TGase 2, was found to diminish binding of the probe to TGase 2 at unphysiologically high concentrations. The binding assay was then applied for the characterization of a small library of GDP and GTP analogs to obtain structure-activity relationships.
In addition, assays quantifying the transamidase [3] and GTP-binding activities, respectively, were subjected to a titration with calcium chloride (Ca²⁺) to elucidate its influence on the conformation of TGase 2. Exclusive interaction of ligands/substrates with the GTP binding site and with the active site were found in the absence of Ca²⁺ and at [Ca²⁺] > 10 mM, respectively. Both assays exhibit an activity of ~60% at [Ca²⁺] = 0.5 mM, with this intermediate calcium concentration being applicable to identify ligands of both the active and the GTP-binding site at the same time. This finding was confirmed in both assays by means of GTPγS and recently reported N⁶-acryloyllysine piperazides [4,5] shown to irreversibly interact with the active-site cysteine residue.

[1] Pietsch et al., Bioorg. Med. Chem. Lett. 2013, 23, 6528.
[2] Schaertl et al., J. Biomol. Screen. 2010, 15, 478.
[3] Hauser et al., Amino Acids 2017, 49, 567.
[4] Wityak et al., ACS Med. Chem. Lett. 2012, 3, 1024.
[5] Wodtke et al., J. Med. Chem. 2018, accepted.