Nucleotide binding kinetics and conformational change analysis of tissue transglutaminase with switchSENSE

Abstract

Function, activity, and interactions of proteins crucially depend on their three-dimensional structure and are often regulated by effector binding and environmental changes. Tissue transglutaminase (Transglutaminase 2, TG2) is a multifunctional protein, allosterically regulated by nucleotides and Ca²⁺ ions, which trigger opposing conformational changes. Here we introduce switchSENSE as a versatile tool for TG2 characterization and provide novel insights into protein conformation as well as analyte binding kinetics. For the first time, we succeeded in measuring the kinetic rate constants and affinities (k_on, k_off, K_D) for guanosine nucleotides (GMP, GDP, GTP, GTPγS). Further, the conformational changes induced by GDP, Ca²⁺ and the covalent inhibitor Z-DON were observed by changes in TG2's hydrodynamic diameter. We confirmed the well-known compaction by guanosine nucleotides and extension by Ca²⁺, and provide evidence for TG2 conformations so far not described by structural analysis. Moreover, we analyze the influence of the peptidic Z-DON inhibitor and the R580A mutation on the conformational responsiveness of TG2 to its natural effectors. In summary, this work shows how the combination of structural and kinetic information obtained by switchSENSE opens new perspectives for the characterization of conformationally active proteins and their interactions with ligands, e.g. potential drug candidates.

Keywords: Biosensor; Conformational changes; Inhibitors; Kinetics; Ligand binding assay; Protein structure; TG2; Tissue transglutaminase; switchSENSE.