Ligand promiscuity in the tryptophan repressor – from structural understanding towards rational design

Abstract

Receptors that promiscuously bind a range of ligands provide insights into how nature mediates affinity and biological functioning. Moreover, such receptors provide vantage points for the rational design of specific binding for biotechnological applications. Here we describe the molecular details of the ligand binding promiscuity of the well-known tryptophan repressor TrpR. We elucidated high-resolution structures of TrpR bound to the co-repressors 5-methyltryptophan and 5-methyl-tryptamine as well as the pseudo-repressors indole-3-propionic and indole-3-acetic acid. Furthermore, using isothermal titration calorimetry we procure the corresponding thermodynamic parameters. Together this data provides molecular explanations for the strongly varied affinities and biological effects of the ligands providing insights into how nature shapes specificity and affinity. Beyond this we use these insights to exemplarily showcase knowledge-based design of TrpR by swapping its specificity from its native ligand tryptophan to indole-3-acetic acid. Finally, we elucidate the structures of the variant bound to indole-3-acetic and indole-3-propionic acid to retrace our design rationale.

Synthetic biology approaches to dissecting linear motor protein function: towards the design and synthesis of artificial autonomous protein walkers

“…One ligand-dependent DNA-binding protein that is currently being used in the tumbleweed design is the Trp repressor (TrpR): it binds to the trp operator DNA site in the presence of the ligand (or co-repressor) tryptophan which forms part of the repressor-DNA interface (Phillips and Stockley 1996). TrpR has already been engineered to accommodate a series of tryptophan-related ligands (Stiel et al 2020). The challenge will be to select an appropriate ligand and use an engineering approach to convert TrpR into an enzyme (Lechner et al 2018), thereby autonomously controlling track binding (see below).…”