The use of isothermal titration calorimetry to unravel chemotactic signalling mechanisms

Abstract

Summary Chemotaxis is based on the action of chemosensory pathways and is typically initiated by the recognition of chemoeffectors at chemoreceptor ligand‐binding domains (LBD). Chemosensory signalling is highly complex; aspect that is not only reflected in the intricate interaction between many signalling proteins but also in the fact that bacteria frequently possess multiple chemosensory pathways and often a large number of chemoreceptors, which are mostly of unknown function. We review here the usefulness of isothermal titration calorimetry (ITC) to study this complexity. ITC is the gold standard for studying binding processes due to its precision and sensitivity, as well as its capability to determine simultaneously the association equilibrium constant, enthalpy change and stoichiometry of binding. There is now evidence that members of all major LBD families can be produced as individual recombinant proteins that maintain their ligand‐binding properties. High‐throughput screening of these proteins using thermal shift assays offer interesting initial information on chemoreceptor ligands, providing the basis for microcalorimetric analyses and microbiological experimentation. ITC has permitted the identification and characterization of many chemoreceptors with novel specificities. This ITC‐based approach can also be used to identify signal molecules that stimulate members of other families of sensor proteins.

Multiple functions of flagellar motility and chemotaxis in bacterial physiology

“… 2017 ; Luu et al . 2019 ; Matilla, Martin-Mora and Krell 2020 ), only a tiny fraction of chemoreceptor ligands are currently known and even fewer have an established mode of binding (Ortega, Zhulin and Krell 2017 ). With the increasing number of characterized ligand-receptor interactions and better understanding of ligand binding by the major structural classes of ligand-binding domains of receptors, computational prediction of ligand specificity should ultimately become possible.…”

The pH Robustness of Bacterial Sensing

“…The central question addressed in this work is to what degree alterations in the pH modulate the capacity of LBDs to recognize their cognate ligands. Much experimental data exists for the microcalorimetric analyses of different ligand – LBD interactions at neutral pH ( 1 , 57 ). However, there has been no systematic evaluation of the pH robustness of these interactions.…”

The structural basis for signal promiscuity in a bacterial chemoreceptor

“…Most chemoreceptors show a restricted and well‐defined ligand spectrum [51], and PcaY_PP is one of the receptors with the broadest ligand range. Minimal requisites for binding are a C‐6 membered ring and a carboxyl group.…”