Membrane Sensor Histidine Kinases: Insights from Structural, Ligand and Inhibitor Studies of Full-Length Proteins and Signalling Domains for Antibiotic Discovery.

Pikyee Ma,Mary K Phillips-Jones

doi:10.3390/molecules26165110

Pikyee Ma, Mary K Phillips-Jones

Open Access

https://doi.org/10.3390/molecules26165110

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Abstract

There is an urgent need to find new antibacterial agents to combat bacterial infections, including agents that inhibit novel, hitherto unexploited targets in bacterial cells. Amongst novel targets are two-component signal transduction systems (TCSs) which are the main mechanism by which bacteria sense and respond to environmental changes. TCSs typically comprise a membrane-embedded sensory protein (the sensor histidine kinase, SHK) and a partner response regulator protein. Amongst promising targets within SHKs are those involved in environmental signal detection (useful for targeting specific SHKs) and the common themes of signal transmission across the membrane and propagation to catalytic domains (for targeting multiple SHKs). However, the nature of environmental signals for the vast majority of SHKs is still lacking, and there is a paucity of structural information based on full-length membrane-bound SHKs with and without ligand. Reasons for this lack of knowledge lie in the technical challenges associated with investigations of these relatively hydrophobic membrane proteins and the inherent flexibility of these multidomain proteins that reduces the chances of successful crystallisation for structural determination by X-ray crystallography. However, in recent years there has been an explosion of information published on (a) methodology for producing active forms of full-length detergent-, liposome- and nanodisc-solubilised membrane SHKs and their use in structural studies and identification of signalling ligands and inhibitors; and (b) mechanisms of signal sensing and transduction across the membrane obtained using sensory and transmembrane domains in isolation, which reveal some commonalities as well as unique features. Here we review the most recent advances in these areas and highlight those of potential use in future strategies for antibiotic discovery. This Review is part of a Special Issue entitled “Interactions of Bacterial Molecules with Their Ligands and Other Chemical Agents” edited by Mary K. Phillips-Jones.

Highlights

Sensor histidine kinases (SHKs) are usually membrane-bound proteins that constitute an integral part of short signalling pathways in bacteria known as two-component signal transduction systems (TCSs)
Whilst the structures and functions of many individual SHK domains, domain hybrids and multi-domain SHKs are known, including natively soluble SHKs that lack transmembrane segments [67,68,69,70,71,72], all of which have contributed significantly to knowledge of these proteins, there remains a lack of structural data on full-length membrane SHKs which results in part from the technical challenges associated with their purification as intact active membrane proteins in sufficient milligram quantities for elucidation of their three-dimensional structures by crystallisation or other methods [73]. These challenges are being overcome and in this Review, we describe some of the emerging successes in the overexpression and purification of full-length active membrane SHKs, the methods used for their solubilisation and reconstitution using detergents, amphipols, liposomes, and nanodiscs, and some of the consequent successes in structural and ligand binding studies, thereby expanding upon and complementing other Reviews of SHKs to date, which have focused less on studies of full-length purified proteins
Use of pTTQ18His as expression plasmid has resulted in a high degree of success for producing intact SHKs compared with members of other membrane protein families, including successful expression of 15 of the 16 genome complement of membrane SHKs of Enterococcus faecalis [34], VanS (A-type) of E. faecium [75,89], BlpH, and ComD2 pheromone-sensing SHKs of Streptococcus pneumoniae [90] and the PrrB redox-sensing SHK protein of the Gram-negative bacterium Rhodobacter sphaeroides [87,91]

Summary

Introduction

Sensor histidine kinases (SHKs) ( known as histidine protein kinases) are usually membrane-bound proteins that constitute an integral part of short signalling pathways in bacteria known as two-component signal transduction systems (TCSs). One advantage of including intact SHKs in drug discovery is being able to identify whether drug candidates bind and disrupt activities of the kinase (rather than, for example, a linked RR in a reporter system), with possible inhibition sites blocking recognition of the in vivo signalling ligand, signal transmission across the membrane, propagation to catalytic domains, dimerization activities, or phosphotransfer or dephosphorylation activities Another advantage is the contribution to knowledge made by understanding how signals are transmitted throughout the entire protein, including the conformational changes that occur. Reliable and consistent methods do exist for the overexpression, purification, and solubilisation of intact SHKs and the section describes some of these methods and how the resulting purified proteins are prepared for investigation

Methodology for Producing Purified Active Forms of Full-Length Membrane SHKs

Identification of Direct Inhibitors of SHKs Using Full-Length Intact Proteins

Binding of the GBAP Pheromone to Purified Intact FsrC

Extracellular Sensing and Transmembrane Signalling Domains

Findings

Conclusions and Perspectives