# Recombinant Cytosolic Truncations of Histidine Kinases Retain Function for Targeted In Vitro Investigations

## TL;DR

Researchers created functional parts of histidine kinases that work in lab tests, helping study bacterial signaling and drug development.

## Contribution

The study introduces improved methods to produce functional cytosolic truncation mutants of histidine kinases from multiple bacterial species.

## Key findings

- Cytosolic truncation mutants of histidine kinases retain autophosphorylation and phosphotransfer capabilities.
- Functional mutants were successfully produced from Escherichia coli, Klebsiella pneumoniae, and other species.
- These mutants are suitable for in vitro biochemical investigations and inhibitor screening.

## Abstract

Histidine kinases are an integral component of bacterial two-component systems (TCSs), playing a pivotal role in signal transduction pathways, resulting in both resistance and virulence. However, their inherent membrane-bound nature often results in poor solubility, making them difficult to isolate and rendering them incompatible with most in vitro biochemical techniques. Consequently, much of the research on two-component systems has centered on response regulators, limiting both drug discovery efforts and our broader understanding of key signal transduction mechanisms. To address these challenges, we sought to straightforwardly generate cytosolic truncation mutants of histidine kinases that retain their autophosphorylation and phosphotransfer capabilities. Previously, we successfully developed a cytosolic truncation mutant of PmrB (PmrBc) that maintained these critical functions, demonstrating its suitability as a viable surrogate for in vitro investigations, including inhibitor compound screening. Building upon this foundation, we have refined our methods and here demonstrate these improvements by producing functional histidine kinase truncation mutants from the following diverse bacterial species: Escherichia coli; PhoQ, BasS and Klebsiella pneumoniae; and PmrB and PhoQ.