Mechanistic Studies of Bioorthogonal ATP Analogues for Assessment of Histidine Kinase Autophosphorylation

Abstract

Phosphorylation is an essential protein modification and is most commonly associated with hydroxyl-containing amino acids via an adenosine triphosphate (ATP) substrate. The last decades have brought greater appreciation to the roles that phosphorylation of myriad amino acids plays in biological signaling, metabolism, and gene transcription. Histidine phosphorylation occurs in both eukaryotes and prokaryotes but has been shown to dominate signaling networks in the latter due to its role in microbial two-component systems. Methods to investigate histidine phosphorylation have lagged behind those to study serine, threonine, and tyrosine modifications due to its inherent instability and the historical view that this protein modification was rare. An important strategy to overcome the reactivity of phosphohistidine is the development of substrate-based probes with altered chemical properties that improve modification longevity but that do not suffer from poor recognition or transfer by the protein. Here, we present combined experimental and computational studies to better understand the molecular requirements for efficient histidine phosphorylation by comparison of the native kinase substrate, ATP, and alkylated ATP derivatives. While recognition of the substrates by the histidine kinases is an important parameter for the formation of phosphohistidine derivatives, reaction sterics also affect the outcome. In addition, we found that stability of the resulting phosphohistidine moieties correlates with the stability of their hydrolysis products, specifically with their free energy in solution. Interestingly, alkylation dramatically affects the stability of the phosphohistidine derivatives at very acidic pH values. These results provide critical mechanistic insights into histidine phosphorylation and will facilitate the design of future probes to study enzymatic histidine phosphorylation.

Evaluation of small molecule kinase inhibitors as novel antimicrobial and antibiofilm agents

“…The Carlson group has developed novel methods and chemical probes for studying HK activity and is using this knowledge in the discovery of HK inhibitors (Chase et al, 2018;Espinasse et al, 2020;Wilke et al, 2012). In one such report, Wilke et al designed a fluorescence polarization (FP) assay using a fluorescent, non-hydrolyzable adenosine diphosphate (ADP) probe that binds to T. maritima HK853 and optimized it for high throughput screening of small molecules that could displace the fluorescent probe (Wilke et al, 2015).…”

Modified nucleoside triphosphates in bacterial research for in vitro and live-cell applications

“…Radioactive [g- 32 P]ATP and less energetic [g- 33 P]ATP have been utilized to investigate the phosphorylation of numerous TCSs, as well as histidine kinase (HK) phosphatase activity since the g-phosphate is transferred between proteins. [116][117][118][119][120][121] Nevertheless, the safety hazards inherent to the use of radioactive materials, as well as their limited scope for use in whole cells, has resulted in the development of modified NTPs with various non-radioactive tags on their phosphate tail.…”

“…Alkylation provided increased stability only under very acidic conditions (pH = 1). 120 Kraatz and coworkers developed a 5 0 -g-ferrocenyl-ATP (g-FE-ATP) containing a g-aminophosphate for monitoring bacterial HK activity electrochemically and by immunodetection (Fig. 11).…”

The Molecular Mechanism of Fludioxonil Action Is Different to Osmotic Stress Sensing

“…That means the phosphate transfer within the MSP occurs from a His to an Asp (in the HK), back to a His (in the histidine-containing phosphotransfer protein), and finally to an Asp (in the regulatory response protein) [ 12 ]. One of the major drawbacks of studies concerning HK signaling is the acid instability of phosphorylated His residues, making it challenging to identify, quantify and study phosphorylation states, especially by modern phospho-proteome analysis [ 13 ]. Additionally, the lack of commercially available antibodies for detecting phosphorylated His residues makes the quantification extremely difficult [ 14 , 15 ].…”