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.

Figure 1.

Autophosphorylation cascade. (A) A stimulus is received by the extracellular domain causing a conformational change in the HK through the transmembrane domain (TM) and, consequently, the autophosphorylation event. First, ATP binds to the catalytic domain (CA) in the ATP-binding pocket. Next, the γ-phosphate is transferred to the conserved histidine on the dimerization and histidine phosphotransfer (DHp) domain. The phosphoryl group is transferred to an aspartic acid on the response regulator (RR), which consequently binds to DNA initiating a cellular response. (B) The structures of ATP and the resulting phosphorylated histidine with the transferred phosphate boxed in pink. Phosphorylation at the τ position is thermodynamically favored.

Figure 2.

Structures of ATP-based probes containing a terminal propargyl phosphate; γ-propargyl-ATP (Probe O), γ-[(propargyl)-thio]-ATP (Probe S), and γ-[(propargyl)-imido]-ATP (Probe N) and their corresponding pHis after the autophosphorylation event. The ATP-based probes bear an alkyne-modified phosphate, thiophosphate, or aminophosphate.

Figure 3.

Comparison of Michaelis–Menten plots of HK853 autophosphorylation with Probes O, S, and N. HK853 (2 μM) was incubated with various concentrations of the probes (1 to 60 μM) at 25 °C. The reactions were quenched at 15 min, and TAMRA-N₃ was conjugated by CuAAC to the propargyl handle (30 min). Formation of the pHis species was quantified by gel-based analysis (n = 2, error bars are standard error of the mean).

Figure 4.

Hydrolysis studies of phosphorylated HK853 species at various pH values. HK853 (2 μM) was incubated with Probe O or Probe S for 15 min. After 3 h of degradation, the media were neutralized, and TAMRA-N₃ was conjugated by CuAAC to the propargyl handle (30 min). Formation of pHis was quantified by fluorescence gel-based analysis. HK853 (5 μM) was incubated for 30 s with [γ−33P]-ATP (60 μM). After 3 h of degradation, the media was neutralized. Formation of pHis was quantified by gel-based analysis and phosphorimaging. Samples that did not undergo acidification or the 3 h incubation period were “100% phosphorylated” controls, to which all samples were compared. Plot indicates the percent of phosphorylated HK remaining in comparison to the 100% control for each probe or ATP as appropriate. n ≥ 3, error bars show standard deviation. Statistical analysis to compare two substrates at a specific pH performed with an unpaired t test (**p ≤ 0.01, *p ≤ 0.05).

Figure 5.

Calculated reaction thermodynamics in kcal·mol⁻¹ (on right) for the hydrolysis of propargyl phosphoimidazole (model of phosphorylated HK853) at pH 1 to 7. Reaction thermodynamics were calculated by taking the average and standard error of double-hydride DFT calculations, and DLPNO–CCSD(T) corresponds to data sets TC1 and TC3 in Supplementary Figure 23.