Exploration of the Effects of γ-Phosphate-Modified ATP Analogues on Histidine Kinase Autophosphorylation

Abstract

While two-component systems (TCSs), composed of a sensor histidine kinase (HK) and a response regulator, are the main signaling pathways in bacteria, global TCS activity remains poorly described. Here, we report the kinetic parameters of the HK autophosphorylation reaction using previously uncharacterized γ-phosphate-modified ATP analogues to further elucidate their utility as activity-based probes for global TCS analysis. Given the increased stability of thiophosphorylated histidine in comparison to that of the native phosphoryl modification, which is attributed to the decreased electrophilicity of this moiety, we anticipated that ATPγS may be turned over much more slowly by the HKs. Surprisingly, we found this not to be the case, with the turnover numbers decreasing <1 order of magnitude. Instead, we found that alkylation of the thiophosphate had a much more dramatic effect on turnover and, in one case, the binding affinity of this substrate analogue (BODIPY-FL-ATPγS).

Figure 1.

(a) HK autophosphorylation event. When activated by an extracellular signal, the conserved histidine on the HK is phosphorylated by ATP. The phosphoryl group is transferred to an aspartic acid on an RR, which triggers a cellular response. (b) Structure of BODIPY-FL-ATPγS, highlighting the portion of the probe that is transferred to the conserved histidine on the HK. (c) Structure of the modified histidine residue.

Figure 2.

Time-dependent autophosphorylation of HKs used to establish the linear range of HK enzyme activity for substrate-dependent assays. (a) [γ-³³P]ATP HK853. (b) [γ-³³P]ATP VicK. (c) [γ-³⁵S]ATP HK853. (d) [γ-³⁵S]ATP VicK. All reactions were performed using 6.25 μM ATP at a specific activity of 2.00 Ci/mmol with 5.0 μM protein. Error bars represent the standard error of the mean from at least three trials.

Figure 3.

[γ-³³P]- and [γ-³⁵S]ATP concentration-dependent autophosphorylation of HKs used to calculate K_m and k_cat as summarized in Table 1. Reactions were quantified as a function of (thio)phosphorylated histidine formed vs ATP. (a) [γ-³³P]ATP HK853. (b) [γ-³³P]ATP VicK. (c) [γ-³⁵S]ATP HK853. (d) [γ-³⁵S]ATP VicK. All reactions were performed at a specific activity of 2.00 Ci/mmol with 5.0 μM protein. [γ-³³P]ATP reactions were quenched at 30 s. [γ-³⁵S]ATP reactions were quenched at 1 min. Error bars represent the standard error of the mean from at least three trials.

Figure 4.

Progress curves of B-ATPγS concentration-dependent autophosphorylation of HKs used to calculate K_m and k_cat as summarized in Table 1. Reactions were quantified as a function of the fluorescently labeled histidine (BtP~His) formed. (a) B-ATPγS HK853. Reactions performed using 1.0 protein and quenched at 15 min. (b) B-ATPγS VicK. Reactions performed using 1.0 μM protein and quenched at 60 min. Error bars represent the standard error of the mean from at least three trials.