Activity-based probe for histidine kinase signaling

Abstract

Bacterial two-component systems (TCSs) are signaling pathways composed of two proteins: a histidine kinase (HK) and a response regulator (RR). Upon stimulation, the HK autophosphorylates at a conserved histidine. The phosphoryl group is subsequently transferred to an aspartate on an RR, eliciting an adaptive response, often up- or downregulation of gene expression. TCS signaling controls many functions in bacteria, including development, virulence, and antibiotic resistance, making the proteins involved in these systems potential therapeutic targets. Efficient methods for the profiling of HKs are currently lacking. For direct readout of HK activity, we sought to design a probe that enables detection of the phosphotransfer event; however, analysis of the phosphohistidine species is made difficult by the instability of the P-N bond. We anticipated that use of a γ-thiophosphorylated ATP analogue, which would yield a thiophosphorylated histidine intermediate, could overcome this challenge. We determined that the fluorophore-conjugated probe, BODIPY-FL-ATPγS, labels active HK proteins and is competitive for the ATP binding site. This activity-based probe provides a new strategy for analysis of TCSs and other HK-mediated processes and will facilitate both functional studies and inhibitor identification.

Figure 1

(A) HK is activated by an extracellular signal, which results in phosphorylation of a conserved histidine by ATP. The phosphoryl group is transferred to the aspartate on a RR. Activated RR then triggers a response, typically acting as a transcription factor. Domains: DHp, dimerization and histidine phosphotransfer; CA, catalytic and ATP-binding. (B) Mechanisms of HK autophosphorylation and phosphotransfer to RR. (C) Structure of B-ATPγS, highlighting the thiophosphate-BODIPY moiety that is transferred during autophosphorylation. (D) Proposed mechanism of B-ATPγS labeling of active HK and cognate RR.

Figure 2

(A) B-ATPγS used as an ABP to show in vitro thiophosphorylation of active HK853 (T. maritima). Top: in-gel fluorescence detection of the BODIPY fluorophore shows signal only with addition of probe (2 µM). Bottom: Coomassie staining reveals even protein loading. Lanes: 1, no probe; 2, B-ATPγS added. (B) Concentration dependence of HK853 labeling by B-ATPγS. Labeling was saturated at ~10 µM and was measured by in-gel fluorescence scanning (integrated band intensities given in arbitrary units). (C) B-ATPγS labeling of HK853 was inhibited when ATP analogs (200 µM, 10 min) were added prior to B-ATPγS (2 µM). Lanes: 1, no inhibitor; 2, ATP; 3, AMP-PNP; 4, ATPγS.

Figure 3

BODIPY-thiophosphate transfers to RR468 (16 kDa) over time only when HK853 (32 kDa) is present. HK853 was first incubated with the probe for 1 h at 25 °C. The RR468 was then added at 7-fold the concentration of HK853. Top: In-gel fluorescence detection of the BODIPY fluorophore. Bottom: Coomassie staining of the same gel reveals even protein loading. The control lane (7) reveals that RR labeling is HK dependent.

Figure 4

(A) His-tagged HK853 was conjugated to NiNTA-bearing liposomes and separated into free and bound HK853. (B) 600 ng of HK853 from purified stock solution (S), free HK853 after ultracentrifugation (F), and liposome-bound HK853 (B) was labeled with 2 µM B-ATPγS. Inhibition of fluorescent labeling was achieved by the addition of ATP prior to B-ATPγS. Use of B-ATPγS reveals that HK853 bound to liposomes is more active than that free in solution.