Interaction Analysis of a Two-Component System Using Nanodiscs

Abstract

Two-component systems are the major means by which bacteria couple adaptation to environmental changes. All utilize a phosphorylation cascade from a histidine kinase to a response regulator, and some also employ an accessory protein. The system-wide signaling fidelity of two-component systems is based on preferential binding between the signaling proteins. However, information on the interaction kinetics between membrane embedded histidine kinase and its partner proteins is lacking. Here, we report the first analysis of the interactions between the full-length membrane-bound histidine kinase CpxA, which was reconstituted in nanodiscs, and its cognate response regulator CpxR and accessory protein CpxP. Using surface plasmon resonance spectroscopy in combination with interaction map analysis, the affinity of membrane-embedded CpxA for CpxR was quantified, and found to increase by tenfold in the presence of ATP, suggesting that a considerable portion of phosphorylated CpxR might be stably associated with CpxA in vivo. Using microscale thermophoresis, the affinity between CpxA in nanodiscs and CpxP was determined to be substantially lower than that between CpxA and CpxR. Taken together, the quantitative interaction data extend our understanding of the signal transduction mechanism used by two-component systems.

Vancomycin does not affect the enzymatic activities of purified VanSA

“…We immobilized VanR A via a C-terminal epitope tag and used the full-length and cytosolic histidine kinases as analytes in the mobile phase. Both constructs bound to VanR A with dissociation constants ( K D ) in the low micromolar range, consistent with the affinities found for other histidine kinase-response regulator pairs [46–53] (Table 1). Full-length VanS A was seen to bind VanR A ~3x more tightly than the isolated cytosolic domain.…”

Nanodiscs in Membrane Biochemistry and Biophysics

“…Another example is the simultaneous interactions of the sensor histidine kinase CpxA with cytosolic partner protein CpxR and the periplasmic protein CpxP. 103 In the latter case, results obtained with SPR were compared with data measured using microscale thermophoresis, and both methods confirmed a 10-fold increase of binding affinity between CpxA in Nanodiscs and the regulator protein CpxR in the presence of ATP. Farnesylated and non-farnesylated versions of full-length cancer signaling protein K-Ras4B as well as peptides corresponding to the hyper-variable region, bind to Nanodiscs assembled with DPPC, DOPC, and DOPS (each containing 5% of DOPE) as was monitored using SPR.…”

“…Examples of successful Nanodisc assembly include cases where all components are solubilized in octyl glucoside, 117118 Triton X-100, 24,119 β-decyl-maltoside, 120 dodecyl maltoside, 103,121–123 and CHAPS. 124 Refolding of membrane proteins in Nanodiscs from an unfolded state via solubilization in SDS detergent was recently demonstrated by successful reconstitution of the homotetramer KcsA channel and bacteriorhodopsin.…”

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

“…We propose to perform detergent removal using hydrophobic beads 48 (step 1.3), while possible alternatives are dialysis or the addition of cyclodextrin 52,53 . Initially, dialysis was used for the reconstitution of HDL 5,34 and is still preferred .…”