TrkA undergoes a tetramer-to-dimer conversion to open TrkH which enables changes in membrane potential

Abstract

TrkH is a bacterial ion channel implicated in K⁺ uptake and pH regulation. TrkH assembles with its regulatory protein, TrkA, which closes the channel when bound to ADP and opens it when bound to ATP. However, it is unknown how nucleotides control the gating of TrkH through TrkA. Here we report the structures of the TrkH-TrkA complex in the presence of ADP or ATP. TrkA forms a tetrameric ring when bound to ADP and constrains TrkH to a closed conformation. The TrkA ring splits into two TrkA dimers in the presence of ATP and releases the constraints on TrkH, resulting in an open channel conformation. Functional studies show that both the tetramer-to-dimer conversion of TrkA and the loss of constraints on TrkH are required for channel gating. In addition, deletion of TrkA in Escherichia coli depolarizes the cell, suggesting that the TrkH-TrkA complex couples changes in intracellular nucleotides to membrane potential.

Fig. 1. Structure of TrkHA in the presence of ADP, ATP, or ATPγS.

a–c Structures of the TrkH (blue and teal)-TrkA (dark blue and purple) in the presence of ADP (a), ATP (b), or ATPγS (c), viewed within the plane of the membrane (top) and from the periplasmic side (bottom). The green outline indicates an individual TrkH protomer.

Fig. 2. Conformational changes in TrkA monomer and tetramer.

a–c TrkA gating ring from TrkHA-ADP (a), TrkHA-ATPγS (b), and TrkHA-ATP (c), viewed from the periplasmic (top), N2-N2 interface (middle), and N1-N1 interface (bottom) sides. The 2Fo-Fc electron density maps of N2-N2 and N1-N1 interfaces are shown as blue mesh contoured at 1.0σ on the right. d Conformational changes within a TrkA protomer. N1 and N2 domains of a TrkA protomer from TrkHA-ADP (blue), TrkHA-ATPγS (orange), TrkHA-ATP (red), or TrkHA-NADH (green) are render in ribbons. The N1 domains were superimposed to show the relative rotations of the N2 domain.

Fig. 3. Interactions between TrkA and different nucleotides.

Ribbon representations of TrkA bound with ADP (a, left panel) or ATPγS (b, left panel), and the magnified views of the N1 (middle panels) and N2 (right panels) nucleotide binding sites. The omit map for nucleotides are shown in green meshes at 3σ. The nucleotide-protein interaction diagrams are drawn using LigPlot⁺.

Fig. 4. The TrkH-TrkA interfaces.

a TrkH-TrkA interactions. TrkHA-ADP (left), TrkHAγS (center), or TrkHA-ATP (right) are shown in colored schematics, viewed within the plane of the membrane. Structural elements involved in TrkH-TrkA interactions are shown as cartoon. Distances from TrkH ILE178 to TrkA ASP25 are marked on the left side of each complex. Distances from the TrkH selectivity filter to TrkH-TrkA-N2 interface (GLY222 to SER389) are marked on the right side. b–d Top: the HN1 interfaces from TrkHA-ADP (a), TrkHA-ATP (b), or TrkHA-ATPγS (c). Bottom: the HN2 interfaces from TrkHA-ADP (a), TrkHA-ATP (b), or TrkHA-ATPγS (c). TrkH (green) and TrkA (hot pink) are shown as cartoon. The 2Fo-Fc electron density maps are shown as blue meshes at 1.0σ cut-off.

Fig. 5. Conformational changes within TrkH.

a–c Top: Surface representations (yellow) of the ion permeation pathways of TrkH from TrkHA-ADP (a), TrkHA-ATPγS (b), or TrkHA-ATP (c), plotted using the program HOLE. Bottom: Stick representations of the selectivity filter and intramembrane loop in TrkH from TrkHA-ADP (a), TrkHA-ATPγS (b), or TrkHA-ATP (c). The 2Fo-Fc electron density maps are shown in blue meshes contoured at 1.0σ. d Radius of the ion permeation pathways, as calculated by HOLE. e TrkH protomers from TrkHA-ADP (blue), TrkHA-ATPγS (orange), or TrkHA-ATP (red) rendered in ribbons superimposed at the selectivity filters, showing either the D1 and D3 domains (top) or the D2 and D4 domains (bottom). f–h Top: TrkH protomers from TrkHA-ADP (f), TrkHA-ATP (g), TrkHA-ATPγS (h), rendered in cylinders viewed from the periplasmic side. Bottom: TrkH protomers from TrkHA-ADP (f), TrkHA-ATP (g), or TrkHA-ATPγS (h) rendered in ribbons viewed within membrane. The D4M1 and D3M2b helices at the dimer interfaces are rendered in cylinders.

Fig. 6. Single-channel activities of TrkHA mutants.

a Current traces of different TrkHA mutants. Top left: Current traces of TrkHA (175A-wt) in the presence of no ligand, 5 mM ATP, or 5 mM ADP. Bottom left: Current traces of TrkHAc (175C-40C), in the presence of no ligand, 10 mM H₂O₂, and both 10 mM H₂O₂ and 5 mM ATP. Top right: Current traces of TrkHA (wt-283V) in the presence of no ligand, 5 mM ATP, or 5 mM ADP. Bottom left: Current traces of TrkHA (wt-309C) in the presence of no ligand, 5 mM ATP, or 5 mM ADP. The scale bar for TrkHA (wt-309C) is 10 pA/10 s, while the scale bar for all other TrkHA mutants are 10 pA/3 s. b Current traces of TrkH 175A (scale bar: 10 pA/3 s). c Open probabilities of TrkHA (wt-wt, 175A-wt, wt-309C, and wt-283V) in the presence of no ligand, 5 mM ATP, or 5 mM ADP plotted in dots overlap with bar graph. Error bars in c–e are standard error of the mean (s.e.m.). Charts in c–e were prepared in GraphPad Prism. d Open probabilities of TrkH (wt) and TrkH (175A) plotted in dots overlap with bar graph. e Open probabilities of TrkHA (175C-40C) in the presence of no ligand, 10 mM H₂O₂, or both 10 mM H₂O₂ and 5 mM ATP plotted in dots overlap with bar graph.

Fig. 7. Deletion of TrkA leads to membrane potential depolarization in E. coli.

The relative membrane potentials of wild-type (wt) and ∆trkA E. coli strains as determined by a fluorescence-based assay. The ∆trkA cells are depolarized relative to wt cells (n = 5, p < 0.001). Points representing each measurement are overlaid with the bar graph. Error bars are s.e.m. The chart was prepared using GraphPad Prism.

Fig. 8. Proposed gating mechanism of TrkHA.

Upper: Cartoon illustration of TrkHA in the closed (left) and open (right) states viewed within the plane of the membrane (gray). Teal and cyan: TrkH; blue: N2 domain; purple: N1 domain; light blue: C2 domain; and light purple: C1. The green outlines mark one TrkH and one TrkA protomer. Lower: the tetrameric TrkA ring in the presence of ATP (left) and the two TrkA dimers in the presence of ATP (right). In the presence of ADP, TrkA forms a tetramer and closes TrkH through interactions at both the HN1 and HN2 interfaces. In the presence of ATP, the tetrameric TrkA gating ring is split into two dimers. Opening of a TrkH channel is achieved by the downward movement of the N2 domains away from the membrane that in turn moves the intramembrane loop from blocking the selectivity filter, and by distruption of the HN1 interfaces that allows dilation of the pore-lining helices. The two TrkH protomers also rotate relative to each other during the gating process.