Conduction pathway for potassium through the E. coli pump KdpFABC
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Curated by eLife
eLife Assessment
This valuable study provides new insights into the movement of ions through the bacterial pump KdpFABC, which regulates intracellular potassium concentration, by solving a 2.1 Å cryo-EM structure of the nanodisc-embedded active wild-type protein, and carrying out mutagenesis and activity assays. Although the structural data and analysis are solid, additional information about other structural classes identified in the EM data, as well as a discussion of relevant work done by others, would further strengthen these findings. The description of the activity assays is currently incomplete because more information is required to rigorously assess these experiments. This work will be of interest to the membrane transporter and channel communities and to microbiologists interested in osmoregulation and potassium homeostasis.
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Abstract
Abstract
Under osmotic stress, bacteria express a heterotetrameric protein complex, KdpFABC, which functions as an ATP-dependent K+ pump to maintain intracellular potassium levels. The subunit KdpA belongs to the Superfamily of K+ Transporters and adopts a pseudo-tetrameric architecture with a membrane embedded selectivity filter as seen in K+ channels. KdpB belongs to the superfamily of P-type ATPases with a conserved binding site for ions within the membrane domain and three cytoplasmic domains that orchestrate ATP hydrolysis via an aspartyl phosphate intermediate. Previous work has hypothesized that K+ moves parallel to the membrane plane through a 40-Å long tunnel that connects the selectivity filter of KdpA with the binding site in KdpB. In the current work, we have reconstituted KdpFABC into lipid nanodiscs and used cryo-EM to image the wild-type pump under turnover conditions. We present a 2.1 Å structure of the E1∼P·ADP conformation, which reveals new features of the conduction pathway. This map shows exceedingly strong densities within the selectivity filter and at the canonical binding site, consistent with K+ bound at each of these sites in this conformation. Many water molecules occupy a vestibule and the proximal end of the tunnel, which becomes markedly hydrophobic and dewetted at the subunit interface. We go on to use ATPase and ion transport assays to assess effects of numerous mutations along this proposed conduction pathway. The results confirm that K+ ions pass through the tunnel and support the existence of a low affinity site in KdpB for releasing these ions to the cytoplasm. Taken together, these data shed new light on the unique partnership between a transmembrane channel and an ATP-driven pump in maintaining the large electrochemical K+ gradient essential for bacterial survival.
Article activity feed
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eLife Assessment
This valuable study provides new insights into the movement of ions through the bacterial pump KdpFABC, which regulates intracellular potassium concentration, by solving a 2.1 Å cryo-EM structure of the nanodisc-embedded active wild-type protein, and carrying out mutagenesis and activity assays. Although the structural data and analysis are solid, additional information about other structural classes identified in the EM data, as well as a discussion of relevant work done by others, would further strengthen these findings. The description of the activity assays is currently incomplete because more information is required to rigorously assess these experiments. This work will be of interest to the membrane transporter and channel communities and to microbiologists interested in osmoregulation and potassium homeostasis.
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Reviewer #1 (Public review):
Summary:
This study on potassium ion transport by the protein complex KdpFABC from E. coli reveals a 2.1 Å cryo-EM structure of the nanodisc-embedded transporter under turnover conditions. The results confirm that K+ ions pass through a previously identified tunnel that connects the channel-like subunit with the P-type ATPase-type subunit.
Strengths:
The excellent resolution of the structure and the thorough analysis of mutants using ATPase and ion transport measurements help to strengthen new and previous interpretations. The evidence supporting the conclusions is solid, including biochemical assays and analysis of mutants. The work will be of interest to the membrane transporter and channel communities and to microbiologists interested in osmoregulation and potassium homeostasis.
Weaknesses:
There is …
Reviewer #1 (Public review):
Summary:
This study on potassium ion transport by the protein complex KdpFABC from E. coli reveals a 2.1 Å cryo-EM structure of the nanodisc-embedded transporter under turnover conditions. The results confirm that K+ ions pass through a previously identified tunnel that connects the channel-like subunit with the P-type ATPase-type subunit.
Strengths:
The excellent resolution of the structure and the thorough analysis of mutants using ATPase and ion transport measurements help to strengthen new and previous interpretations. The evidence supporting the conclusions is solid, including biochemical assays and analysis of mutants. The work will be of interest to the membrane transporter and channel communities and to microbiologists interested in osmoregulation and potassium homeostasis.
Weaknesses:
There is insufficient credit and citation of previous work.
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Reviewer #2 (Public review):
Summary:
The paper describes the high-resolution structure of KdpFABC, a bacterial pump regulating intracellular potassium concentrations. The pump consists of a subunit with an overall structure similar to that of a canonical potassium channel and a subunit with a structure similar to a canonical ATP-driven ion pump. The ions enter through the channel subunit and then traverse the subunit interface via a long channel that lies parallel to the membrane to enter the pump, followed by their release into the cytoplasm.
Strengths:
The work builds on the previous structural and mechanistic studies from the authors' and other labs. While the overall architecture and mechanism have already been established, a detailed understanding was lacking. The study provides a 2.1 Å resolution structure of the E1-P state of …
Reviewer #2 (Public review):
Summary:
The paper describes the high-resolution structure of KdpFABC, a bacterial pump regulating intracellular potassium concentrations. The pump consists of a subunit with an overall structure similar to that of a canonical potassium channel and a subunit with a structure similar to a canonical ATP-driven ion pump. The ions enter through the channel subunit and then traverse the subunit interface via a long channel that lies parallel to the membrane to enter the pump, followed by their release into the cytoplasm.
Strengths:
The work builds on the previous structural and mechanistic studies from the authors' and other labs. While the overall architecture and mechanism have already been established, a detailed understanding was lacking. The study provides a 2.1 Å resolution structure of the E1-P state of the transport cycle, which precedes the transition to the E2 state, assumed to be the rate-limiting step. It clearly shows a single K+ ion in the selectivity filter of the channel and in the canonical ion binding site in the pump, resolving how ions bind to these key regions of the transporter. It also resolves the details of water molecules filling the tunnel that connects the subunits, suggesting that K+ ions move through the tunnel transiently without occupying well-defined binding sites. The authors further propose how the ions are released into the cytoplasm in the E2 state. The authors support the structural findings through mutagenesis and measurements of ATPase activity and ion transport by surface-supported membrane (SSM) electrophysiology.
Weaknesses:
While the results are overall compelling, several aspects of the work raised questions. First, the authors determined the structure of the pump in nanodiscs under turnover conditions and observed several structural classes, including E1-P, which is detailed in the paper. Two other structural classes were identified, including one corresponding to E2. It is unclear why they are not described in the paper. Notably, the paper considers in some detail what might occur during the E1-P to E2 state transition, but does not describe the 3.1 Å resolution map for the E2 state that has already been obtained. Does the map support the proposed structural changes?
The paper relies on the quantitative activity comparisons between mutants measured using SSM electrophysiology. Such comparisons are notoriously tricky due to variability between SSM chips and reconstitution efficiencies. The authors should include raw traces for all experiments in the supplementary materials, explain how the replicates were performed, and describe the reproducibility of the results. Related to this point above, size exclusion chromatography profiles and reconstitution efficiencies for mutants should be shown to facilitate comparison between measured activities. For example, could it be that the inactive V496R mutant is misfolded and unstable?
Similarly, are the reduced activities of V496W and V496H (and many other mutants) due to changes in the tunnel or poor biochemical properties of these variants? Without these data, the validity of the ion transport measurements is difficult to assess.
The authors propose that the tunnel connecting the subunits is filled with water and lacks potassium ions. This is an important mechanistic point that has been debated in the field. It would be interesting to calculate the volume of the tunnel and estimate the number of ions that might be expected in it, given their concentration in bulk. It may also be helpful to provide additional discussion on whether some of the observed densities correspond to bound ions with low occupancy.
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Reviewer #3 (Public review):
Summary:
By expressing protein in a strain that is unable to phosphorylate KdpFABC, the authors achieve structures of the active wild-type protein, capturing a new intermediate state, in which the terminal phosphoryl group of ATP has been transferred to a nearby Asp, and ADP remains covalently bound. The manuscript examines the coupling of potassium transport and ATP hydrolysis by a comprehensive set of mutants. The most interesting proposal revolves around the proposed binding site for K+ as it exits the channel near T75. Nearby mutations to charged residues cause interesting phenotypes, such as constitutive uncoupled ATPase activity, leading to a model in which lysine residues can occupy/compete with K+ for binding sites along the transport pathway.
Strengths:
Although this structure is not so different …
Reviewer #3 (Public review):
Summary:
By expressing protein in a strain that is unable to phosphorylate KdpFABC, the authors achieve structures of the active wild-type protein, capturing a new intermediate state, in which the terminal phosphoryl group of ATP has been transferred to a nearby Asp, and ADP remains covalently bound. The manuscript examines the coupling of potassium transport and ATP hydrolysis by a comprehensive set of mutants. The most interesting proposal revolves around the proposed binding site for K+ as it exits the channel near T75. Nearby mutations to charged residues cause interesting phenotypes, such as constitutive uncoupled ATPase activity, leading to a model in which lysine residues can occupy/compete with K+ for binding sites along the transport pathway.
Strengths:
Although this structure is not so different from previous structures, its high resolution (2.1 Å) is impressive and allows the resolution of many new densities in the potassium transport pathway. The authors are judicious about assigning these as potassium ions or water molecules, and explain their structural interpretations clearly. In addition to the nice structural work, the mechanistic work is thorough. A series of thoughtful experiments involving ATP hydrolysis/transport coupling under various pH and potassium concentrations bolsters the structural interpretations and lends convincing support to the mechanistic proposal.
Weaknesses:
The structures are supported by solid membrane electrophysiology. These data exhibit some weaknesses, including a lack of information to assess the rigor and reproducibility (i.e., the number of replicates, the number of sensors used, controls to assess proteoliposome reconstitution efficiency, and the stability of proteoliposome absorption to the sensor).
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