Structure and ion-release mechanism of PIB-4-type ATPases
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Evaluation Summary:
This paper presents crystal structures of sCoaT, a heavy metal transporting P-type ATPase. These structures and complementary functional data define the overall fold of this protein and provide insight into several mechanistic features, including a conserved histidine proposed to act as a novel counter-ion during transport. The study will be of interest to biochemists and microbiologists interested in the transport of transition metals, structural biology of membrane proteins and drug development.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Transition metals, such as zinc, are essential micronutrients in all organisms, but also highly toxic in excessive amounts. Heavy-metal transporting P-type (P IB ) ATPases are crucial for homeostasis, conferring cellular detoxification and redistribution through transport of these ions across cellular membranes. No structural information is available for the P IB-4 -ATPases, the subclass with the broadest cargo scope, and hence even their topology remains elusive. Here, we present structures and complementary functional analyses of an archetypal P IB-4 -ATPase, sCoaT from Sulfitobacter sp. NAS14-1. The data disclose the architecture, devoid of classical so-called heavy-metal-binding domains (HMBDs), and provide fundamentally new insights into the mechanism and diversity of heavy-metal transporters. We reveal several novel P-type ATPase features, including a dual role in heavy-metal release and as an internal counter ion of an invariant histidine. We also establish that the turnover of P IB -ATPases is potassium independent, contrasting to many other P-type ATPases. Combined with new inhibitory compounds, our results open up for efforts in for example drug discovery, since P IB-4 -ATPases function as virulence factors in many pathogens.
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Evaluation Summary:
This paper presents crystal structures of sCoaT, a heavy metal transporting P-type ATPase. These structures and complementary functional data define the overall fold of this protein and provide insight into several mechanistic features, including a conserved histidine proposed to act as a novel counter-ion during transport. The study will be of interest to biochemists and microbiologists interested in the transport of transition metals, structural biology of membrane proteins and drug development.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The manuscript by Gronberg et al. discloses the crystal structures of the P1B4-type ATPase sCoaT in a late E2P state (E2P*) and a transition state of E2P dephosphorylation (E2.Pi). As P1B-ATPases maintains heavy metal homeostasis in the cell, its inhibitors are the candidate of antibiotics. Two crystal structures with phosphate analogs BeF and AlF reported in this study appear to represent the same conformation of outward-occluded E2-P state. Determined structures of sCoaT define its TM topology and absence of HMBD in its N-terminus. Tight configurations in the TM metal-binding site centered by H657 and C327 in E2P state suggest the role of H657 as a build-in counter ion as is observed in the SsZutA Lys-Asp pair. The ATPase assays address the role of the N-terminus, an inter-domain salt-bridge, as well as …
Reviewer #1 (Public Review):
The manuscript by Gronberg et al. discloses the crystal structures of the P1B4-type ATPase sCoaT in a late E2P state (E2P*) and a transition state of E2P dephosphorylation (E2.Pi). As P1B-ATPases maintains heavy metal homeostasis in the cell, its inhibitors are the candidate of antibiotics. Two crystal structures with phosphate analogs BeF and AlF reported in this study appear to represent the same conformation of outward-occluded E2-P state. Determined structures of sCoaT define its TM topology and absence of HMBD in its N-terminus. Tight configurations in the TM metal-binding site centered by H657 and C327 in E2P state suggest the role of H657 as a build-in counter ion as is observed in the SsZutA Lys-Asp pair. The ATPase assays address the role of the N-terminus, an inter-domain salt-bridge, as well as residues hypothesized to contribute to substrate binding and release. MD simulation suggests the exoplasmic gate opening to release cargo mental. They also found a salt bridge formation at the cytoplasmic regulatory site that mimic K+-binding hence this enzyme is K+-independent. The authors further explored the specific inhibitors for sCoaT, and two candidate compound shows antibiotic activity.
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Reviewer #2 (Public Review):
In this study, the authors determined the high-resolution structure of the ATP-driven metal transporter (P1B-4 ATPase), characterized unique structural and mechanistic features of this transporter, and developed inhibitors for this transporter. Transition metals such as Zn, Co, and Cu play important roles in many aspects of cell biology as cofactors of enzymes, integral components of protein structure and signaling molecules. The concentration of transition metals is regulated by the P1B-type ATPases, which also serve as important virulence factors in microorganisms. This new structure of the P1B-4 type ATPase with specificity to zinc, cobalt, and several other metals resolves the controversy about this protein topology and add new important information about its transport mechanism.
The work is carefully …
Reviewer #2 (Public Review):
In this study, the authors determined the high-resolution structure of the ATP-driven metal transporter (P1B-4 ATPase), characterized unique structural and mechanistic features of this transporter, and developed inhibitors for this transporter. Transition metals such as Zn, Co, and Cu play important roles in many aspects of cell biology as cofactors of enzymes, integral components of protein structure and signaling molecules. The concentration of transition metals is regulated by the P1B-type ATPases, which also serve as important virulence factors in microorganisms. This new structure of the P1B-4 type ATPase with specificity to zinc, cobalt, and several other metals resolves the controversy about this protein topology and add new important information about its transport mechanism.
The work is carefully executed. Generation of the structural model is accompanied by mutational studies that test the functional significance of several amino-acid residues and by molecular dynamic simulations, which explore the mechanism of metal release. This approach offers solid support to the authors' claims about a distinctness of the P1B-4 ATPases' transport mechanism. The following findings are particularly significant. The authors demonstrated that the Cys-xx-Cys motif, which in other P1B ATPases is found in the N-terminus, in P1B-4 ATPases is the part of the membrane domain. The authors also identified the conserved histidine residue as a potential built-in counter ion and demonstrated independence of the transporter activity of potassium. These new findings expand our understanding of how transition metal transporters work. The authors also made the first step towards identifying specific inhibitors of this transporter, which show efficacy in vivo against Mycobacterium, and thus provided foundation for drug discovery. Overall, the work is an important milestone in studies of the transition metal transporters.
The structural models were built for two similar conformations that do not have bound metal. This limits the authors ability to discuss the structural basis of metal selectivity, which is an important topic for future studies.
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Reviewer #3 (Public Review):
The authors provide important new insights into a heavy-metal transporting P-type ATPase, sCoaT from Sulfitobacter sp. NAS14-1. The functional and structural data presented are high quality and novel insights are provided. The overall structure, the internal counterion, the potential ion-release pathway, and the A-domain modulatory site are all interesting insights into this subclass of transport proteins. However, despite assertions that the PIB-4 P-type ATPases are distinct with uncertain topologies, the structure of sCoaT is similar to previous structures of PIB-2 P-type ATPases CopA and ZntA.
The authors describe sCoaT as a zinc transporter, yet the ATPase activity is almost three times higher for cadmium. The authors do not discuss this or why cadmium is not considered a preferred transport ion.
Two …
Reviewer #3 (Public Review):
The authors provide important new insights into a heavy-metal transporting P-type ATPase, sCoaT from Sulfitobacter sp. NAS14-1. The functional and structural data presented are high quality and novel insights are provided. The overall structure, the internal counterion, the potential ion-release pathway, and the A-domain modulatory site are all interesting insights into this subclass of transport proteins. However, despite assertions that the PIB-4 P-type ATPases are distinct with uncertain topologies, the structure of sCoaT is similar to previous structures of PIB-2 P-type ATPases CopA and ZntA.
The authors describe sCoaT as a zinc transporter, yet the ATPase activity is almost three times higher for cadmium. The authors do not discuss this or why cadmium is not considered a preferred transport ion.
Two structures of sCoaT are presented in the presence of BeF3- (3.1Å) and AlF4- (3.2Å). The authors claim that these structures represent different transport intermediates (late E2P and E2.Pi, respectively), yet the structures are identical to one another and they appear to be in an E2.Pi conformation. The density surrounding the phosphomimetic ligands are not shown and the densities are quite similar in the electron density maps provided. Stronger evidence or supporting figures need to be provided to show that BeF3- and AlF4- are in fact bound. It is possible that the crystal lattice traps the same conformation of sCoaT despite the use of ligands that should trap E2P and E2.Pi intermediates. The assertion that the two structures represent different, closely related conformational states, is not yet supported by the current presentation.
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