Author response: Structure and ion-release mechanism of PIB-4-type ATPases (2021)

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Transition metals, such as zinc, are essential micronutrients in all organisms, but also highly toxic in excessive amounts. Heavy-metal transporting P-type (PIB) 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 PIB-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 PIB-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 PIB-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 PIB-4-ATPases function as virulence factors in many pathogens. Editor's evaluation 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. This work will be of interest to biochemists and microbiologists interested in the transport of transition metals, structural biology of membrane proteins and drug development. https://doi.org/10.7554/eLife.73124.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Heavy metals such as zinc and cobalt are toxic at high levels, yet most organisms need tiny amounts for their cells to work properly. As a result, proteins studded through the cell membrane act as gatekeepers to finetune import and export. These proteins are central to health and disease; their defect can lead to fatal illnesses in humans, and they also help bacteria infect other organisms. Despite their importance, little is known about some of these metal-export proteins. This is particularly the case for PIB-4-ATPases, a subclass found in plants and bacteria and which includes, for example, a metal transporter required for bacteria to cause tuberculosis. Intricate knowledge of the three-dimensional structure of these proteins would help to understand how they select metals, shuttle the compounds in and out of cells, and are controlled by other cellular processes. To reveal this three-dimensional organisation, Grønberg et al. used X-ray diffraction, where high-energy radiation is passed through crystals of protein to reveal the positions of atoms. They focused on a type of PIB-4-ATPases found in bacteria as an example. The work showed that the protein does not contain the metal-binding regions seen in other classes of metal exporters; however, it sports unique features that are crucial for metal transport such as an adapted pathway for the transport of zinc and cobalt across the membrane. In addition, Grønberg et al. tested thousands of compounds to see if they could block the activity of the protein, identifying two that could kill bacteria. This better understanding of how PIB-4-ATPases work could help to engineer plants capable of removing heavy metals from contaminated soils, as well as uncover new compounds to be used as antibiotics. Introduction The ability to adapt to environmental changes in heavy-metal levels is paramount for all cells, as these elements are essential for a range of cellular processes and yet toxic at elevated concentrations (Waldron et al., 2009; Kozlowski et al., 2009). Transition metal transporting P-type (PIB) ATPase proteins are critical for cellular heavy-metal homeostasis, providing efflux of for example copper, zinc, and cobalt from the intracellular milieu. Indeed, malfunctioning of the human PIB-members, ATP7A and ATP7B, cause the fatal neurological Menkes disease and Wilson disease (Bull et al., 1993; Vulpe et al., 1993). The PIB-ATPases belong to the P-type ATPase superfamily of integral membrane proteins, which exploit energy from ATP hydrolysis for transport of cargo across cellular membranes. These proteins share an overall mechanism described by the so-called Post-Albers cycle (Albers et al., 1963; Post and Sen, 1965), as established by decades of structural and functional investigations of primarily Ca2+-, Na+/K+-, and H+-specific P-type ATPases (Toyoshima et al., 2000; Toyoshima and Nomura, 2002; Toyoshima et al., 2004; Olesen et al., 2004; Olesen et al., 2007; Winther et al., 2013; Toyoshima et al., 2013; Morth et al., 2007; Shinoda et al., 2009). In summary, four cornerstone states, E1–E1P–E2P–E2, provide alternating access and affinity for the transported ions (and counterions, if present). Inward facing (e.g. cytosolic) E1 and outward facing (e.g. extracellular) E2P conformations are coupled to ATP-dependent phosphorylation (yielding ion-occluded E1P) and dephosphorylation (to occluded E2) of an invariant catalytical aspartate, respectively. PIB-ATPases are subdivided into groups based on conserved sequence motifs and the selectivity towards transported transition metal ions (Smith et al., 2014; Argüello, 2003; Zielazinski et al., 2012; Zhitnitsky and Lewinson, 2014). Whereas Cu+- and Zn2+-transporting PIB-1 and PIB-2 ATPases are relatively well characterized, little is known regarding the PIB-4 proteins, which comprise some of the simplest and shortest proteins within the entire P-type ATPase superfamily (Smith et al., 2014). They are present in plants, archaea, and prokaryotes, and have been assigned a role as virulence factors in pathogens, as for example the PIB-4-ATPase MtCtpD is required for tuberculosis infections (Sassetti and Rubin, 2003; Joshi et al., 2006), and therefore represent attractive targets for novel antibiotics. The PIB-4-ATPases are classically referred to as cobalt transporters. However, the metal specificity of the PIB-4-ATPases remains elusive as some members have a confirmed cobalt specificity, while others seemingly have broader or altered ion transport profiles, also transporting ions such as Zn2+, Ni2+, Cu+, and even Ca2+ (Zielazinski et al., 2012; Raimunda et al., 2012; Moreno et al., 2008; Scherer and Nies, 2009; Seigneurin-Berny et al., 2006). Thus, the PIB-4-ATPases appear to have the widest scope of transported ions of the PIB-ATPases, and it is possible that further subclassification principles and sequence motifs will be identified. Due to the broad ion transport range, they have been proposed to serve as multifunctional emergency pumps that can be exploited under extreme environmental stress to maintain heavy-metal homeostasis (Smith et al., 2017). Hitherto, the available high-resolution structural information of full-length PIB-ATPases is limited to two structures each of ion-free conformations of the Cu+-transporting PIB-1-ATPase from Legionella pneumophila (LpCopA) (Gourdon et al., 2011b; Wang et al., 2014), and the Zn2+-transporting PIB-2-ATPase from Shigella sonnei (SsZntA) (Wang et al., 2014). Thus, the principal architecture of the PIB-4-ATPases remains debated, as sequence analyses have proposed different topologies for the N-terminus: with or without (1) the so-called HMBDs, and (2) the first two transmembrane helices, MA and MB (Smith et al., 2014; Andersson et al., 2014; Rosenzweig and Argüello, 2012; Drees et al., 2015), which both are present in other PIB-ATPases (Figure 1—figure supplement 1a). These represent structural features that have been suggested to be important for ion-uptake and/or regulation in other PIB-ATPases (Gourdon et al., 2011b; Wang et al., 2014; González-Guerrero and Argüello, 2008; Mattle et al., 2013), raising questions if similar levels of protein control are absent or replaced in the PIB-4 group. In addition, despite a shared overall architecture, the PIB-1 and PIB-2 structures suggested significantly different types of entry and exit pathways, hinting at unique translocation mechanisms for each PIB group (Sitsel et al., 2015). However, it remains unknown if similar molecular adaptions have taken place in PIB-4-ATPases to handle the unique array of cargos. To address these fundamental questions, we determined structures of a PIB-4-ATPase in different states and validated our findings using in vitro functional characterization. Results and discussion Metal specificity We employed the established PIB-4 model sCoaT (UniProt ID A3T2G5) to shed further light on the structure and mechanism of the entire PIB-4-class. As the metal ion specificity of the PIB-4-ATPases is known to be wide, the ATPase activity was assessed in vitro in lipid–detergent solution using the so-called Baginski assay, in the presence of a range of different heavy metals. The protein exhibited clear Zn2+- and Cd2+-dependent ATPase activity, while Co2+ only stimulated ATP hydrolysis at high ion concentrations (Figure 2—figure supplement 1). This is in partial agreement with the ion range profile previously reported for sCoaT, as higher Co2+ sensitivity has been detected using different functional assays and different experimental conditions (Zielazinski et al., 2012; Figure 2—figure supplement 1). The fact that the KM value for the Co2+-dependent sCoaT activity reported previously is lower than measured in this study is unexpected (Figure 2—figure supplement 1b; Zielazinski et al., 2012). We therefore assessed if this observation relates to lower available concentration of Co2+ consequent to chelation by buffer solution components, or if this metal interferes with the colour development in the ATPase assays determining Pi concentrations (Figure 2—figure supplement 1c). However, Co2+ and Zn2+ display similar Baginski colour development as determined by calibration with separate standard curves. Moreover, neither exclusion of azide and molybdate to avoid possible Co2+ binding of these compounds, nor supplementation of the reducing agent TCEP (to avoid possible oxidation of Co2+ from molecular oxygen) has a significant effect on turnover. We also investigated if the type of assay may affect the outcome (Figure 2—figure supplement 1c). However, employment of the alternative Malachite Green Phosphate Assay essentially reproduced the relative activity in the presence of Zn2+ and Co2+, respectively (Lanzetta et al., 1979). sCoaT is purified in a buffer containing 5 mM β-mercaptoethanol, and even following dilution into the assay buffer the concentration is still approximately 100 µM, and as thiols can act as ligands for Co2+ it may explain part of the differences in the KM values. However, this still does not explain why Zn2+- and Cd2+-dependent ATPase activity has not been observed for sCoaT in the previously study (Smith et al., 2017), although other PIB-4-members have been associated with Zn2+ activity. While not detected, the reported KM and Vmax may nevertheless be influenced by numerous environmental factors not tested for here, such as lipids, detergents, presence/absence/location of metal-binding his-tags, or other settings. Despite that higher sensitivity has been measured for Zn2+ compared to Co2+, it cannot be excluded that Co2+, rather than Zn2+, is the preferred cargo in vivo as the relative intracellular availability of Co2+ is more than three orders of magnitude higher than that of Zn2+ in certain bacterial cells (Osman et al., 2019). Structure determination We determined structures of sCoaT in metal-free conditions supplemented with two different phosphate analogues, BeF3− and AlF4−, respectively, which previously have been exploited to stabilize E2 reaction intermediates of the transport cycle of PIB-ATPases (Wang et al., 2014; Gourdon et al., 2011b; Andersson et al., 2014). The structures were determined at 3.1 and 3.2 Å resolution, using molecular replacement (MR) as phasing method and SsZntA as search model, and the final models yielded R/Rfree of 24.4/26.8 and 21.8/25.5 (Table 1). The two crystal forms were obtained using the HiLiDe method (crystallization in the presence of high concentrations of detergent and lipids) (Gourdon et al., 2011a). Surprisingly however, the crystal packing for both structures reveal only minor contacts between adjacent membrane-spanning regions, which are critical for the crystals obtained of most other P-type ATPase proteins (Wang et al., 2014; Gourdon et al., 2011b; Sørensen et al., 2006; Figure 1—figure supplement 2). Hence, some crystal forming interactions likely take place through lipid–detergent molecules. To our knowledge, this is the first time that type I crystals with unrestrained transmembrane domains are reported, but a consequence is that peripheral parts of the membrane domain are less well resolved (Figure 1—figure supplement 3). While this caused difficulty in modelling some transmembrane (TM) helices, satisfying solutions were found with the aid of the software ISOLDE (Croll, 2018) due to its use of AMBER forcefield which helped to maintain physical sensibility in the lowest resolution regions. In addition, root means square deviation, secondary structure as well as centre-of-mass of the transmembrane helices, only showed minor variation over time in MD simulations, indicative of a stable structure (Figure 1—figure supplements 4 and 5). The TM helices also showed lowered backbone root mean square fluctuation compared to more dynamic regions, such as the soluble domains and loop regions (Figure 1—figure supplement 4b). Table 1 Data collection and refinement statistics. Statistics for the highest resolution shell are shown in parentheses. E2-BeF3−E2-AlF4−Data collectionWavelength (Å)1.01.0Space groupP 21 21 2P 21 21 2Cell dimensionsa, b, c (Å)89.0 94.5 128.889.6 93.7 128.3a, b, g (°)90 90 9090 90 90Resolution (Å)47.3–3.1(3.22–3.11)45.6–3.3(3.37–3.25)Rmerge (%)11.4 (276.3)15.5 (246)I / σI17.8 (1.12)8.5 (0.98)CC1/21 (0.475)0.99 (0.37)Completeness (%)97.3 (99.8)99.2 (99.9)Redundancy13.3 (13.8)6.1 (6.6)RefinementResolution (Å)47.3–3.1(3.22–3.11)45.6–3.3(3.37–3.25)No. reflections19,643 (1963)17,466 (1714)Rwork / Rfree (%)24.4/26.821.8/25.5No. of atomsProtein4,6954,695Ligand/ion56Water100Average B-factorsProtein135.91152.54Ligand/ion84.1586.47Solvent79.62R.m.s. deviationsBond lengths (Å)0.0040.003Bond angles (°)0.770.83Ramachandran statisticsFavoured (%)97.896.9Allowed (%)2.23.1Outliers (%)ClashscoreMolProbity score0.01.050.850.07.891.62 Overall structure, without classical HMBD Examination of the structures reveals that the PIB-4-ATPase architecture is reminiscent to that of other P-type ATPases, with three cytosolic domains, A (actuator), N (nucleotide-binding), and P (phosphorylation), as well as a membrane-spanning M-domain (Figure 1a). Furthermore, the core of the soluble portions, including the nucleotide-binding pocket and catalytic phosphorylation site at D369, are well conserved. Figure 1 with 5 supplements see all Download asset Open asset Overall architecture and reaction cycle. The sCoaT structures reveal that PIB-4-ATPases comprise soluble A-, P-, and N-domains, shown in yellow, blue, and red, respectively, as well as a transmembrane domain with eight helices: MA and MB, in cyan, and M1–M6, in grey, and that the PIB-4-topology lacks classical so-called heavy-metal-binding domain. The transport mechanism of P-type ATPases depends on ATP-dependent phosphorylation and auto-dephosphorylation, and includes four principal conformations, E1, E1P, E2P, and E2, where P denotes phosphorylation. The determined structures are trapped in two transition states following ion release – an occluded late E2P (E2P*) and an occluded transition state of dephosphorylation, E2.Pi. The topology of PIB-4-ATPases has been a conundrum as sequence analyses have proposed different arrangements, with variable number of transmembrane segments and different sizes of the N-termini (Smith et al., 2014; Gourdon et al., 2011b; Wang et al., 2014; Rosenzweig and Argüello, 2012; Drees et al., 2015; Andersson et al., 2014). However, our data unambiguously demonstrate that PIB-4-ATPases possess eight transmembrane helices, MA and MB followed by M1–M6. As previously observed for PIB-1- and PIB-2-ATPases, MB is kinked by a conserved Gly–Gly motif (G82 and G83), forming an amphipathic 'platform', MB', immediately prior to M1, see further below (Figure 1—figure supplement 3). Are then HMBDs present in PIB-4-ATPases as in the other PIB subclasses? As only the first 47 residues remain unmodelled in the final structures (Table 1), it is clear that many PIB-4-ATPases including sCoaT are lacking a classical HMBD ferredoxin-like fold (typically 70 residues long). In agreement with this observation, the cysteine pair (CGIC in the sequence) in the N-terminus of sCoaT is rather positioned in MA, facing M1 (Figure 1—figure supplements 1 and 3), in contrast to the surface-exposed, metal-binding CXXC hallmark-motif detected in classical HMBDs. Functional analysis of mutant forms lacking these cysteines in vitro also support that they are unimportant for function (Figure 2a). We note that there are PIB-4-ATPases with extended N-termini that, in contrast to sCoaT, may harbour HMBDs (Smith et al., 2014). Conversely, the sCoaT N-terminus is rich in metal-binding methionine, cysteine, histidine, aspartate, and glutamate residues, and this feature is conserved among PIB-4-ATPases (Figure 2—figure supplement 2). We therefore explored the role of this N-terminal tail through assessment of an sCoaT form lacking the first 33 residues. However, in vitro characterization suggests only minor differences compared to wild-type, indicating that the residues upstream of MA are not essential for catalytic activity (Figure 2a). Aggregated, this hints at that no classical HMBD is present, and hence that this level of regulation is absent in many PIB-4-ATPases, although it cannot be excluded that the N-termini are important in vivo. Figure 2 with 4 supplements see all Download asset Open asset Mechanistic insight into the function of PIB-4-ATPases. (a) Functional ATPase assay in lipid–detergent solution with targeted residues in sequential order. The wild-type (WT)-specific activity using the employed experimental conditions in the presence of 50 μM metal is 1.00 ± 0.01 μmol mg−1 min−1 with Zn2+ and 2.80 ± 0.06 μmol mg−1 min−1 with Cd2+, comparable to the activity previously measured for PIB-4-ATPases. For biological averages and SD, see Figure 2—figure supplement 1e. (b) Comparisons of E2-AlF4− and structures of sCoaT and the of SsZntA ID of SsZntA and were based on the and the for the overall structures are in the structure using the software The and the E2-AlF4− structures are lacking between the site to the outward The changes that likely for of the release as from the structure of SsZntA to the E2-AlF4− structures of sCoaT or of and residues in the M-domain of sCoaT and The of is with and is likely to ion likely place and The between and of including the final The of in sCoaT with the of in and both likely serve as it has been shown that the in vivo transport specificity of the sCoaT from can be from Co2+ to Zn2+ by the N-terminal to that of the Zn2+ transporting PIB-2 ATPase from et al., This that PIB-4-ATPases not only in vitro but also in vivo are to transport Zn2+, if the M-domain access to the possible for the of specificity for the is that the N-terminal as also suggested for et al., 2017), ATP hydrolysis through binding to the soluble domains, and this is then binding of the metal to the N-terminal and/or However, it is also possible that the role of the N-terminal of PIB-4 proteins is to Zn2+ an ability that is with the N-terminal part of assessment of the metal specificity of the N-terminal tail of sCoaT suggests it has little or no effect on between Co2+ and Zn2+ in vitro (Figure 2—figure supplement this it is clear that further are to shed light on the function of the N-terminal in PIB-ATPases, also in PIB-4-ATPases. this questions also on the role of the which has been proposed to serve as an site for HMBDs in PIB-1- and PIB-2-ATPases, and for the as a site for metal (Gourdon et al., 2011b; Wang et al., 2014; González-Guerrero and Argüello, 2008; et al., 2009). As there are no known for PIB-4-ATPases, and classical HMBDs appear to be in at some proteins of the the function may need to be the N-terminus may have been through without conferring functional or in a transition state of dephosphorylation The classical of P-type ATPases is that the E2P state is outward open and that the following transition state of dephosphorylation, is and that these conformations can be using the phosphate employed for structure BeF3− and AlF4−, respectively. Furthermore, have been proposed among PIB-ATPases (Gourdon et al., 2011b; Wang et al., 2014; Andersson et al., 2014; Mattle et al., 2015), including a exit pathway by MA, and that remains open also in the state for the In a from the of the metal in the M-domain of ion-occluded states to the has been observed for the PIB-2-ATPases, and this group with the E2P to Surprisingly however, analysis of the two obtained structures suggests that the significant domain are absent in sCoaT (Figure and the models are in contrast rather The of the soluble domains and the of the the the conserved motif for dephosphorylation towards the phosphorylation are associated with of dephosphorylation, indicating that the two structures are trapped in an transition state (Figure 2—figure supplement This observation from the structures of the other determined PIB-ATPases, in which the phosphorylation site of the E2P state by is from the loop as also observed for the (Figure 2—figure supplement highly similar and structures have also been observed for the P-type ATPase from was proposed that for dephosphorylation in a late E2P state by in with its and of dephosphorylation ion release may also be the case for sCoaT, and the structure may a late E2P state Comparisons of the sCoaT structures to the structure of SsZntA a unique of the (Figure 2—figure supplement The well with the in but the of the is towards the – approximately and Å (Figure 2—figure supplement However, it cannot be excluded that this is due to crystal contacts as the two peripheral of the are with parts of a we that the of sCoaT a similar to but this feature is not present in PIB-1- and and it is not a conserved in the PIB-4 group (Figure 2—figure supplements 2 and Conversely, the of the two sCoaT structures are overall similar and appear outward occluded (Figure as also by with the structures of contrasting to the observed in PIB-1- and release to shed light on ion we compared the sCoaT structures to the E2P state of in which the of and from the proposed metal-binding an exit pathway to be (Figure that and PIB-4-ATPases have cargo range, share overall topology and that they release ions in form to the in contrast to their PIB-1 we it likely that they similar exit pathways, primarily by and (Figure Wang et al., 2014; Andersson et al., 2014). The site in PIB-4-ATPases has previously been suggested to be by residues from the conserved from and motifs of and based on X-ray and (Zielazinski et al., 2012; et al., however, how the ion is then to the the that likely the of is from the the motif (Figure and Figure 1—figure supplement 3). We that from its in the to E2P to lower the cargo affinity to release the and (Figure The conserved of replaced with an in is this exit The also with the conserved in SsZntA (Figure which has been suggested to serve as a metal release from the motif of (Wang et al., 2014). We a similar role for in sCoaT as further by the activity of sCoaT form (Figure 2a). A unique internal P-type ATPases and counter and hence the reaction cycle cannot be without The of the counter transport has been in for example as Na+/K+-, and et al., et al., et al., et al., In the of counter transport has been proposed for (Wang et al., 2014), et al., and et al., which rather exploit a for the PIB-2-ATPases, a conserved of in as the through with the conserved metal-binding of in in E2 are not but a likely is not conserved in the group et al., it is possible that the for translocation is by the exit of the ion and et al., For the PIB-4-ATPases, have proposed an of (Zielazinski et al., 2012; Raimunda et al., 2012; et al., et al., no information is available regarding the presence or of counter transport. In the sCoaT structure, we a of between the residues, from the observed in (Figure Despite the packing of the clear is for indicating a (Figure Moreover, activity of an of demonstrate that it is crucial for function (Figure 2a). In light of these findings and an that a of the of in MtCtpD the ion affinity et al., we this histidine as an internal as for the invariant in