Structure and ion-release mechanism of PIB-4-type ATPases
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 .
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 ) . 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 ) . 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 . 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 . Interestingly , it has been shown that the in vivo transport specificity of the sCoaT homolog from Synechocystis PCC 6803 ( CoaT ) can be switched from Co2+ to Zn2+ by exchanging the N-terminal region to that of the Zn2+ transporting PIB-2 ATPase ZiaA from same organism ( Borrelly et al . , 2004 ) . This demonstrates that PIB-4-ATPases not only in vitro ( our data ) , but also in vivo are able to transport Zn2+ , if the M-domain gain access to the metal . One possible explanation for the change of specificity for the CoaT chimeric construct is that the N-terminal peptide tail , as also suggested for ATP7B ( Yu et al . , 2017 ) , prevents ATP hydrolysis through binding to the soluble domains , and this inhibition is then released upon binding of the cognate metal to the N-terminal and/or HMBD . However , it is also possible that the role of the N-terminal region of PIB-4 proteins is to impair Zn2+ acquisition , an ability that is lost when exchanged with the N-terminal part of ZiaA . Preliminary assessment of the metal specificity influence of the N-terminal tail of sCoaT suggests it has little or no effect on distinguishing between Co2+ and Zn2+ in vitro ( Figure 2—figure supplement 1d ) . From this it is clear that further studies are needed to shed light on the function of the N-terminal region in PIB-ATPases , also in PIB-4-ATPases . Associated , this raises questions also on the role of the above-mentioned MB’ platform , which has been proposed to serve as an interaction site for HMBDs in PIB-1- and PIB-2-ATPases , and for the Cu+-ATPases as a docking site for metal delivering chaperones ( Gourdon et al . , 2011b; Wang et al . , 2014; González-Guerrero and Argüello , 2008; Morin et al . , 2009 ) . As there are no known zinc/cadmium chaperones for PIB-4-ATPases , and because classical HMBDs appear to be missing in at least some proteins of the group , the MB’ function may need to be revisited . Alternatively , the N-terminus may have merely been maintained through evolution without conferring functional benefits or disadvantages . The classical view of P-type ATPases is that the E2P state is outward open and that the following transition state of dephosphorylation , E2 . Pi , is occluded , and that these conformations can be stabilized using the phosphate analogues employed here for structure determination , BeF3− and AlF4− , respectively . Furthermore , distinct ion-release pathways have been proposed among PIB-ATPases ( Gourdon et al . , 2011b; Wang et al . , 2014; Andersson et al . , 2014; Mattle et al . , 2015 ) , including a narrow exit pathway lined by MA , M2 , and M6 that remains open also in the E2 . Pi state for the PIB-1-ATPases . In contrast , a wide opening extending from the location of the bound metal in the M-domain of ion-occluded states to the non-cytoplasmic side has been observed for the PIB-2-ATPases , and this group becomes reoccluded with the E2P to E2 . Pi shift . Surprisingly however , analysis of the two obtained structures suggests that the anticipated significant domain reorientations are absent in sCoaT ( Figure 2b ) , and the models are in contrast rather similar . The compact assembly of the soluble domains and the position of the A-domain near the P-domain , placing the conserved TGE motif responsible for dephosphorylation towards the phosphorylation site , are typically associated with commencement of dephosphorylation , indicating that the two structures are trapped in an E2 . Pi like transition state ( Figure 2—figure supplement 3a , c , e ) . This observation differs from the equivalent structures of the other structurally determined PIB-ATPases , in which the phosphorylation site of the E2P state ( stabilized by BeF3− ) is shielded from the TGE loop as also observed for the well-studied sarcoendoplasmic reticulum Ca2+-ATPase ( SERCA ) ( Figure 2—figure supplement 3d ) . Notably , analogous highly similar BeF3−- and AlF4-stablized structures have recently also been observed for the Ca2+-specific P-type ATPase from Listeria monocytogenes ( LMCA1 ) ( Hansen , 2020 ) . It was proposed that LMCA1 preorganizes for dephosphorylation already in a late E2P state ( E2P* , stabilized by BeF3− ) , in accordance with its rapid dephosphorylation . Favoured occlusion and activation of dephosphorylation directly upon ion release may thus also be the case for sCoaT , and consequently the E2-BeF3− structure captured here may represents a late ( or quasi ) E2P state ( E2P* ) . Comparisons of the sCoaT structures to the equivalent structure of SsZntA ( E2 . Pi ) revealed a unique arrangement of the A-domain ( Figure 2—figure supplement 4 ) . The TGE-loop region superposes well with the corresponding area in SsZntA , but the rest of the A-domain is rotated towards the P-domain – approximately 14° and 5 . 3 Å ( Figure 2—figure supplement 4 ) . However , it cannot be excluded that this rotation is due to crystal contacts as the two peripheral β-sheets of the A-domain are interacting tightly with parts of a neighbouring molecule . Additionally , we noticed that the A-domain of sCoaT possesses a surface-exposed extension similar to SERCA , but this feature is not present in PIB-1- and PIB-2-ATPases and it is not a conserved property in the PIB-4 group either ( Figure 2—figure supplements 2 and 4 ) . Conversely , the M-domains of the two sCoaT structures are overall similar and appear outward occluded ( Figure 2c ) , as also supported by comparisons with the equivalent structures of SsZntA , again contrasting to the situation observed in PIB-1- and PIB-2-ATPases . Next , to shed light on ion release , we compared the sCoaT structures to the E2P state of SsZntA , in which the extracellular ends of M5 and M6 shifts away from the proposed metal-binding site , allowing an exit pathway to be formed ( Figure 2d ) . Considering that PIB-2- and PIB-4-ATPases have overlapping cargo range , share overall topology and that they release ions in free form to the extracellular environment , in contrast to their PIB-1 counterparts , we find it likely that they employ similar exit pathways , lined primarily by M2 , M4 , M5 , and M6 ( Figure 2d; Wang et al . , 2014; Andersson et al . , 2014 ) . The high-affinity-binding site in PIB-4-ATPases has previously been suggested to be formed by residues from the conserved SPC- ( starting from S325 ) and HEGxT- ( from H657 ) motifs of M4 and M6 , based on X-ray absorption spectroscopy and mutagenesis studies ( Zielazinski et al . , 2012; Patel et al . , 2016 ) . An outstanding remaining question is , however , how the ion is then discharged to the extracellular site ? Among the resides that likely constitute the high-affinity-binding site , remarkably E658 of M6 is pointing away from the ion-binding region around the SPC motif ( Figure 2e and Figure 1—figure supplement 3 ) . We anticipate that E658 rotates away from its ion-binding configuration in the E1P to E2P transition , thereby assisting to lower the cargo affinity to permit release via the M2 , M4 , M5 , and M6 cavity ( Figure 2e ) . The conserved E120 of M2 ( sometimes replaced with an aspartate in PIB-4-ATPases ) is located along this exit pathway . The residue also overlays with the conserved E202 in SsZntA ( Figure 2f ) , which has been suggested to serve as a transient metal ligand , stimulating substrate release from the CPC motif of PIB-2-ATPases ( Wang et al . , 2014 ) . We propose a similar role for E120 in sCoaT as further supported by the decreased activity of E120A sCoaT form ( Figure 2a ) . Many P-type ATPases couple ion- and counter transport , and hence the reaction cycle cannot be completed without counterions . The importance of the counter transport has been demonstrated in for example Ca2+/H+- ( such as SERCA ) , Na+/K+- , and H+/K+-ATPases ( Moller et al . , 2010; Faxén et al . , 2011; Abe et al . , 2018; Dyla et al . , 2020 ) . In contrast , the absence of counter transport has been proposed for PIB-2-ATPases ( Wang et al . , 2014 ) , H+-ATPases ( Pedersen et al . , 2007 ) , and P4-ATPases ( Nakanishi et al . , 2020 ) , which rather exploit a built-in counterion . Specifically for the PIB-2-ATPases , a conserved lysine of M5 ( K693 in SsZntA ) serves as the counterion , through interaction with the conserved metal-binding aspartate of M6 ( D714 in SsZntA ) in E2 states . Similarly , PIB-1-ATPases are not Cu+/H+ antiporters , but a likely built-in counterion residue is not conserved in the group ( Abeyrathna et al . , 2020 ) . Instead , it is possible that the requirement for counterion translocation is prevented by the narrow exit pathway , preventing backtransfer of the released ion and perhaps rendering complete occlusion unnecessary ( Abeyrathna et al . , 2020 ) . For the PIB-4-ATPases , biochemical studies have proposed an ion-binding stoichiometry of one ( Zielazinski et al . , 2012; Raimunda et al . , 2012; Smith et al . , 2017; Patel et al . , 2016 ) , however no information is available regarding the presence or absence of counter transport . In the E2-BeF3− sCoaT structure , we identify a tight configuration of HEGxT-motif H657 , being sandwiched between the SPC residues , distinct from the M5 lysine–M6 aspartate interaction observed in PIB-2-ATPases ( Figure 2g , h ) . Despite the packing issues of the generated crystals , clear electron density is visible for H657 , indicating a rigid conformation ( Figure 2g ) . Moreover , activity measurements of an alanine substitution of H657 demonstrate that it is crucial for function ( Figure 2a ) . In light of these findings and an earlier report suggesting that a mutation of the equivalent of H657 in MtCtpD leaves the ion affinity unaffected ( Patel et al . , 2016 ) , we suggest this histidine serves as an internal counterion , similarly as for the invariant lysine in SsZntA , perhaps preventing backtransfer of released ions and for charge stabilization , however we cannot exclude that H657 is also part of the high-affinity-binding site in sCoaT . The rigid conformation observed for H657 in the E2-BeF3− structure is also observed in the E2-AlF4− structure ( Figure 1—figure supplement 3b ) . In contrast , for SsZntA the interaction between K693 and D714 is only detected in the E2 . Pi state . Thus , the interaction pattern is consistent with the idea that sCoaT preorganizes for dephosphorylation already in the ( late ) E2P state , with the associated occlusion and internal counter–ion interaction taking place earlier than for SsZntA . A conserved K+ site , which cross-links between the A- and P-domains in E2 states and thereby allosterically stimulates the E2P to E2 process ( Sørensen et al . , 2004; Schack et al . , 2008 ) , has been suggested to be present also in PIB-ATPases ( Sørensen et al . , 2004 ) . However , our new E2 structures and available structures of PIB-1- and PIB-2-ATPases suggest that the A-/P-domain linker is maintained without K+ in PIB-ATPases , and instead is established directly between R273/D601 in sCoaT , as also supported by potassium titration experiments monitoring sCoaT ATPase activity ( Figure 3a–d ) . Nevertheless , the A-/P-domain point-of-interaction appears critical for PIB-ATPases , as functional characterization of R273A , D601A , and D601K result in a marked reduction of turnover ( Figure 2a ) . This differs from similar mutations of classical P-type ATPases , where only minor effects are observed ( Sørensen et al . , 2004; Schack et al . , 2008 ) . Furthermore , substitution of D601 with glutamate suggests that even the A-/P-domain distance is critical ( Figure 2a ) . It is possible that PIB-ATPases are more reliant on this particularly tight , ion-independent stabilization , as the A–M1/A-domain linker is absent , and because many other P-type ATPases also have a complementary A-/P-domain interaction ( Figure 1—figure supplement 1c ) . Thus , our data indicate that this regulation is a general feature of many P-type ATPase classes , yet featuring unique properties for PIB-ATPases . PIB-2- and PIB-4-ATPases serve as virulence factors and are critical for the disease caused by many microbial pathogens , as underscored by the frequent presence of several redundant genes ( Sassetti and Rubin , 2003; Joshi et al . , 2006; Botella et al . , 2011; Pi et al . , 2016 ) . In this light and because these P-type ATPases are missing in humans , they represent putative targets for novel antibiotics . The shared mechanistic principles identified here suggest that compounds can be identified that inhibit both PIB groups , for example directed against the common release pathway , thereby increasing efficacy . Indeed , screening of a 20 , 000-substance library using a complementary in vitro assay , uncovers several compounds that abrogate function of sCoaT and SsZntA ( Figure 3e , f , data only shown for sCoaT ) . Furthermore , initial tests of two of these suggest they have a potent effect against mycobacteria , which previously have been shown to be PIB-4 dependent for infection ( Patel et al . , 2016 ) ; 90% of the mycobacteria were killed at mean concentrations of 18 . 75 and above 50 µM , respectively , using either of these two separate molecules ( Figure 3g ) . In contrast , investigation of cytotoxic effects on primary human macrophages at concentrations up to 25 µM demonstrated considerably less impact on cell survival for both blockers ( Figure 3h ) . Evidently downstream in-depth studies , ranging from investigations of the target specificity , the detailed effect on human cells as well as antibiotic potency in human , are required to fully understand the value of these putative PIB-2- and PIB-4 inhibitors . Nevertheless , the substances outlined here represent promising leads for drug-discovery efforts or to aid the development of tools to manipulate heavy-metal accumulation in plants to prevent accumulation or for enrichment . Collectively , the first structure of a PIB-4-type ATPase reveals the topology of PIB-4-ATPases , displaying an eight helix M-domain configuration , and likely no HMBDs , at least in members without extended N-termini . Major findings include the observation of an ion-release pathway similar as in the related PIB-2-ATPases , a previously not observed counterion principle for P-type ATPases , and a unique potassium-independent regulation of the PIB-transport cycle ( Figure 4 ) . Thus , our results significantly increase the understanding of heavy-metal homeostasis in cells . The novel identified putative inhibitors and the partially overlapping mechanistic principles of PIB-2- and PIB-4-ATPases also open up a novel avenue for development of compounds accessible from outside the cell against these PIB groups , to combat global threats such as multidrug resistance and/or tuberculosis or for biotechnological purposes .
Forms of the 72 kDa sCoaT from Sulfitobacter sp . NAS14-1 ( UniProt ID A3T2G5 ) were transformed into E . coli ( C41 strain ) cells . The cells were cultured in LB medium at 37°C with shaking at 175 rpm in baffled flasks until the optical density ( 600 nm ) reached 0 . 6–1 , cooled to 18°C , and then induced with 1 mM IPTG for 16 hr . Harvested cells were resuspended in buffer A ( 1 g cells per 5 ml buffer ) containing 20 mM Tris–HCl , pH = 7 . 6 , 200 mM KCl , 20% ( vol/vol ) glycerol and frozen at −80°C until further use . Cells were disrupted by two runs in a high-pressure homogenizer ( Constant System ) at 25 , 000 psi following addition of 5 mM of fresh β-mercaptoethanol ( BME ) , 5 mM MgCl2 , 1 mM phenylmethanesulphonyl fluoride , 2 μg/ml DNase I and Roche protease inhibitor cocktail ( 1 tablet for 6 l cells ) . The sample was kept at 4°C throughout the purification . Cellular debris was pelleted via centrifugation at 20 , 000 × g for 20 min . Membranes were isolated by ultracentrifugation for 3 hr at 185 , 500× g , and resuspended in 10 ml buffer B ( 20 mM Tris–HCl , pH = 7 . 6 , 200 mM KCl , 1 mM MgCl2 , 5 mM BME , and 20% [vol/vol] glycerol ) per g membranes and frozen at −80°C until further use . The protein concentration in the membranes was estimated using the Bradford , 1976 assay . Proteins were solubilized through supplementation of 1% ( wt/vol ) final concentration n-dodecyl-β-D-maltopyranoside ( DDM ) and 3 mg/ml final total protein concentration in buffer B with gentle stirring for 2 hr . Unsolubilized material was removed by ultracentrifugation for 1 hr at 185 , 500 × g . The supernatant was supplemented with imidazole to a final concentration of 30 mM and solid KCl ( 500 mM final concentration ) , filtered ( 0 . 22 mm ) , and then applied to 5 ml HiTrap Chelating HP columns ( GE Healthcare , protein from 6 l cells per column ) charged with Ni2+ and equilibrated with four column volumes of buffer C ( 20 mM Tris–HCl , pH = 7 . 6 , 200 mM KCl , 1 mM MgCl2 , 5 mM BME , 150 mg/ml octaethylene glycol monododecyl ether [C12E8] and 20% [vol/vol] glycerol ) . Proteins were eluted using a gradient , ending with buffer C containing 500 mM imidazole . Eluted protein was assessed using sodium dodecyl sulphate–polyacrylamide gel electrophoresis ( SDS–PAGE ) , and the fractions containing sCoaT concentrated to approximately 20 mg/ml using VivaSpin concentrators ( MWCO = 50 kDa ) . 10 mg concentrated protein was subjected to size-exclusion chromatography using a Superose six gel-filtration column ( GE-Healthcare ) , pre-equilibrated with 50 ml buffer E ( 20 mM Tris–HCl , pH = 7 . 6 , 80 mM KCl , 1 mM MgCl2 , 5 mM BME , 150 mg/ml C12E8 and 20% [vol/vol] glycerol ) . Fractions containing purified sCoaT were pooled , and concentrated to approximately 10 mg/ml , flash frozen in liquid nitrogen , and stored at −80°C until further use . For the experiments to assess K+ dependence , the buffer E was replaced with 20 mM Tris–HCl , pH = 7 . 5 , 1 mM MgCl2 , 5 mM BME , 0 . 15 mg/ml C12E8 , and 20% ( vol/vol ) glycerol . 10 mg/ml sCoaT was supplemented with 3 mg/ml ( final concentration ) DOPC and 6 mg/ml ( final concentration ) C12E8 , incubated at 4°C and stirring for 16–48 hr ( modified HiLiDe method Gourdon et al . , 2011a ) . Aggregates and insoluble DOPC were then removed by ultracentrifugation at 50 , 000 ×g for 10 min . 2 mM AlCl3 or BeSO4 , 10 mM NaF , and 2 mM EGTA ( final concentrations ) were supplemented and incubated on ice for 30 min . Crystals were grown using the hanging drop vapour diffusion method at 19°C . E2-AlF4− crystals were grown with a reservoir solution containing 200 mM MgCl2 , 14% ( vol/vol ) PEG1500 , 10 mM tris ( 2-carboxyethyl ) phosphine , 10% ( vol/vol ) glycerol , 3% 2-methyl-2 , 4-pentanediol , and 100 mM sodium acetate , pH = 5 . 0 . The E2-BeF3− crystals were grown with a reservoir solution containing 200 mM magnesium formate , 14% ( vol/vol ) PEG5000 , 100 mM sodium acetate , pH = 4 . 0 , and 0 . 5% ( vol/vol ) 2-propanol was added as an additive . Crystals were fished using litholoops ( Molecular Dimensions ) , flash cooled in liquid nitrogen , and tested at synchrotron sources . Complete final data sets were collected at the Swiss Light Source , the Paul Scherrer Institute , Villigen , beam line X06SA . Collected data were processed and scaled with XDS ( Table 1 ) . For the E2-AlF4− structure , initial phases were obtained by the MR method using software PHASER ( McCoy et al . , 2007 ) of the Phenix package ( Liebschner et al . , 2019 ) , and using the AlF4−-stabilized structure of SsZntA ( PDB ID: 4UMW ) as a search model . The E2-BeF3− structure was solved using the generated E2-AlF4− structure as a MR model . Both crystal forms display poor crystal packing between the membrane domains ( Figure 1—figure supplement 2 ) , deteriorating the quality of the electron density maps in these regions ( Figure 1—figure supplement 3 ) . In this light , model building of the membrane domains was executed with particular prudence , taking into consideration the connectivity to the well-resolved soluble domains , distinct structural features as well as sequence and structure conservation patterns . Examples of such include the conserved GG motif that forms the kink in MB helix , which is clearly identified also at low length , the SPC motif that twists the M4 helix and the conserved and functionally important well-resolved residue H657 that assisted assigning nearby residues . Initial manual model building was performed primarily using COOT ( Emsley et al . , 2010 ) . ISOLDE ( Croll , 2018 ) in ChimeraX ( Goddard et al . , 2018 ) was employed for model building and analysis , and was critical for obtainment of the final models with reasonable chemical restraints and low clash score . In particular , ISOLDE’s interactive register shifting tool was instrumental in determining the register of the most weakly resolved TM helices . Secondary structure restraints were applied in some flexible regions , also taking into consideration homology to sCoaT and other models . During final refinements with phenix . refine ( Afonine et al . , 2012 ) , the geometry was restrained in torsion space to ISOLDE’s output . Molprobity was exploited for structure validation ( Williams et al . , 2018 ) . The final models are lacking the first 40 residues only , which is shorter than a classical MBD of 67 amino acids . All structural figures were generated using Pymol ( DeLano , 2002 ) . Statistics for the final models were 96 . 70 , 3 . 30 , 0 . 20 , and 0 , 74 for E2-BeF3− and 93 . 24 , 6 . 13 , 0 . 63 , and 8 . 31 for E2-AlF4− in Ramachandran favored and allowed regions , and for rotamer outliers and clash score , respectively . Activity assay sCoaT forms were functionally characterized using the Baginski method to assess the amount of released inorganic phosphate ( Baginski et al . , 1967 ) . Briefly , 0 . 5 μg of purified sCoaT mixed with reaction buffer containing 40 mM MOPS–KOH , pH = 6 . 8 , 5 mM KCl , 5 mM MgCl2 , 150 mM NaCl , 0 . 3 mg/ml C12E8 , 0 . 12 mg/ml soybean lipid , 5 mM NaN3 , and 0 . 25 mM Na2MoO4 in a total volume of 50 μl . For metal stimulation assays , different heavy-metal ions or EGTA were supplemented the reaction buffer to a final concentration of 50 µM . For inhibitor screening ( see how inhibitors were identified below ) , different concentrations of inhibitors were added to the reaction buffer containing 50 μM ZnCl2 . The samples were then incubated at 37°C with 500 rpm shaking for 5 min , and then supplemented with 5 mM ATP ( final concentration ) to start the reaction , and incubated at 37°C with 1000 rpm shaking for 10 min . 50 μl freshly prepared stop solution containing 2 . 5% ( wt/vol ) ascorbic acid , 0 . 4 M ( vol/vol ) HCl , and 1% SDS was then supplemented to stop the reaction and start colour development . 75 μl colour solution ( 2% [wt/vol] arsenite , 2% [vol/vol] acetic acid , and 3 . 5% [wt/vol] sodium citrate ) was added to the mixture following 10-min incubation at room temperature . Absorbance was measured at 860 nm after another 30-min incubation at room temperature . For the experiments to assess K+ dependence , the reaction buffer was replaced with 40 mM Tris–HCl , pH = 7 . 5 , 5 mM MgCl2 , 3 . 0 mg/ml C12E8 , and 1 . 2 mg/ml soybean lipid in a total volume of 50 μl . The inhibitor screening experiments were initially carried out on the zinc transporting PIB-2-type ATPase ZntA from Shigella sonnei ( SsZntA ) . SsZntA was produced and purified as described previously ( Wang et al . , 2014 ) and the inhibitory effect of approximately 20 , 000 compounds was assessed by the Chemical biology Consortium Sweden ( CBCS ) . Briefly , the ATPase activity of 0 . 7 µM highly pure protein was measured in the presence of 50 µM inhibitor through the release of inorganic phosphate ( Pi ) by the Baginski assay ( Baginski et al . , 1967 ) in a total volume of 200 nl as reported earlier ( Wang et al . , 2014 ) . The inorganic phosphate was detected with Malachite Green reagent ( 0 . 005% Carbinol hydrochloride , 1 . 7% sulfuric acid , 0 . 14% ammonium molybdate , 0 . 025% Triton-X ) at an absorbance of 620 nm . Mycobacterium bovis bacillus Calmette–Guerin ( BCG ) Montreal containing the pSMT1-luxAB plasmid was prepared as previously described ( Snewin et al . , 1999 ) . Briefly , the mycobacteria were grown in Middlebrook 7H9 broth , supplemented with 10% ADC enrichment ( Middlebrook Albumin Dextrose Catalase Supplement , Becton Dickinson , Oxford , UK ) and hygromycin ( 50 mg/l; Roche , Lewes , UK ) , the culture was washed twice with sterile PBS , and resuspended in broth and then dispensed into vials . Glycerol was added to a final concentration of 25% and the vials were frozen at −80°C . Prior to each experiment , a vial was defrosted , added to 9 ml of 7H9/ADC/hygromycin medium , and incubated with shaking for 72 hr at 37°C . Mycobacteria were then centrifuged for 10 min at 3000 × g , washed twice with PBS , and resuspended in 10 ml of PBS . Resazurin microtiter assay was used to determine the minimum inhibitory concentration ( MIC90 ) for the inhibitors against the mycobacterial strain . The inhibitors ( 10 µl ) were added to bacterial suspensions ( 90 µl ) on a 96-well plate at a concentration range between 0 . 4 and 50 . 0 µM . MIC was determined by the colour change using resazurin ( 1:10 vol/vol , PrestoBlue Cell viability reagent , Thermo Scientific ) . MIC was determined after 1 week by adding 10 µl resazurin followed by incubation overnight , corresponding to 90% inhibition . Human venous blood mononuclear cells were obtained from healthy volunteers using a Lymphoprep density gradient ( Axis-Shield , Oslo , Norway ) according to the manufacturer’s instructions . To obtain pure monocytes , CD14 microbeads were applied to the cell suspension , washed , and passed through a LS column according to the manufacturer’s description ( 130-050-201 , 130-042-401 , Miltenyi Biotec , USA ) . The monocytes were counted ( Sysmex ) , diluted in RPMI 1640 supplemented with 5% FCS , NEAA , 1 mM sodium pyruvate , 0 . 1 mg/ml gentamicin ( 11140-035 , 111360-039 , 15710-49 , Gibco , Life Technologies ) and 50 ng/ml GM-CSF ( 215 GM , R&D Systems ) and seeded in 96-well plates ( 105/well ) for a week to differentiate into macrophages . Infection experiments were performed in RPMI 1640 without Gentamicin . The medium was replaced with fresh medium containing 6 . 3 , 12 . 5 , 25 , or 50 μM inhibitor or DMSO and incubated 24 hr in 5% CO2 atmosphere . For cytotoxicity measurement , 10 μl 3- ( 4 , 5-dimethylthiazol-2-yl ) –2 , 5 diphenyltetrazolium bromide solution ( Sigma ) was added to each well according to the manufacturer’s instructions and analysed in a spectrophotometer at 580 nm . NZX cytotoxicity was further examined by ATPlite assays . Primary macrophages were treated with 6 . 3 , 12 . 5 , 25 , or 50 μM inhibitor or DMSO ( Sigma ) for 24 hr . Cell viability was assessed with cellular ATP levels using ATPlite kit ( 6016943 , Perkin Elmer ) compared to untreated controls , according to the manufacturer’s instructions . The two crystal structures , E2-AlF4− and E2-BeF3− , were inserted into a DOPC ( 1 , 2-dioleoyl-sn-glycero-3-phosphocholine ) membrane patch using the CHARMM-GUI membrane builder ( Wu et al . , 2014 ) . The membrane positions were predicted by the Orientations of Proteins in Membranes ( OPM ) server ( Lomize et al . , 2012 ) . During the simulation equilibration phase , position restraints were gradually released from the water and lipids for a total of 30 ns followed by 500 ns non-restrained production runs . Each protein state was simulated in independent repeat simulations starting from a different set of initial velocities , adding up to a sampling total of 500 ns × 4 . A Nose–Hoover temperature coupling ( Nosé and Klein , 2006 ) was applied using a reference temperature of 310 K . A Parrinello–Rahman pressure coupling ( Parrinello and Rahman , 1981 ) was applied with a reference pressure of 1 bar and compressibility of 4 . 5e−5 bar−1 in a semi-isotropic environment . The TIP3P water model was used and the system contained 0 . 15 M NaCl . The E2-AlF4− system was composed of 256 lipids and 29 , 429 water molecules while E2-BeF3− system was composed of 254 lipids and 30 , 535 water molecules . The systems were equilibrated and simulated using the GROMACS-2021 simulation package ( Abraham et al . , 2015 ) and CHARMM36 all-atom force fields ( Best et al . , 2012 ) for the protein and lipids . The membrane domain was used as alignment reference for the root means square deviation and centre-of-mass calculations , and the protein backbone was used as alignment reference for calculating the root mean square fluctuation . The secondary structure was assessed with the do_dssp tool in GROMACS-2021 ( Abraham et al . , 2015 ) . Atomic coordinates and structure factors for the sCoaT AlF4−- and BeF3−-stabilized crystal structures have been deposited at the Protein Data Bank ( PDB ) under accession codes 7QBZ and 7QC0 . The authors declare no competing financial interests . Correspondence and requests for materials should be addressed to P . G . ( pontus@sund . ku . dk ) .
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 .
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 .