Acidic residues in the lactococcal multidrug efflux pump LmrP play critical roles in transport of lipophilic cationic compounds

The proton motive force-driven efflux pump LmrP confers multidrug resistance on Lactococcus lactis cells by extruding a wide variety of lipophilic cationic compounds from the inner leaflet of the cytoplasmic membrane to the exterior of the cell. LmrP contains one cysteine (Cys(270)), which was replaced by alanine. This cysteine-less variant was used in a cysteine scanning accessibility approach. All 19 acidic residues in LmrP were replaced one by one by cysteine and subsequently challenged with the large thiol reagent fluorescein maleimide. The labeling pattern strongly indicates that only three acidic residues (Asp(142), Glu(327), and Glu(388)) are membrane-embedded. The roles of these residues in drug recognition were evaluated based on transport experiments with two cationic substrates, ethidium and Hoechst 33342, after replacing each of these residues with cysteine, alanine, lysine, glutamate, or aspartate. The obtained results suggest that the negative charges at positions 142 and 327 are not critical for the transport function but are important for drug recognition by LmrP. Surprisingly, the residues Cys(142) and Cys(327) become accessible for fluorescein maleimide upon binding of substrates, indicating a movement of these residues from a nonpolar to a polar environment. Substrate binding apparently results in a conformational change in this region of the protein and a reorientation of a lipid-embedded, hydrophobic substrate-binding site to an aqueous substrate translocation pathway.     

Facilitated drug influx by an energy-uncoupled secondary multidrug transporter

The majority of bacterial multidrug resistance transporters belong to the class of secondary transporters. LmrP is a proton/drug antiporter of Lactococcus lactis that extrudes positively charged lipophilic substrates from the inner leaflet of the membrane to the external medium. This study shows that LmrP is a true secondary transporter. In the absence of a proton motive force, LmrP facilitates downhill fluxes of ethidium in both directions. These fluxes are inhibited by other substrates of LmrP. The cysteine-reactive agent p-chloromercuri-benzene sulfonate inhibits these fluxes in wild type LmrP but not in the cysteine-less LmrP C270A mutant. Cysteine mutagenesis of LmrP resulted in three mutants, D68C/C270A, D128C/C270A, and E327C/C270A, with an energy-uncoupled phenotype. Asp68 is located in the conserved motif GXXX(D/E)(R/K)XGRK for the major facilitator superfamily of secondary transporters and was found to play an important role in energy coupling, whereas the negatively charged residues Asp128 and Glu327 have indirect effects on the transport process. L. lactis strains expressing these uncoupled mutants of LmrP show an increased rate of ethidium influx and an increased drug susceptibility compared with cells harboring an empty vector. The rate of influx in these mutants is enhanced by a transmembrane electrical potential, inside negative. These observations suggest a new strategy for eliminating drug-resistant microbial pathogens, i.e. the design and use of modulators of secondary multidrug resistance transporters that uncouple drug efflux from proton influx, thereby allowing transmembrane electrical potential-driven influx of cationic drugs.     

Two proton translocation pathways in a secondary active multidrug transporter

LmrP is a secondary active multidrug transporter from Lactococcus lactis. The protein belongs to the major facilitator superfamily and utilizes the electrochemical proton gradient (inside negative and alkaline) to extrude a wide range of lipophilic cations from the cell. Previous work has indicated that ethidium, a monovalent cationic substrate, is exported by LmrP by electrogenic antiport with two (or more) protons. This observation raised the question whether these protons are translocated sequentially along the same pathway, or through different routes. To address this question, we constructed a 3-D homology model of LmrP based on the high-resolution structure of the glycerol-3P/Pi antiporter GlpT from Escherichia coli, and we tested by mutagenesis the possible proton conduction points suggested by this model. Similar to the template, LmrP is predicted to contain an internal cavity formed at the interface between the two halves of the transporter. On the surface of this cavity lie two clusters of polar, aromatic and carboxylate residues with potentially important function in proton shuttling. Cluster 1 in the C-terminal half contains D235 and E327 in immediate proximity of each other, and is located near the apex of the cavity. Cluster 2 in the N-terminal half contains D142. Analyses of LmrP mutants containing charge-conservative or carboxyl-to-amide replacements at positions 142, 235 and 327 suggest that D142 is part of a dedicated proton translocation pathway in the ethidium translocation reaction. In contrast, D235 and E327 are part of an independent pathway, in which D235 interacts with protons. E327 appears to modulate the pKa of D235 and plays a role in the interaction with ethidium. These results are consistent with the proposal that major facilitator superfamily proteins consist of two membrane domains, one of which is involved in substrate binding and the other in ion coupling, and they indicate that there are two proton conduction pathways at play in the transport mechanism.     

Basic residues R260 and K357 affect the conformational dynamics of the major facilitator superfamily multidrug transporter LmrP

Secondary-active multidrug transporters can confer resistance on cells to pharmaceuticals by mediating their extrusion away from intracellular targets via substrate/H(+)(Na(+)) antiport. While the interactions of catalytic carboxylates in these transporters with coupling ions and substrates (drugs) have been studied in some detail, the functional importance of basic residues has received much less attention. The only two basic residues R260 and K357 in transmembrane helices in the Major Facilitator Superfamily transporter LmrP from Lactococcus lactis are present on the outer surface of the protein, where they are exposed to the phospholipid head group region of the outer leaflet (R260) and inner leaflet (K357) of the cytoplasmic membrane. Although our observations on the proton-motive force dependence and kinetics of substrate transport, and substrate-dependent proton transport demonstrate that K357A and R260A mutants are affected in ethidium-proton and benzalkonium-proton antiport compared to wildtype LmrP, our findings suggest that R260 and K357 are not directly involved in the binding of substrates or the translocation of protons. Secondary-active multidrug transporters are thought to operate by a mechanism in which binding sites for substrates are alternately exposed to each face of the membrane. Disulfide crosslinking experiments were performed with a double cysteine mutant of LmrP that reports the substrate-stimulated transition from the outward-facing state to the inward-facing state with high substrate-binding affinity. In the experiments, the R260A and K357A mutations were found to influence the dynamics of these major protein conformations in the transport cycle, potentially by removing the interactions of R260 and K357 with phospholipids and/or other residues in LmrP. The R260A and K357A mutations therefore modify the maximum rate at which the transport cycle can operate and, as the transitions between conformational states are differently affected by components of the proton-motive force, the mutations also influence the energetics of transport.     

The Multidrug Transporter LmrP Protein Mediates Selective Calcium Efflux

LmrP is a major facilitator superfamily multidrug transporter from Lactococcus lactis that mediates the efflux of cationic amphiphilic substrates from the cell in a proton-motive force-dependent fashion. Interestingly, motif searches and docking studies suggested the presence of a putative Ca(2+)-binding site close to the interface between the two halves of inward facing LmrP. Binding experiments with radioactive (45)Ca(2+) demonstrated the presence of a high affinity Ca(2+)-binding site in purified LmrP, with an apparent K(d) of 7.2 μm, which is selective for Ca(2+) and Ba(2+) but not for Mn(2+), Mg(2+), or Co(2+). Consistent with our structure model and analogous to crystal structures of EF hand Ca(2+)-binding proteins, two carboxylates (Asp-235 and Glu-327) were found to be critical for (45)Ca(2+) binding. Using (45)Ca(2+) and a fluorescent Ca(2+)-selective probe, calcium transport measurements in intact cells, inside-out membrane vesicles, and proteoliposomes containing functionally reconstituted purified protein provided strong evidence for active efflux of Ca(2+) by LmrP with an apparent K(t) of 8.6 μm via electrogenic exchange with three or more protons. These observations demonstrate for the first time that LmrP mediates selective calcium/proton antiport and raise interesting questions about the functional and physiological links between this reaction and that of multidrug transport.     

Hoechst 33342 Is a Hidden “Janus” amongst Substrates for the Multidrug Efflux Pump LmrP

Multidrug transporters mediate the active extrusion of antibiotics and toxic ions from the cell. This reaction is thought to be based on a switch of the transporter between two conformational states, one in which the interior substrate binding cavity is available for substrate binding at the inside of the cell, and another in which the cavity is exposed to the outside of the cell to enable substrate release. Consistent with this model, cysteine cross-linking studies with the Major Facilitator Superfamily drug/proton antiporter LmrP from Lactococcus lactis demonstrated binding of transported benzalkonium to LmrP in its inward-facing state. The fluorescent dye Hoechst 33342 is a substrate for many multidrug transporters and is extruded by efflux pumps in microbial and mammalian cells. Surprisingly, and in contrast to other multidrug transporters, LmrP was found to actively accumulate, rather than extrude, Hoechst 33342 in lactococcal cells. Consistent with this observation, LmrP expression was associated with cellular sensitivity, rather than resistance to Hoechst 33342. Thus, we discovered a hidden “Janus” amongst LmrP substrates that is translocated in reverse direction across the membrane by binding to outward-facing LmrP followed by release from inward-facing LmrP. These findings are in agreement with distance measurements by electron paramagnetic resonance in which Hoechst 33342 binding was found to stabilize LmrP in its outward-facing conformation. Our data have important implications for the use of multidrug exporters in selective targeting of “Hoechst 33342-like” drugs to cells and tissues in which these transporters are expressed.     

Identification of essential amino acid residues of the NorM Na+/multidrug antiporter in Vibrio parahaemolyticus

NorM is a member of the multidrug and toxic compound extrusion (MATE) family and functions as a Na+/multidrug antiporter in Vibrio parahaemolyticus, although the underlying mechanism of the Na+/multidrug antiport is unknown. Acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM are conserved in one of the clusters of the MATE family. In this study, we investigated the role(s) of acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM by site-directed mutagenesis. Wild-type NorM and mutant proteins with amino acid replacements D32E (D32 to E), D32N, D32K, E251D, E251Q, D367A, D367E, D367N, and D367K were expressed and localized in the inner membrane of Escherichia coli KAM32 cells, while the mutant proteins with D32A, E251A, and E251K were not. Compared to cells with wild-type NorM, cells with the mutant NorM protein exhibited reduced resistance to kanamycin, norfloxacin, and ethidium bromide, but the NorM D367E mutant was more resistant to ethidium bromide. The NorM mutant D32E, D32N, D32K, D367A, and D367K cells lost the ability to extrude ethidium ions, which was Na+ dependent, and the ability to move Na+, which was evoked by ethidium bromide. Both E251D and D367N mutants decreased Na+-dependent extrusion of ethidium ions, but ethidium bromide-evoked movement of Na+ was retained. In contrast, D367E caused increased transport of ethidium ions and Na+. These results suggest that Asp32, Glu251, and Asp367 are involved in the Na+-dependent drug transport process.     

Role of a conserved membrane-embedded acidic residue in the multidrug transporter MdfA

According to the current topology model of the Escherichia coli multidrug transporter MdfA, it contains a membrane-embedded negatively charged residue, Glu26, which was shown to play an important role in substrate recognition. To further elucidate the role of this substrate recognition determinant, various Glu26 replacements were characterized. Surprisingly, studies with neutral MdfA substrates showed that, unlike many enzymatic systems where the size and chemical properties of binding site residues are relatively defined, MdfA tolerates a variety of changes at position 26, including size, hydrophobicity, and charge. Moreover, although efficient transport of positively charged substrates requires a negative charge at position 26 (Glu or Asp), neutralization of this charge does not always abrogate the interaction of MdfA with cationic drugs, thus demonstrating that the negative charge does not play an essential role in the multidrug transport mechanism. Collectively, these results suggest a link between the broad substrate specificity profile of multidrug transporters and the structural and chemical promiscuity at their substrate recognition pockets.     

Role of glutamate residues in substrate recognition by human MATE1 polyspecific H+/organic cation exporter

Human multidrug and toxic compound extrusion 1 (hMATE1) is an electroneutral H(+)/organic cation exchanger responsible for the final excretion step of structurally unrelated toxic organic cations in kidney and liver. To elucidate the molecular basis of the substrate recognition by hMATE1, we substituted the glutamate residues Glu273, Glu278, Glu300, and Glu389, which are conserved in the transmembrane regions, for alanine or aspartate and examined the transport activities of the resulting mutant proteins using tetraethylammonium (TEA) and cimetidine as substrates after expression in human embryonic kidney 293 (HEK-293) cells. All of these mutants except Glu273Ala were fully expressed and present in the plasma membrane of the HEK-293 cells. TEA transport activity in the mutant Glu278Ala was completely absent. Both Glu300Ala and Glu389Ala and all aspartate mutants exhibited significantly decreased activity. Glu273Asp showed higher affinity for cimetidine, whereas it has reduced affinity to TEA. Glu278Asp showed decreased affinity to cimetidine. Both Glu300Asp and Glu389Asp had lowered affinity to TEA, whereas the affinity of Glu389Asp to cimetidine was fourfold higher than that of the wild-type transporter with about a fourfold decrease in V(max) value. Both Glu273Asp and Glu300Asp had altered pH dependence for TEA uptake. These results suggest that all of these glutamate residues are involved in binding and/or transport of TEA and cimetidine but that their individual roles are different.     

The key residue for substrate transport (Glu14) in the EmrE dimer is asymmetric

Transport proteins exhibiting broad substrate specificities are major determinants for the phenomenon of multidrug resistance. The Escherichia coli multidrug transporter EmrE, a 4-transmembrane, helical 12-kDa membrane protein, forms a functional dimer to transport a diverse array of aromatic, positively charged substrates in a proton/drug antiport fashion. Here, we report (13)C chemical shifts of the essential residue Glu(14) within the binding pocket. To ensure a native environment, EmrE was reconstituted into E. coli lipids. Experiments were carried out using one- and two-dimensional double quantum filtered (13)C solid state NMR. For an unambiguous assignment of Glu(14), an E25A mutation was introduced to create a single glutamate mutant. Glu(14) was (13)C-labeled using cell-free expression. Purity, labeling, homogeneity, and functionality were probed by mass spectrometry, NMR spectroscopy, freeze fracture electron microscopy, and transport assays. For Glu(14), two distinct sets of chemical shifts were observed that indicates structural asymmetry in the binding pocket of homodimeric EmrE. Upon addition of ethidium bromide, chemical shift changes and altered line shapes were observed, demonstrating substrate coordination by both Glu(14) in the dimer.     

Cholate resistance in Lactococcus lactis is mediated by an ATP-dependent multispecific organic anion transporter

The cholate-resistant Lactococcus lactis strain C41-2, derived from wild-type L. lactis MG1363 through selection for growth on cholate-containing medium, displayed a reduced accumulation of cholate due to an enhanced active efflux. However, L. lactis C41-2 was not cross resistant to deoxycholate or cationic drugs, such as ethidium and rhodamine 6G, which are typical substrates of the multidrug transporters LmrP and LmrA in L. lactis MG1363. The cholate efflux activity in L. lactis C41-2 was not affected by the presence of valinomycin plus nigericin, which dissipated the proton motive force. In contrast, cholate efflux in L. lactis C41-2 was inhibited by ortho-vanadate, an inhibitor of P-type ATPases and ATP-binding cassette transporters. Besides ATP-dependent drug extrusion by LmrA, two other ATP-dependent efflux activities have previously been detected in L. lactis, one for the artificial pH probe 2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF) and the other for the artificial pH probe N-(fluorescein thio-ureanyl)-glutamate (FTUG). Surprisingly, the efflux rate of BCECF, but not that of FTUG, was significantly enhanced in L. lactis C41-2. Further experiments with L. lactis C41-2 cells and inside out membrane vesicles revealed that cholate and BCECF inhibit the transport of each other. These data demonstrate the role of an ATP-dependent multispecific organic anion transporter in cholate resistance in L. lactis.     

Multidrug resistance in lactic acid bacteria: molecular mechanisms and clinical relevance

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. The secondary multidrug transporter LmrP and the ATP-binding cassette (ABC) type multidrug transporter LmrA in Lactococcus lactis are representatives of the two major classes of multidrug transporters found in pro- and eukaryotic organisms. Therefore, knowledge of the molecular properties of LmrP and LmrA will have a wide significance for multidrug transporters in all living cells, and may enable the development of specific inhibitors and of new drugs which circumvent the action of multidrug transporters. Interestingly, LmrP and LmrA are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by t he same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA will represent an intriguing new area of research.     

A membrane-embedded glutamate is required for ligand binding to the multidrug transporter EmrE

EmrE is an Escherichia coli multidrug transporter that confers resistance to a variety of toxins by removing them in exchange for hydrogen ions. The detergent-solubilized protein binds tetraphenylphosphonium (TPP(+)) with a K(D) of 10 nM. One mole of ligand is bound per approximately 3 mol of EmrE, suggesting that there is one binding site per trimer. The steep pH dependence of binding suggests that one or more residues, with an apparent pK of approximately 7.5, release protons prior to ligand binding. A conservative Asp replacement (E14D) at position 14 of the only membrane-embedded charged residue shows little transport activity, but binds TPP(+) at levels similar to those of the wild-type protein. The apparent pK of the Asp shifts to <5.0. The data are consistent with a mechanism requiring Glu14 for both substrate and proton recognition. We propose a model in which two of the three Glu14s in the postulated trimeric EmrE homooligomer deprotonate upon ligand binding. The ligand is released on the other face of the membrane after binding of protons to Glu14.     

Interactions between phosphatidylethanolamine headgroup and LmrP, a multidrug transporter: a conserved mechanism for proton gradient sensing?

In a number of cases, the function of membrane proteins appears to require the presence of specific lipid species in the bilayer. We have shown that the secondary multidrug transporter LmrP requires the presence of phosphatidylethanolamine (PE), as its replacement by phosphatidylcholine (PC) inhibits transport activity and directly affects its structure, although the underlying mechanism was unknown. Here, we show that the effect of PE on the structure and the function of LmrP is mediated by interactions between the lipid headgroup and the protein. We used methyl-PE and dimethyl-PE analogs of PE to show that only replacement of the three hydrogens by methyl moieties leads to changes in the biochemical and biophysical properties of the reconstituted protein. This suggests that LmrP does not depend on the bulk properties of the phospholipids tested but solely on the hydrogen bonding ability of the headgroup. We then show that a single point mutation in LmrP, D68C, is sufficient to recapitulate precisely every biochemical and biophysical effect observed when PE is replaced by PC, including energy transfer between the protein tryptophan residues and the lipid headgroups. We conclude that the negatively charged Asp-68 is likely to participate in the interaction with PE and that such interaction is required for proton gradient sensing, substrate binding, and transport. Because Asp-68 belongs to a highly conserved motif in the Major Facilitator Superfamily (which includes LacY and EmrD), this interaction might be a general feature of these transporters that is involved in proton gradient sensing and lipid dependence.     

Asymmetric protonation of EmrE

The small multidrug resistance transporter EmrE is a homodimer that uses energy provided by the proton motive force to drive the efflux of drug substrates. The pKa values of its “active-site” residues—glutamate 14 (Glu14) from each subunit—must be poised around physiological pH values to efficiently couple proton import to drug export in vivo. To assess the protonation of EmrE, pH titrations were conducted with ¹H-¹⁵N TROSY-HSQC nuclear magnetic resonance (NMR) spectra. Analysis of these spectra indicates that the Glu14 residues have asymmetric pKa values of 7.0 ± 0.1 and 8.2 ± 0.3 at 45°C and 6.8 ± 0.1 and 8.5 ± 0.2 at 25°C. These pKa values are substantially increased compared with typical pKa values for solvent-exposed glutamates but are within the range of published Glu14 pKa values inferred from the pH dependence of substrate binding and transport assays. The active-site mutant, E14D-EmrE, has pKa values below the physiological pH range, consistent with its impaired transport activity. The NMR spectra demonstrate that the protonation states of the active-site Glu14 residues determine both the global structure and the rate of conformational exchange between inward- and outward-facing EmrE. Thus, the pKa values of the asymmetric active-site Glu14 residues are key for proper coupling of proton import to multidrug efflux. However, the results raise new questions regarding the coupling mechanism because they show that EmrE exists in a mixture of protonation states near neutral pH and can interconvert between inward- and outward-facing forms in multiple different protonation states.     

Towards the molecular mechanism of prokaryotic and eukaryotic multidrug transporters.

Due to their ability to extrude structurally dissimilar cytotoxic drugs out of the cell, multidrug transporters are able to reduce the cytoplasmic drug concentration, and, hence, are able to confer drug resistance on human cancer cells and pathogenic microorganisms. This review will focus on the molecular properties of two bacterial multidrug transporters, the ATP-binding cassette transporter LmrA and the proton motive force-dependent major facilitator superfamily transporter LmrP, which each represent a major class of multidrug transport proteins encountered in pro- and eukaryotic cells. In spite of the structural differences between LmrA and LmrP, the molecular bases of their drug transport activity may turn out to be more similar than might currently appear.     

Structure-function analysis of multidrug transporters in Lactococcus lactis

The active extrusion of cytotoxic compounds from the cell by multidrug transporters is one of the major causes of failure of chemotherapeutic treatment of tumor cells and of infections by pathogenic microorganisms. A multidrug transporter in Lactococcus lactis, LmrA, is a member of the ATP-binding cassette (ABC) superfamily and a bacterial homolog of the human multidrug resistance P-glycoprotein. Another multidrug transporter in L. lactis, LmrP, belongs to the major facilitator superfamily, and is one example of a rapidly expanding group of secondary multidrug transporters in microorganisms. Thus, LmrA and LmrP are transport proteins with very different protein structures, which use different mechanisms of energy coupling to transport drugs out of the cell. Surprisingly, both proteins have overlapping specificities for drugs, are inhibited by the same set of modulators, and transport drugs via a similar transport mechanism. The structure-function relationships that dictate drug recognition and transport by LmrP and LmrA represent an intriguing area of research.     

Mutagenic analysis of functional residues in putative substrate-binding site and acidic domains of vacuolar H+-pyrophosphatase

Vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase) uses PP(i) as an energy donor and requires free Mg(2+) for enzyme activity and stability. To determine the catalytic domain, we analyzed charged residues (Asp(253), Lys(261), Glu(263), Asp(279), Asp(283), Asp(287), Asp(723), Asp(727), and Asp(731)) in the putative PP(i)-binding site and two conserved acidic regions of mung bean V-PPase by site-directed mutagenesis and heterologous expression in yeast. Amino acid substitution of the residues with alanine and conservative residues resulted in a marked decrease in PP(i) hydrolysis activity and a complete loss of H(+) transport activity. The conformational change of V-PPase induced by the binding of the substrate was reflected in the susceptibility to trypsin. Wild-type V-PPase was completely digested by trypsin but not in the presence of Mg-PP(i), while two V-PPase mutants, K261A and E263A, became sensitive to trypsin even in the presence of the substrate. These results suggest that the second acidic region is also implicated in the substrate hydrolysis and that at least two residues, Lys(261) and Glu(263), are essential for the substrate-binding function. From the observation that the conservative mutants K261R and E263D showed partial activity of PP(i) hydrolysis but no proton pump activity, we estimated that two residues, Lys(261) and Glu(263), might be related to the energy conversion from PP(i) hydrolysis to H(+) transport. The importance of two residues, Asp(253) and Glu(263), in the Mg(2+)-binding function was also suggested from the trypsin susceptibility in the presence of Mg(2+). Furthermore, it was found that the two acidic regions include essential common motifs shared among the P-type ATPases.     

Elucidating the role of conserved glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

H(+)-pyrophosphatase (H(+)-PPase) catalyzes pyrophosphate-driven proton transport against the electrochemical potential gradient in various biological membranes. All 50 of the known H(+)-PPase amino acid sequences contain four invariant glutamate residues. In this study, we use site-directed mutagenesis in conjunction with functional studies to determine the roles of the glutamate residues Glu(197), Glu(202), Glu(550), and Glu(649) in the H(+)-PPase of Rhodospirillum rubrum (R-PPase). All residues were replaced with Asp and Ala. The resulting eight variant R-PPases were expressed in Escherichia coli and isolated as inner membrane vesicles. All substitutions, except E202A, generated enzymes capable of PP(i) hydrolysis and PP(i)-energized proton translocation, indicating that the negative charge of Glu(202) is essential for R-PPase function. The hydrolytic activities of all other PPase variants were impaired at low Mg(2+) concentrations but were only slightly affected at high Mg(2+) concentrations, signifying that catalysis proceeds through a three-metal pathway in contrast to wild-type R-PPase, which employs both two- and three-metal pathways. Substitution of Glu(197), Glu(202), and Glu(649) resulted in decreased binding affinity for the substrate analogues aminomethylenediphosphonate and methylenediphosphonate, indicating that these residues are involved in substrate binding as ligands for bridging metal ions. Following the substitutions of Glu(550) and Glu(649), R-PPase was more susceptible to inactivation by the sulfhydryl reagent mersalyl, highlighting a role of these residues in maintaining enzyme tertiary structure. None of the substitutions affected the coupling of PP(i) hydrolysis to proton transport.     

Structure-function relationships of Na(+), K(+), ATP, or Mg(2+) binding and energy transduction in Na,K-ATPase

The focus of this article is on progress in establishing structure-function relationships through site-directed mutagenesis and direct binding assay of Tl(+), Rb(+), K(+), Na(+), Mg(2+) or free ATP at equilibrium in Na,K-ATPase. Direct binding may identify residues coordinating cations in the E(2)[2K] or E(1)P[3Na] forms of the ping-pong reaction sequence and allow estimates of their contributions to the change of Gibbs free energy of binding. This is required to understand the molecular basis for the pronounced Na/K selectivity at the cytoplasmic and extracellular surfaces. Intramembrane Glu(327) in transmembrane segment M4, Glu(779) in M5, Asp(804) and Asp(808) in M6 are essential for tight binding of K(+) and Na(+). Asn(324) and Glu(327) in M4, Thr(774), Asn(776), and Glu(779) in 771-YTLTSNIPEITP of M5 contribute to Na(+)/K(+) selectivity. Free ATP binding identifies Arg(544) as essential for high affinity binding of ATP or ADP. In the 708-TGDGVND segment, mutations of Asp(710) or Asn(713) do not interfere with free ATP binding. Asp(710) is essential and Asn(713) is important for coordination of Mg(2+) in the E(1)P[3Na] complex, but they do not contribute to Mg(2+) binding in the E(2)P-ouabain complex. Transition to the E(2)P form involves a shift of Mg(2+) coordination away from Asp(710) and Asn(713) and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp(369).