Escherichia coli was used to overexpress the large cytoplasmic loop of the rat Na,K-ATPase. A 1260-base DNA segment encoding Lys354–Lys774 of the rat α1-subunit was constructed via polymerase chain reaction. The polymerase chain reaction product was successfully subcloned into the expression vector pET-28 (Novagen), which produces an N-terminal 6-histidine-tagged fusion protein. The pET-28 vector containing rat α-loop, i.e. pAN, was used to transform calcium-competent E. coli BL21(DE3) cells, and positive clones were selected by kanamycin resistance. Bacterial cultures were grown, and protein synthesis was induced with isopropyl β-d-thiogalactoside. Cells were harvested and lysed, revealing production of the His-tagged fusion protein (∼46 kDa). The fusion protein was affinity-purified from other soluble cellular proteins via a Ni-NTA column, which routinely yielded ∼20 mg of soluble His6-α-loop/L cell culture. The His6-α-loop retained significant native structure, as evidenced by the ability of ATP and ADP (but not AMP, CTP, GTP, or UTP) to protect against chemical modification by either fluorescein isothiocyanate or maleimidylanilinonapthalene sulfonic acid. More specifically, circular dichroism spectroscopy was used to estimate the secondary structure of the His6 loop, revealing an ordered folding composed of 23% α-helix, 23% antiparallel β-sheet, 4% parallel β-sheet, 19% β-turn, and 32% random coil. The 6-histidine loop bound the fluorescent ATP analog trinitrophenyl-ATP with high affinity, as determined by measuring the fluorescence changes associated with binding. Affinities for ATP (∼350 μm) and ADP (∼550 μm) were determined by their ability to compete with and displace 2′,3′-O-[2,4,6,-trinitrophenyl]-ATP. These nucleotide affinities are similar to those observed for the E2conformation of the intact Na,K-ATPase. Escherichia coli was used to overexpress the large cytoplasmic loop of the rat Na,K-ATPase. A 1260-base DNA segment encoding Lys354–Lys774 of the rat α1-subunit was constructed via polymerase chain reaction. The polymerase chain reaction product was successfully subcloned into the expression vector pET-28 (Novagen), which produces an N-terminal 6-histidine-tagged fusion protein. The pET-28 vector containing rat α-loop, i.e. pAN, was used to transform calcium-competent E. coli BL21(DE3) cells, and positive clones were selected by kanamycin resistance. Bacterial cultures were grown, and protein synthesis was induced with isopropyl β-d-thiogalactoside. Cells were harvested and lysed, revealing production of the His-tagged fusion protein (∼46 kDa). The fusion protein was affinity-purified from other soluble cellular proteins via a Ni-NTA column, which routinely yielded ∼20 mg of soluble His6-α-loop/L cell culture. The His6-α-loop retained significant native structure, as evidenced by the ability of ATP and ADP (but not AMP, CTP, GTP, or UTP) to protect against chemical modification by either fluorescein isothiocyanate or maleimidylanilinonapthalene sulfonic acid. More specifically, circular dichroism spectroscopy was used to estimate the secondary structure of the His6 loop, revealing an ordered folding composed of 23% α-helix, 23% antiparallel β-sheet, 4% parallel β-sheet, 19% β-turn, and 32% random coil. The 6-histidine loop bound the fluorescent ATP analog trinitrophenyl-ATP with high affinity, as determined by measuring the fluorescence changes associated with binding. Affinities for ATP (∼350 μm) and ADP (∼550 μm) were determined by their ability to compete with and displace 2′,3′-O-[2,4,6,-trinitrophenyl]-ATP. These nucleotide affinities are similar to those observed for the E2conformation of the intact Na,K-ATPase. The Na,K-ATPase (3.6.1.37) is an integral membrane protein that is responsible for maintaining ionic homeostasis in animal cells by mediating the active translocation of sodium and potassium ions against their electrochemical gradients across the plasma membrane. The ion gradients produced by the sodium pump are important for cell excitability and contractility, as well as for regulation of other intracellular ion and solute concentrations. The current model for the sodium pump cycle is conserved for other ion pumps, e.g. the plasma membrane and sarcoplasmic reticulum calcium pumps, the gastric hydrogen/potassium pump, and the Neurospora proton pump. These ion pumps make up the enzyme class known as the P-type ATPases. Although much information has been gathered about the kinetic mechanism of the sodium pump, there is still rather little detail known about the structure of the protein. The sodium pump exists as a functional heterodimeric protein consisting of a catalytic α-subunit (∼110 kDa) and a smaller, glycosylated β-subunit (∼55 kDa). Both subunits have been cloned, and the primary structure has been determined in several isoforms from a variety of species (1Lingrel J.B Kuntzweiler T. J. Biol. Chem. 1994; 269: 19659-19662Abstract Full Text PDF PubMed Google Scholar). However, even after extensive study for several decades, information about the specific amino acids involved in formation of the binding sites for physiological ligands is rudimentary. Although the detailed topology of the α-subunit of the sodium pump is still the subject of investigation, an overall consensus on structure is emerging; it shows 10 transmembrane segments (3Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (418) Google Scholar). All the residues implicated with ATP binding thus far have been localized to the major cytoplasmic loop, which is composed of about 430 amino acid residues between transmembrane segments M4 and M5 (2Kaplan J.H. Kaplan J.H. De Weer P. The Sodium Pump: Structure, Mechanism, and Regulation. The Rockefeller University Press, New York1991: 117-128Google Scholar). Moreover, this loop contains four of the most highly conserved P-type ATPase sequences (3Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (418) Google Scholar). Much of the information suggesting the involvement of various amino acid residues with pump function comes from chemical modification experiments. Such studies have identified a number of residues in the M4M5 loop thought to be involved in ATP binding. For example, Lys480, Lys501, Gly502, Asp710, Asp714 and Lys719 are all modified by a variety of chemical agents in the absence of ATP but not in its presence, suggesting a role in ATP binding (4Pedemonte C.H. Kaplan J.H. Am. J. Physiol. 1990; 258: C1-C23Crossref PubMed Google Scholar). In subsequent studies, some of these residues were changed via mutagenesis with little or no effect on enzyme activity (1Lingrel J.B Kuntzweiler T. J. Biol. Chem. 1994; 269: 19659-19662Abstract Full Text PDF PubMed Google Scholar). However, measurements of overall enzyme activity only provide information on whether the mutated residue is absolutely essential for function, but they do not necessarily address whether the residue is involved directly in ATP binding. For example, mutation of Asp369 (i.e.the site of phosphorylation), not surprisingly, abolishes enzyme activity (5Johnson C.L. Kuntzweiler T.A. Lingrel J.B Johnson C.G. Wallick E.T. Biochem. J. 1995; 309: 187-194Crossref PubMed Scopus (21) Google Scholar), but interestingly, mutation of this residue increases the ATP binding affinity (6Pedersen P.A. Rasmussen J.H. Jorgensen P.L. J. Biol. Chem. 1996; 271: 2514-2522Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Consequently, to determine whether a residue is important for ATP binding one must directly measure ATP binding and characterize its affinity. The only approach that will identify ATP contact residues is to crystallize the protein in the presence of substrate. Unfortunately, crystallographic analysis of integral membrane proteins still remains difficult because generally applicable strategies for obtaining x-ray quality crystals of these molecules do not yet exist. As an approach to achieving this aim, we describe the bacterial production and purification of a soluble polypeptide corresponding to the large cytoplasmic loop of the Na,K-ATPase. This soluble protein is able to bind ATP, as evidenced by the ability of ATP to prevent modification by both fluorescein 5′-isothiocyanate (FITC) 1The abbreviations used are: FITC, fluorescein 5′-isothiocyanate; TNP-ATP, 2′,3′-O-[2,4,6,-trinitrophenyl]adenosine 5′-triphosphate; MIANS, 2-[4′maleimidylanilino]napthalene-6-sulfonic acid; IPTG, isopropyl β-d-thiogalactoside; MOPS, morpholinepropanesulfuric acid; LB, Luria broth; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; His6 loop, 6-histidine-tagged M4M5 cytoplasmic loop from the rat Na,K-ATPase α1-subunit; CAPS, 3-(cyclohexylamino)propanesulfonic acid; SITS, 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid. and 2-[4′maleimidylanilino]napthalene-6-sulfonic acid (MIANS), two fluorescent probes previously demonstrated to label the M4M5 loop of the full-length Na,K-ATPase in an ATP-protectable fashion. In addition, we used 2′,3′-O-[2,4,6,-trinitrophenyl]adenosine 5′-triphosphate (TNP-ATP) fluorescence to estimate, by competition with ATP, the ATP binding affinity to this segment of the sodium pump. A preliminary report of some of this work was presented at the Eighth International Meeting on the sodium pump J.H. S. S. PubMed Scopus Google Scholar). ATP, AMP, CTP, GTP, and were from SDS, DNA and were from was from and were from were from FITC, MIANS, and were from membrane was from and were from and were from The and DNA polymerase were from The pET-28 expression vector and Escherichia coli BL21(DE3) cells were from and kanamycin were from the University E. coli cells were a from and DNA DNA IPTG, and were from DNA was at the at The of the rat α1-subunit encoding the M4M5 cytoplasmic loop was via polymerase chain reaction in the presence of 10 of the four and of DNA polymerase in of the The was a from at at and at were in a of the a DNA of The was as The was as purification of the a was with the to the and contains a of the encoding the product of T. E. J. A E. coli cells were with the and positive were selected on containing and were selected and in and the DNA was via and DNA positive which were to that no random the The M4M5 loop was from by with and these sites were into the The 6-histidine fusion protein pET-28 (Novagen), was with and A reaction was after both DNA at a of to DNA of at for The pAN, was into cells and selected by the kanamycin by cells have a for with do we used cells for and of as we BL21(DE3) cells for protein The constructed expression vector was used to transform calcium The E. coli were selected on A was to in of and this was used to of a of the to an A of was to the synthesis of protein from the and at to an A of Cells were and in of a containing with a A of was in the presence of and the was on for with of the of mg of the was to with the and difficult to of was and the was at the was no A of was and the cell was for an at The soluble was from the by at for at of the His6 loop was by a of the soluble cell on a and membrane CAPS, The membrane was with a of a 6-histidine revealing a protein (∼46 kDa) from the of cells A of was into a after the a of The was with 10 of and with by of was with of binding of the soluble cell was to the The was with of binding to bound E. coli The His6 loop protein was from the with a from to in a containing and were and for protein by the of Biochem. PubMed Scopus Google Scholar). were via The product was routinely from to The between only that were of the were used for of the Na,K-ATPase or the produced His6 loop was with either or for 10 at in of a containing and of either ATP, AMP, CTP, GTP, or as in the corresponding and For whether or label the protein was in the presence of to the chemical The chemical modification were by the of of containing a SDS, and and a of The was and on a to the of PubMed Scopus Google Scholar). were and proteins were and with in acid. binding to the was as by and J. Biol. Chem. Full Text PDF PubMed Google for binding to Na,K-ATPase with fluorescence changes were in on a model The was and the was of a were into a of containing of His6 loop at The were into The between the and the fluorescence increases to between and the with was by from a of ATP, or into a of containing and 10 His6 The high protein to was to that all of the was bound to the nucleotide there is a significant of in it is difficult to the that is from the His6 loop by the of the loop with were to determine the fluorescence changes by of the effect from the nucleotide the fluorescence changes associated with the from the were on a A were a cell at a of were on an the was by and were the of 10 at a of and a The protein was determined by amino acid analysis and was for secondary structure the was and the was the by The secondary structure of the His6 loop was the and of Johnson J. Biol. Chem. Full Text PDF PubMed Google Scholar). was to characterize the ATP binding of the Na,K-ATPase. As a to this we a to large of a soluble corresponding to the cytoplasmic loop between transmembrane segments M4 and M5 from to rat The was constructed by the encoding the rat M4M5 cytoplasmic loop into pET-28 site at the and the site from the was used to transform a BL21(DE3) E. coli For of cells were in of containing a of and fusion protein production was induced with presence of the of fusion protein in the soluble This routinely yielded significant production of the His6 loop fusion protein kDa) with associated with the soluble The His6 loop was via a Ni-NTA affinity is a fluorescent that in the this reaction is by the presence of ATP J. Biol. Chem. Full Text PDF PubMed Google Wallick E.T. Biochem. PubMed Scopus Google Scholar). In this we used ATP against as a to that ATP to His6 loop is that after with FITC, both the Na,K-ATPase and the His6 loop were by Moreover, the was in the presence of either ATP or AMP, only ATP modification of both the intact sodium pump and the His6 loop the nucleotide of the His6 loop, we the ability of several to protect against modification His6 loop was with in the presence of ATP, AMP, GTP, and is that both ATP and ADP against FITC, AMP, CTP, GTP, and were to prevent These are in with the nucleotide demonstrated for the which has been to bind ATP and ADP with high affinity, J. Biochem. PubMed Scopus Google Scholar). it that the His6 loop the that nucleotide in the intact The of the His6 loop was directly by the for after we the His6 loop with SDS, with to the were observed we Na,K-ATPase with These are not to an effect of on the reaction; was to the His6 loop after with or by and not More the overall secondary structure of the fusion protein was by As in the which the residue as a function of a at and two one at and the other at The is the of 10 the of which not of the of the His6 loop, the Johnson J. Biol. Chem. Full Text PDF PubMed Google Scholar), secondary as 23% α-helix, 23% antiparallel β-sheet, 4% parallel β-sheet, 19% β-turn, and 32% random coil. The of the in this is not to be but rather between and The that the to to that the fusion protein is highly as by this the fluorescent was to the Na,K-ATPase by a residue in the M4M5 loop of the Na,K-ATPase J.H. Physiol. Scholar). Both and enzyme were by the presence of J. J. P. and J. in In this we whether ATP was able to protect against modification of the His6 ATP, but not of the produced His6 loop However, FITC, label the but ATP no against its modification The for a of ATP is that there is no significant structure to bind The of the His6 loop and were This fluorescent ATP analog has been to bind with high affinity to the native as well as to other of the P-type ATPase J. Biol. Chem. Full Text PDF PubMed Google J. Biol. Chem. Full Text PDF PubMed Google Biochem. PubMed Scopus Google Scholar). is a fluorescent for the nucleotide binding site because its fluorescence changes on binding. an of His6 loop was to a containing TNP-ATP, it in an of the fluorescence with binding to the protein The of ATP to a containing His6 loop and in a in the fluorescent as be ATP from the His6 loop The binding affinity of the His6 loop for was determined by of into a containing His6 the fluorescence against that the binding of to the His-tagged was composed of two a and a of the from all the the fluorescence changes to specific binding The affinity of the His6 loop for was affinities of for the for His6 of binding to of binding to in a The affinities for ATP, and were determined from their ability to displace bound from the His6 For these a of the protein with was used to that all of the was bound and that the from the binding was PubMed Scopus Google Scholar). The competition of binding by is evidenced by a in fluorescence the of from the His6 A of of in fluorescence from a is in The for ATP and ADP were and no binding in the For the binding were on of His6 loop with have produced a soluble polypeptide by bacterial that is in with the loop of the rat have that the ATP and but not other with the as native Na,K-ATPase. This highly ordered shows and of the intact Na,K-ATPase. modification (4Pedemonte C.H. Kaplan J.H. Am. J. Physiol. 1990; 258: C1-C23Crossref PubMed Google J.H. Kaplan J.H. De Weer P. The Sodium Pump: Structure, Mechanism, and Regulation. The Rockefeller University Press, New York1991: 117-128Google of the as well as mutagenesis studies (1Lingrel J.B Kuntzweiler T. J. Biol. Chem. 1994; 269: 19659-19662Abstract Full Text PDF PubMed Google Scholar), that several amino acids between the and transmembrane segments in the of However, in some the from these two to be For example, with a number of FITC, SITS, and the the binding of ATP both modification and with a role in ATP binding. However, was changed to via mutagenesis S. 1990; PubMed Scopus Google Scholar), the enzyme retained that is not essential for enzyme in ATP binding and yet not be for enzyme is that is one of several residues that the ATP binding of a contact residue in a ATP affinity, but not in a of ATP binding. in the presence of ATP, Na,K-ATPase activity However, a chemical to a contact residue or a residue not only that residue from but in the of that modification of prevent ATP binding via Consequently, it is that a detailed structure is to identify the contact sites for ATP and to describe its binding approach the major cytoplasmic between M4 and M5 of the Na,K-ATPase was in E. have been by other to the ATP binding of the proton pump E. J. Biol. Chem. Full Text PDF PubMed Google Scholar), the sarcoplasmic reticulum calcium pump J. Biol. Chem. 269: Full Text PDF Google Scholar), the sodium pump E. J. 1996; Scholar), and the transmembrane P.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The from the rat encoding amino acids from was into a fusion protein vector and into E. This routinely mg of soluble of cell culture. demonstrated that this and an ordered structure, be by both and MIANS, both ATP and and the fluorescent ATP are to the His6 topology of the sodium pump x-ray crystallographic However, some have been that an α-subunit structure with 10 transmembrane all the residues associated with ATP binding have been localized to the major cytoplasmic loop between transmembrane segments M4 and M5 (2Kaplan J.H. Kaplan J.H. De Weer P. The Sodium Pump: Structure, Mechanism, and Regulation. The Rockefeller University Press, New York1991: 117-128Google Scholar). the residues thus far implicated in nucleotide binding all the of the P-type ATPase have been to this of the nucleotide binding of P-type have been with and J. Biochem. PubMed Scopus Google Scholar). to these the large cytoplasmic loop is into a a nucleotide binding and a the and of the nucleotide binding of the Na,K-ATPase cytoplasmic loop from the previously J. Biochem. PubMed Scopus Google Scholar). to their the of and Biochemistry. Scholar), we a of about and These well with current of 23% and for the secondary structure of the M4M5 The in that the and the in analysis and not in that of and J. Biochem. PubMed Scopus Google have structure the nucleotide binding it the between analysis and primary structure J. Biochem. PubMed Scopus Google or a retained structure of His6 loop with native intact Na,K-ATPase. For two decades, it has been known that the Na,K-ATPase in an ATP-protectable J. PubMed Scopus Google Scholar). The site of modification was identified as J. Biol. Chem. Full Text PDF PubMed Google Wallick E.T. Biochem. PubMed Scopus Google Scholar). that in the ATP binding site of the sodium this the ATP modification of J. PubMed Scopus Google Scholar), the of is to binding in a similar to the effect that has on ATP binding C.H. Kaplan J.H. Biochemistry. PubMed Scopus Google Kaplan J.H. J. Biol. Chem. 1990; Full Text PDF PubMed Google S. Kaplan J.H. Biochem. PubMed Scopus Google J.H. Physiol. Scholar), and S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (418) Google an residue exists in a conserved most of the P-type ATPase (3Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (418) Google Scholar). In a similar to the intact ATP and ADP (but not protect against modification of Moreover, that do not bind with high affinity to the native enzyme do not protect the from modification These that the His6 loop has to formation of a nucleotide binding that shows the His6 loop to in no the was observed with the native Na,K-ATPase it that the of is a product of the in the The folding of this ATP binding loop a highly at This is in with the of and have observed that ATP, but not AMP, protect the loop against modification by the has been to a specific residue in the large cytoplasmic loop of ATP against and N-terminal amino acid has identified the residue as either or ATP against modification at and that a folding of the loop the segments of the is that after of this loop, modification still with MIANS, but modification is no by the presence of The sodium pump and most other P-type have a ATP a high affinity ATP effect and a affinity ATP In the sodium pump, of to and The for for ATP μm) well with the ATP binding affinity J. Biochem. PubMed Scopus Google J. Biol. Chem. Full Text PDF PubMed Google Scholar). ATP the of is and by the of potassium S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). However, ATP μm) the of This affinity effect is as the for ATP ATP of the sodium pump, with both high and has been as to either two ATP sites or to a ATP binding that its affinity in pump the fluorescent ATP TNP-ATP, we were able to estimate the ATP affinity of the His6 In to determine the His6 loop affinity for TNP-ATP, we that there were two to the binding of this ATP a and and PubMed Scopus Google a similar binding for binding to the native Na,K-ATPase. the binding from the a specific with an of for This is about an of the Na,K-ATPase binding affinity for at J. Biol. Chem. Full Text PDF PubMed Google Scholar), i.e. it to a affinity affinity of was for ATP by competition and of This is in with the ATP affinity for the of the native Na,K-ATPase associated with potassium In addition, the has a similar affinity for This is with the that in the affinity of the sodium pump, ADP has been to potassium and J. Biol. Chem. Full Text PDF PubMed Google J. Physiol. Scopus Google J.H. PubMed Scopus Google Scholar). up to were not to displace and of up to were to protect against modification the nucleotide binding of the sarcoplasmic reticulum calcium pump J. Biol. Chem. 269: Full Text PDF Google Scholar), the proton pump E. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and the sodium pump E. J. 1996; have been in E. In the sodium ATP binding was demonstrated by measuring against with to protect as well as E. J. 1996; Scholar). All were in the absence of the are to the ATP binding to from the proton pump and sarcoplasmic reticulum calcium pump was by ATP competition of TNP-ATP, as in this The affinities for binding to the calcium pump and proton pump were and similar to of for the sodium pump In the ATP affinity for the proton pump was E. J. Biol. Chem. Full Text PDF PubMed Google Scholar), from that for the calcium pump J. Biol. Chem. 269: Full Text PDF Google and for the sodium pump The affinity for the proton pump ATP binding be to an of the or because the proton pump was a fusion the calcium pump and sodium pump were both His-tagged we were to successfully measure the ATP affinity for the of even we were able to that ATP against that ATP against of J. and J. that expression of the of these P-type produces a protein that is able to bind ATP but with an affinity to that in the loop has secondary structure, it to that the high affinity binding is by with other of the ATPase the binding of these changes in the ATP binding site be to that there are two or an and a the affinity for ATP and the affinity The changes in structure of the intact protein that we identify as sodium affinity and high ATP and potassium affinity and ATP are by changes in and in the ATP binding is between the ATP binding in the loop and other segments of the protein that the loop in the which has a high affinity for In the loop is not by these and a exists with affinity. The study of ATP binding to the M4M5 loop of the Na,K-ATPase a for mutagenesis studies of the residues thought to a role in ATP binding. the ability to large of soluble protein this for detailed of the and ATP binding of P-type ATPases. are to and for In addition, we for and the of both the and fusion for with the and for this is for both and with the measurements and
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