NORMAL GENOMIC

• Scanzano R, Segall L, Blostein R
• Specific sites in the cytoplasmic N terminus modulate conformationaltransitions of the Na,K-ATPase.
• J Biol Chem. 2007; 282: 33691-7
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• The cytoplasmic N terminus of the Na,K-ATPase is a highly charged andflexible structure that comprises three predicted helical regionsincluding H1 spanning residues 27 to 33 and H2 spanning residues 42 to 50.Previous deletion mutagenesis experiments showed that deletion of residuesup to and including most of H2 shifts the E(1)/E(2) conformationalequilibrium toward E(1). The present study describes a clusteredcharge-to-alanine mutagenesis approach designed to delineate specificsites within the N terminus that modulate the steady-state E(1) <--> E(2)and E(1)P <--> E(2)P poise. Criteria to assess shifts in poise include (i)sensitivity to inhibition by inorganic orthovanadate to assess overallpoise; (ii) K(+)-sensitivity of Na-ATPase measured at micromolar ATP toassess changes in the E(2)(K) + ATP --> E(1) x ATP + K(+) rate; (iii)K'(ATP) for low-affinity ATP binding at the latter step; (iv) overallcatalytic turnover, and (v) the E(1)P --> E(2)P transition. The results ofalanine replacements in H1 (31KKE) suggest that this site stabilizes E(2)Pand to a lesser extent E(2). In H2, residues within 47HRK have a role instabilizing E(2) but not E(2)P as revealed with double mutants 31KKE -->AAA/47H --> A and 31KKE --> AAA/47HRK --> AAA. Taken together, theseobservations suggest that sites 31KKE in H1 and 47HRK in H2 have distinctroles in modulating the enzyme's conformational transitions during thecatalytic cycle of the enzyme.

• Zouzoulas A, Blostein R
• Regions of the catalytic alpha subunit of Na,K-ATPase important forfunctional interactions with FXYD 2.
• J Biol Chem. 2006; 281: 8539-44
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• The gamma modulator (FXYD 2) is a member of the FXYD family of singletransmembrane proteins that modulate the kinetic behavior of Na,K-ATPase.This study concerns the identification of regions in the alpha subunitthat are important for its functional interaction with gamma. An importanteffect of gamma is to increase K+ antagonism of cytoplasmic Na+ activationapparent as an increase in KNa' at high [K+]. We show that although gammaassociates with alpha1, alpha2, and alpha3 isoforms, it increases the KNa'of alpha1 and alpha3 but not alpha2. Accordingly, chimeras of alpha1 andalpha2 were used to identify regions of alpha critical for the increasedKNa'. As with alpha1 and alpha2, all chimeras associate with gamma.Kinetic analysis of alpha2front/alpha1back chimeras indicate that theC-terminal (Lys907-Tyr1018) region of alpha1, which includes transmembrane(TM)9 close to gamma, is important for the increase in KNa'. However,similar experiments with alpha1front/alpha2back chimeras indicate amodulatory role of the loop between TMs 7 and 8. Thus, as long as thealpha1 L7/8 loop is present, replacement of TM9 of alpha1 with that ofalpha2 does not abrogate the gamma effect on KNa'. In contrast, as long asTM9 is that of alpha1, replacement of L7/8 of alpha1 with that of alpha2does not abolish the effect. It is suggested that structural associationof the TM regions of alpha and FXYD 2 is not the sole determinant of thiseffect of FXYD on KNa' but is subject to long range modulation by theextramembranous L7/8 loop of alpha.

• Toustrup-Jensen M, Vilsen B
• Interaction between the catalytic site and the A-M3 linker stabilizesE2/E2P conformational states of Na+,K+-ATPase.
• J Biol Chem. 2005; 280: 10210-8
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• The consequences of mutations Ile(265) --> Ala, Thr(267) --> Ala, Gly(271)--> Ala, and Gly(274) --> Ala for the partial reaction steps of theNa(+),K(+)-ATPase transport cycle were analyzed. The mutated residues arepart of the long loop ("A-M3 linker") connecting the cytoplasmic A-domainwith transmembrane segment M3. It was found that mutation Ile(265) --> Aladisplaces the E(1)-E(2) and E(1)P-E(2)P equilibria in favor of E(1)/E(1)P,whereas mutations Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) --> Aladisplace these conformational equilibria in favor of E(2)/E(2)P. Themutations affect both the rearrangement of the cytoplasmic domains (seenby changes in phosphoenzyme properties and apparent ATP/vanadateaffinities) and the membrane sector (indicated by change in K(+)/Rb(+)deocclusion rate). Destabilization of E(2)/E(2)P in Ile(265) --> Ala, aswell as a direct effect on the intrinsic affinity of the E(2) form forvanadate, may be explained on the basis of the E(2) crystal structures ofthe Ca(2+)-ATPase, showing interaction of the equivalent isoleucine withconserved residues near the catalytic region of the P-domain. The rate ofphosphorylation from ATP was unaffected in Ile(265) --> Ala, indicating alack of interference with the catalytic function in E(1)/E(1)P. Theeffects of mutations Thr(267) --> Ala, Gly(271) --> Ala, and Gly(274) -->Ala provide the first evidence in the literature of a relativestabilization of E(2)/E(2)P resulting from perturbation of the A-M3 linkerregion. These mutations may lead to increased strain of the A-M3 linker inE(1)/E(1)P, increased stability of the A3 helix of the A-M3 linker inE(2)/E(2)P, and/or a change of the orientation of the A3 helix,facilitating its interaction with the P-domain.

• Imagawa T, Yamamoto T, Kaya S, Sakaguchi K, Taniguchi K
• Thr-774 (transmembrane segment M5), Val-920 (M8), and Glu-954 (M9) areinvolved in Na+ transport, and Gln-923 (M8) is essential for Na,K-ATPaseactivity.
• J Biol Chem. 2005; 280: 18736-44
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• The highly conserved amino acids of rat Na,K-ATPase, Thr-774 in thetransmembrane helices M5, Val-920 and Gln-923 in M8, and Glu-953 andGlu-954 in M9, the side chains of which appear to be in close proximity,were mutated, and the resulting proteins, T774A, E953A/K, and E954A/K,V920E and Q923N/E/D/L, were expressed in HeLa cells. Ouabain-resistantcell lines were obtained from T774A, V920E, E953A, and E954A, whereasQ923N/E/D/L, E953K, and E954K could only be transiently expressed asfusion proteins with an enhanced green fluorescent protein. The apparentK0.5 values for Na+, as estimated by the Na+-dependent phosphoenzymeformation (K0.5(Na,EP)) or Na,K-ATPase activity (K(0.5)(Na,ATPase)), wereincreased by around 2 approximately 8-fold in the case of T774A, V920E,and E954A. The apparent K0.5 values for K+, as estimated by theNa,K-ATPase (K0.5(K,ATPase)) or p-nitrophenylphosphatase activity(K0.5(K,pNPPase)), were affected only slightly by the 3 mutations, exceptthat V920E showed a 1.7-fold increase in the K0.5(K,ATPase). The apparentK0.5 values for ATP (K0.5(EP)), as estimated by phosphorylation (a highaffinity ATP effect), were increased by 1.6 approximately 2.6-fold in thecase of T774A, V920E, and E954A. Those estimated by Na,K-ATPase activity(K0.5(ATPase)) and ATP-induced inhibition (K(i,0.5)(pNPPase)) of K-pNPPaseactivity (low affinity ATP effects) were, respectively, increased by1.8-fold and unchanged in the case of T774A but decreased by 2- and4.8-fold in the case of V920E and were slightly changed and increased by1.7-fold in the case of E954A. The E953A showed little significant changein the apparent affinities. These results suggest that Gln-923 in M8 iscrucial for the active transport of Na+ and/or K+ across membranes andthat the side chain oxygen atom of Thr-774 in M5, the methyl group(s) ofVal-920 in M8, and the carboxyl oxygen(s) of Glu-954 in M9 mainly playsome role in the transport of Na+ and also in the high and low affinityATP effects rather than the transport of K+.

• Imagawa T, Kaya S, Taniguchi K
• The amino acid sequence 442GDASE446 in Na/K-ATPase is an important motifin forming the high and low affinity ATP binding pockets.
• J Biol Chem. 2003; 278: 50283-92
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• A highly conserved amino acid sequence 442GDASE446 in the ATP bindingpocket of rat Na/K-ATPase was mutated, and the resulting proteins, G442A,G442P, D443A, S445A, and E446A, were expressed in HeLa cells toinvestigate the effect of individual ligands on Na/K-ATPase. The apparentKm for the high and low affinity ATP effects was estimated by ATPconcentration dependence for the formation of the Na-dependentphosphoenzyme (Kmh) and Na/K-ATPase activity (Kml). The apparent Km forp-nitrophenylphosphate (pNPP) for K-dependent-pNPPase (KmP) and itsinhibition by ATP (Ki,0.5) and the apparent Km for Mg2+, Na+, K+, andvanadate in Na/K-ATPase were also estimated. For all the mutants, thevalue for ATP was approximately 2-10-fold larger than that of the wildtype. While the turnover number for Na/K-ATPase activity were unaffectedor reduced by 20 approximately 50% in mutants G442(A/P) and D443A.Although both affinities for ATP effects were reduced as a result of themutations, the ratio, Kml Kmh, for each mutant was 1.3 approximately 3.7,indicating that these mutations had a greater impact on the low affinityATP effect than on the high affinity effect. Each KmP value with theturnover number suggests that these mutations favor the binding of pNPPover that of ATP. These data and others indicate that the sequence442GDASE446 in the ATP binding pocket is an important motif that it isinvolved in both the high and low affinity ATP effects rather than in freeMg2+, Na+, and K+ effects.

• Segall L, Lane LK, Blostein R
• New insights into the role of the N terminus in conformational transitionsof the Na,K-ATPase.
• J Biol Chem. 2002; 277: 35202-9
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• The deletion of 32 residues from the N terminus of the alpha1 catalyticsubunit of the rat Na,K-ATPase (mutant alpha1M32) shifts the E(1)/E(2)conformational equilibrium toward E(1), and the combination of thisdeletion with mutation E233K in the M2-M3 loop acts synergistically toshift the conformation further toward E(1) (Boxenbaum, N., Daly, S. E.,Javaid, Z. Z., Lane, L. K., and Blostein, R. (1998) J. Biol. Chem. 273,23086-23092). To delimit the region of the cytoplasmic N terminus involvedin these interactions, the consequences of a series of N-terminaldeletions of alpha1 beyond Delta32 were evaluated. Criteria to assessshifts in conformational equilibrium were based on effects of perturbationof the entire catalytic cycle ((i) sensitivity to vanadate inhibition,(ii) K(+) sensitivity of Na-ATPase measured at micromolar ATP, (iii)changes in K'(ATP), and (iv) catalytic turnover), as well as estimates ofthe rates of the conformational transitions of phospho- anddephosphoenzyme (E(1)P --> E(2)P and E(2)(K(+)) --> E(1) + K(+)). Theresults show that, compared with alpha1M32, the deletion of up to 40residues (alpha1M40) further shifts the poise toward E(1). Remarkably,further deletions (mutants alpha1M46, alpha1M49, and alpha1M56) reversethe effect, such that these mutants increasingly resemble the wild typealpha1. These results suggest novel intramolecular interactions involvingdomains within the N terminus that impact the manner in which the Nterminus/M2-M3 loop regulatory domain interacts with the M4-M5 catalyticloop to effect E(1) <--> E(2) transitions.

• Vila R, Ponte I, Jimenez MA, Rico M, Suau P
• An inducible helix-Gly-Gly-helix motif in the N-terminal domain of histoneH1e: a CD and NMR study.
• Protein Sci. 2002; 11: 214-20
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• Knowledge of the structural properties of linker histones is important tothe understanding of their role in higher-order chromatin structure andgene regulation. Here we study the conformational properties of thepeptide Ac-EKTPVKKKARKAAGGAKRKTSG-NH(2) (NE-1) by circular dichroism and(1)H-NMR. This peptide corresponds to the positively charged region of theN-terminal domain, adjacent to the globular domain, of mouse histone H1e(residues 15-36). This is the most abundant H1 subtype in many kinds ofmammalian somatic cells. NE-1 is mainly unstructured in aqueous solution,but in the presence of the secondary-structure stabilizer trifluoroethanol(TFE) it acquires an alpha-helical structure. In 90% TFE solution thealpha-helical population is approximately 40%. In these conditions, NE-1is structured in two alpha-helices that comprise almost all the peptide,namely, from Thr17 to Ala27 and from Gly29 to Thr34. Both helical regionsare highly amphipathic, with the basic residues on one face of the helixand the apolar ones on the other. The two helical elements are separatedby a Gly-Gly motif. Gly-Gly motifs at equivalent positions are found inmany vertebrate H1 subtypes. Structure calculations show that the Gly-Glymotif behaves as a flexible linker between the helical regions. The widerange of relative orientations of the helical axes allowed by the Gly-Glymotif may facilitate the tracking of the phosphate backbone by the helicalelements or the simultaneous binding of two nonconsecutive DNA segments inchromatin.

• Kanagawa M et al.
• Membrane enzyme systems responsible for the Ca(2+)-dependentphosphorylation of Ser(27), the independent phosphorylation of Tyr(10) andTyr(7), and the dephosphorylation of these phosphorylated residues in thealpha-chain of H/K-ATPase.
• J Biochem. 2000; 127: 821-8
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• H/K-ATPase preparations (the G1 membrane) from pig stomach contain bothkinases and phosphatases and show reversible phosphorylation of Tyr(7),Tyr(10), and Ser(27) residues of the alpha-chain of H/K-ATPase. TheTyr-kinase is sensitive to genistein and quercetin and recognized byanti-c-Src antibody. The Ser-kinase is dependent on Ca(2)(+) (K(0.5) = 0.9microM), sensitive to a PKC inhibitor, and recognized by antibodiesagainst PKCalpha and PKCbetaII. The addition of3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid (CHAPS)caused a dramatic increase in the phosphorylation of added syntheticcopolymer substrates and permitted the phosphorylation of maltose-bindingproteins fused with the N-terminal domain of alpha-chains. Thephosphotyrosine phosphatase was inhibited by vanadate. The phosphoserinephosphatase was inhibited by okadaic acid and by inhibitor-2. The presenceof protein phosphatase-1 was immunologically detected. Columnchromatographic separation of CHAPS-solubilized G1 membrane and othersindicate the apparent molecular weight of the Src-kinase to beapproximately 60 kDa, the PKCalpha and/or PKCbII to be approximately 80kDa, the Tyr-phosphatase to be 200 kDa, and PP-1 to be approximately 35kDa. These data show that these membrane-bound enzyme systems are insufficiently close proximity to be responsible for reversiblephosphorylation of Tyr(7), Tyr(10), and Ser(27) of the catalytic subunitof membrane H/K-ATPase in parietal cells, the physiological role of whichis unknown.

• Yoon MK, Park SH, Won HS, Na DS, Lee BJ
• Solution structure and membrane-binding property of the N-terminal taildomain of human annexin I.
• FEBS Lett. 2000; 484: 241-5
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• The conformational preferences of AnxI(N26), a peptide corresponding toresidues 2-26 of human annexin I, were investigated using CD and NMRspectroscopy. CD results showed that AnxI(N26) adopts a mainlyalpha-helical conformation in membrane-mimetic environments, TFE/water andSDS micelles, while a predominantly random structure with slight helicalpropensity in aqueous buffer. The helical region of AnxI(N26) showed anearly identical conformation between in TFE/water and in SDS micelles,except for the orientation of the Trp-12 side-chain, which was quitedifferent between the two. The N-terminal region of the AnxI(N26) helixshowed a typical amphipathic nature, which could be stabilized by theneighboring hydrophobic cluster. The helical stability of the peptide inSDS micelles was increased by addition of calcium ions. These resultssuggest that the N-terminal tail domain of human annexin I interacts withbiological membranes in a partially calcium-dependent manner.

• Kanagawa M et al.
• Direct evidence for in vivo reversible tyrosine phosphorylation of theN-terminal domain of the H/K-ATPase alpha-subunit in mammalian stomachcells.
• J Biochem. 1999; 126: 266-70
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• In vivo reversible phosphorylation of Tyr-7 and Tyr-10 of the pig stomachH/K-ATPase alpha-chain was initially demonstrated in mammals, rat, rabbit,and pig, in the presence of vanadate + H(2)O(2). In vitro phosphorylationhas also been unequivocally demonstrated via the use of proteaseinhibitors during membrane H/K-ATPase preparation. An amphoretic detergentpermitted each intrinsic kinase to phosphorylate each fusion proteincontaining the requisite Tyr residues, along with a reduction inalpha-chain phosphorylation. These and other data suggest that someimportant enzyme systems are present in the apical membrane and that theyare in sufficient proximity to participate in the reversiblephosphorylation of the amino terminal soluble domain of the alpha-chainwith an unknown physiological function in the membrane embeddedH/K-ATPase.

• Carpenter KA et al.
• The glycine residue in cyclic lactam analogues of galanin(1-16)-NH2 isimportant for stabilizing an N-terminal helix.
• Biochemistry. 1999; 38: 15295-304
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• The neuropeptide galanin is a 29- or 30-residue peptide whosephysiological functions are mediated by G-protein-coupled receptors.Galanin's agonist activity has been shown to be associated with theN-terminal sequence, galanin(1-16). Conformational investigationspreviously carried out on full-length galanin have, furthermore, indicatedthe presence of a helical conformation in the neuropeptide's N-terminaldomain. Several cyclic lactam analogues of galanin(1-16)-NH2 were preparedin an attempt to stabilize an N-terminal helix in the peptide. Here wedescribe and compare the solution conformational properties of theseanalogues in the presence of SDS micelles as determined by NMR, CD, andfluorescence spectroscopy. Differences in CD spectral profiles wereobserved among the compounds that were studied. Both c[D4,K8]Gal(1-16)-NH2 and c[D4,K8]Gal(1-12)-NH2 adopted stable helicalconformations in the micelle solution. On the basis of the analyses oftheir respective alpha H chemical shifts and NOE patterns, this helix waslocalized to the first 10 residues. The distance between the aromaticrings of Trp2 and Tyr9 in c[D4, K8]Gal(1-16)-NH2 was determined to be 10.8+/- 3 A from fluorescence resonance energy transfer measurements. Thisinterchromophore spacing was found to be more consistent with a helicalstructure than an extended one. Removal of the Gly1 residue in compoundsc[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2 resulted in a loss ofhelical conformation and a concomitant reduction in binding potency at theGalR1 receptor but not at the GalR2 receptor. The nuclear Overhauserenhancements obtained for the Gly1 deficient analogues did, however,reveal the presence of nascent helical structures within the N-terminalsequence. Decreasing the ring structure size in c[D4, K8]Gal(1-16)-NH2 byreplacing Lys8 with an ornithine residue or by changing the position ofthe single lysine residue from eight to seven was accompanied by acomplete loss of helical structure and dramatically reduced receptoraffinity. It is concluded from the data obtained for the series of cyclicgalanin(1-16)-NH2 analogues that both the ring structure size and thepresence of an N-terminal glycine residue are important for stabilizing anN-terminal helix in these compounds. However, although an N-terminal helixconstitutes a predominant portion of the conformational ensemble forcompounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2, these peptidesnevertheless are able to adopt other conformations in solution.Consequently, the correlation between the ability of the cyclic galaninanalogues to adopt an N-terminal helix and bind to the GalR1 receptor maybe considered as a working hypothesis.

• Coppi MV, Compton LA, Guidotti G
• Isoform-specific effects of charged residues at borders of the M1-M2 loopof the Na,K-ATPase alpha subunit.
• Biochemistry. 1999; 38: 2494-505
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• The Na,K-ATPase is specifically inhibited by the cardiac glycoside,ouabain. Via a largely undefined mechanism, the ouabain affinity of theNa,K-ATPase can be manipulated by mutating the residues at the borders ofthe first extracellular (M1-M2) loop of the alpha subunit [Price, E. M.,Rice, D. A., and Lingrel, J. B. (1990) J. Biol. Chem. 265, 6638-6641]. Toaddress this issue, we compared the effects of two combinations of chargedresidues at the M1-M2 loop border, R113, D124 and D113,R124 (numberedaccording to the rat alpha1 subunit), on the ouabain sensitivity of thealpha1 and alpha2 isoforms. We report that ouabain sensitivity isdependent not only upon the identity of the residues at the M1-M2 loopborder but also upon the context into which they are introduced.Furthermore, at low concentrations of ATP, the identity of the residues atthe M1-M2 loop border affects the regulation of ATP hydrolysis bypotassium in an isoform-specific manner. Analysis of chimeric alphasubunits reveals that the effects of potassium are determined primarily bythe interaction of the N-terminus and M1-M2 loop with the C-terminal thirdof the alpha subunit. M1-M2 loop border residues may, therefore, influenceouabain sensitivity indirectly by altering the stability or structure ofthe intermediate of the Na,K-ATPase catalytic cycle which is competent tobind ouabain.

• Naganagowda GA, Gururaja TL, Levine MJ
• Delineation of conformational preferences in human salivary statherin by1H, 31P NMR and CD studies: sequential assignment and structure-functioncorrelations.
• J Biomol Struct Dyn. 1998; 16: 91-107
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• Membrane-induced solution structure of human salivary statherin, a 43amino acid residue acidic phosphoprotein, has been investigated bytwo-dimensional proton nuclear magnetic resonance (2D 1H NMR)spectroscopy. NMR assignments and structural analysis of thisphosphoprotein was accomplished by analyzing the pattern of sequential andmedium range NOEs, alphaCH chemical shift perturbations and deuteriumexchange measurements of the amide proton resonances. The NMR datarevealed three distinct structural motifs in the molecule: (1) analpha-helical structure at the N-terminal domain comprising Asp1-Tyr16,(2) a polyproline type II (PPII) conformation predominantly occurring atthe middle proline-rich domain spanning Gly19-Gln35, and (3) a3(10)-helical structure at the C-terminal Pro36-Phe43 sequence. Presenceof a few weak dalphaN(i,i+2) NOEs suggests that N-terminus also possessesminor population of 3(10)-helical conformation. Of the three secondarystructural elements, helical structure formed by the N-terminal residues,Asp1-Ile11 appears to be more rigid as observed by the relatively veryslow exchange of amide hydrogens of Glu5-Ile11. 31P NMR experimentsclearly indicated that N-terminal domain of statherin exists mainly indisordered state in water whereas, upon addition of structure stabilizingco-solvent, 2,2,2-trifluorethanol (TFE), it showed a strong propensity forhelical conformation. Calcium ion interaction studies suggested that thedisordered N-terminal region encompassing the two vicinal phosphoserinesis essential for the binding of calcium ions in vivo. Results from thecircular dichroism (CD) experiments were found to be consistent with andcomplimentary to the NMR data and provided an evidence that non-aqueousenvironment such as TFE, could induce the protein to fold into helicalconformation. The findings that the statherin possesses blended solventsensitive secondary structural elements and the requirement ofnon-structured N-terminal region under aqueous environment in calcium ioninteraction may be invaluable to understand various physiologicalfunctions of statherin in the oral fluid.

• Boxenbaum N, Daly SE, Javaid ZZ, Lane LK, Blostein R
• Changes in steady-state conformational equilibrium resulting fromcytoplasmic mutations of the Na,K-ATPase alpha-subunit.
• J Biol Chem. 1998; 273: 23086-92
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• Mutations comprising either deletion of 32 amino acids from the NH2terminus (alpha1M32) or a Glu233 --> Lys substitution in the first M2-M3cytoplasmic loop (E233K) of the alpha1-subunit of the Na, K-ATPase resultin a shift in the steady-state E1 left arrow over right arrow E2conformational equilibrium toward E1 form(s). In the present study, thefunctional consequences of both NH2-terminal deletion and Glu233substitution provide evidence for mutual interactions of these cytoplasmicregions. Following transfection and selection of HeLa cells expressing theouabain-resistant alpha1M32E233K double mutant, growth was markedlyreduced unless the K+ concentration in the culture medium was increased toat least 10 mM. Marked changes effected by this double mutation included1) a 15-fold reduction in catalytic turnover (Vmax/EPmax), 2) a 70-foldincrease in apparent affinity for ATP, 3) a marked decrease in vanadatesensitivity, and 4) marked (approximately 10-fold) K+ activation of theNa-ATPase activity measured at micromolar ATP under which condition theE2(K) --> --> E1 pathway is normally (alpha1) rate-limiting and K+ isinhibitory. The decrease in catalytic turnover was associated with a5-fold decrease in Vmax and a compensatory approximately 3-fold increasein expressed alpha1M32E233K protein. In contrast to the behavior of eitheralpha1M32 or E233K, alpha1M32E233K also showed alterations in apparentcation affinities. K'Na was decreased approximately 2-fold and K'K wasincreased approximately 2-fold. The importance of the charge at residue233 is underscored by the consequences of single and double mutationscomprising either a conservative change (E233D) or neutral substitution(E233Q). Thus, whereas mutation to a positively charged residue (E233K)causes a drastic change in enzymatic behavior, a conservative changecauses only a minor change and the neutral substitution, an intermediateeffect. Overall, the combined effects of the NH2-terminal deletion and theGlu233 substitutions are synergistic rather than additive, consistent withan interaction between the NH2-terminal region, the first cytoplasmicloop, and possibly the large M4-M5 cytoplasmic loop bearing the nucleotidebinding and phosphorylation sites.

• Daly SE, Blostein R, Lane LK
• Functional consequences of a posttransfection mutation in the H2-H3cytoplasmic loop of the alpha subunit of Na,K-ATPase.
• J Biol Chem. 1997; 272: 6341-7
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• During kinetic studies of mutant rat Na,K-ATPases, we identified aspontaneous mutation in the first cytoplasmic loop between transmembranehelices 2 and 3 (H2-H3 loop) which results in a functional enzyme withdistinct Na,K-ATPase kinetics. The mutant cDNA contained a single G950 toA substitution, which resulted in the replacement of glutamate at 233 witha lysine (E233K). E233K and alpha1 cDNAs were transfected into HeLa cellsand their kinetic behavior was compared. Transport studies carried outunder physiological conditions with intact cells indicate that the E233Kmutant and alpha1 have similar apparent affinities for cytoplasmic Na+ andextracellular K+. In contrast, distinct kinetic properties are observedwhen ATPase activity is assayed under conditions (low ATP concentration)in which the K+ deocclusion pathway of the reaction is rate-limiting. At 1microM ATP K+ inhibits Na+-ATPase of alpha1, but activates Na+-ATPase ofE233K. This distinctive behavior of E233K is due to its faster rate offormation of dephosphoenzyme (E1) from K+-occluded enzyme (E2(K)), as wellas 6-fold higher affinity for ATP at the low affinity ATP binding site. Alower ratio of Vmax to maximal level of phosphoenzyme indicates that E233Khas a lower catalytic turnover than alpha1. These distinct kinetics ofE233K suggest a shift in its E1/E2 conformational equilibrium toward E1.Furthermore, the importance of the H2-H3 loop in coupling conformationalchanges to ATP hydrolysis is underscored by a marked (2 orders ofmagnitude) reduction in vanadate sensitivity effected by this Glu233 -->Lys mutation.

• Togawa K et al.
• Ser-27, Tyr-10 and Tyr-7 in the alpha-chain of pig stomach H+,K(+)-ATPaseas Ca(2+)-dependent phosphorylatable sites by intrinsic and extrinsicprotein kinases.
• Biochem Biophys Res Commun. 1996; 227: 810-5
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• When pig stomach membrane H+,K(+)-ATPase preparations were incubated with[gamma-32P]ATP, Mg2+ and Ca2+, reversible phosphorylation of specific Tyrand Ser residues in the N-terminal alpha-chain of H+,K(+)-ATPase occurredwithout any detectable phosphorylation in other regions of thealpha-chain. Mild tosylphenylalanyl chloromethyl ketone trypsin treatmentfollowed by reverse-phase column chromatography yielded three radioactivepeptide peaks. The first peak contained both Tyr10(32P) and Tyr7(32P) andthe second peak contained Tyr10(32P). The third peak contained Ser27(32P)which was also obtained after trypsin treatment of partially purifiedH+,K(+)-ATPase preparations phosphorylated with protein kinase-C + Ca2+ orprotein kinase-A. This is the first demonstration of Ca2(+)-dependentphosphorylation of the alpha-chain of H+,K(+)-ATPase by protein kinases.

• Daly SE, Lane LK, Blostein R
• Structure/function analysis of the amino-terminal region of the 1 and 2subunits of Na,K-ATPase.
• J Biol Chem. 1996; 271: 23683-9
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• The alpha2 isoform of the Na,K-ATPase exhibits kinetic behavior distinctfrom that of the alpha1 isoform. The distinctive behavior is apparent whenthe reaction is carried out under conditions (micromolar ATPconcentration) in which the K+ deocclusion pathway of the reaction cycleis rate-limiting; the alpha1 activity is inhibited by K+, whereas alpha2is stimulated. When 32 NH2-terminal amino acid residues are removed fromalpha1, the kinetic behavior of the mutant enzyme (alpha1M32) is similarto that of alpha2 (Daly, S. E., Lane, L. K., and Blostein, R. (1994) J.Biol. Chem. 269, 23944-23948). In the current study, the region of thealpha1 NH2 terminus involved in modulating this kinetic behavior has beenlocalized to the highly charged sequence comprising residues 24-32. Withinthis nonapeptide, differences between alpha1 and alpha2 are conservativeand are confined to residues 25-27. The behavior of two chimeric enzymes:(i) alpha1 with the first 32 residues identical to the alpha2 sequence,alpha1 (1-32alpha2), and (ii) alpha2 with the first 32 residues identicalto the alpha1 sequence, alpha2(1-32alpha1), indicates that the distinctivekinetic behavior of alpha1 and alpha2 is not due to the 24-32 NH2-terminaldomain, per se, but rather to its interaction with other, isoform-specificregion(s) of the alpha1 protein. We also demonstrate that the distinct K+activation profiles of either alpha2 or alpha1M32, compared to alpha1 isdue to a faster release of K+ from the K+-occluded enzyme, and to a higheraffinity for ATP. This was determined in studies using two approaches: (i)kinetic analysis of the reaction modeled according to a branched pathwayof K+ deocclusion through low and high affinity ATP pathways and, (ii)measurements of the (rapid) phosphorylation of the enzyme (E1conformation) by [gamma-32P]ATP following the rate-limiting formation ofthe K+-free enzyme from the K+-occluded state (E2(K) --> E1 + K+). Theobserved kinetic differences between alpha2 and alpha1 suggest that theseNa,K-ATPase isoforms differ in the steady-state distribution of E1 and E2conformational states.

• Togawa K, Ishiguro T, Kaya S, Shimada A, Imagawa T, Taniguchi K
• Reversible phosphorylation of both Tyr7 and Tyr10 in the alpha-chain ofpig stomach H+,K(+)-ATPase by a membrane-bound kinase and a phosphatase.
• J Biol Chem. 1995; 270: 15475-8
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• When pig stomach membrane H+,K(+)-ATPase preparations were incubated with[gamma-32P]ATP and Mg2+ with vanadate, 32P was incorporated into thealpha-chain of H+,K(+)-ATPase to a steady-state level of approximately 0.7mol of phosphotyrosine (Tyr(P))/mol of phosphoenzyme intermediates. Theaddition of a membrane H+,K(+)-ATPase preparation with Mg2+ acceleratedthe liberation of 32P from Tyr(P) residues in the alpha-chain. Mildtosylphenylalanyl chloromethyl ketone-trypsin treatment solubilized32P-containing peptides from the alpha-chain almost completely. Areverse-phase column chromatography of the supernatant gave two peaks of32P-peptide with similar total radioactivities. The amino acid sequence ofboth peaks was shown to be Gly-Lys-Ala-Glu-Asn-Tyr-Glu-Leu-Tyr-Gln--,which is consistent with the amino-terminal sequence of the alpha-chain ofH+,K(+)-ATPase deduced from cDNA from pig stomach except that the initialMet was absent. The comparison of the recovery of amino acid from eachEdman cycle showed that the phosphorylation of Tyr10 occurred precedingthe phosphorylation of Tyr7. These data and others suggested the presenceof a novel membrane-bound enzyme system to participate in reversiblephosphorylation of both Tyr residues in the alpha-chain of H+,K(+)-ATPase.

• Blanco FJ, Jimenez MA, Pineda A, Rico M, Santoro J, Nieto JL
• NMR solution structure of the isolated N-terminal fragment of protein-G B1domain. Evidence of trifluoroethanol induced native-like beta-hairpinformation.
• Biochemistry. 1994; 33: 6004-14
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• The solution structure of the isolated N-terminal fragment ofstreptococcal protein-G B1 domain has been investigated in H2O and TFE/H2Osolution by CD and NMR to gain insight into the possible role that nativebeta-hairpin secondary structure elements may have in early proteinfolding steps. The fragment also has been studied under denaturingconditions (6 M urea), and the resulting NMR chemical shifts were used asa reference for the disordered state. On the basis of CD and NMR data, itis concluded that in aqueous solution the fragment is basically flexible,with two local low populated chain bends involving residues 8-9 and 14-15,respectively, in close agreement with secondary structure predictions, astructure that is different from the final folded state of that segment ofthe protein. The changes in the CD spectrum, the presence of severalmedium-range NOEs plus two long-range NOEs, and the sign of the H alphaconformational shifts reveal that the addition of TFE facilitates theformation of a set of transient beta-hairpins involving essentially thesame residues that form the native beta-hairpin found in the finalthree-dimensional structure of the B1 domain. The stabilization ofnative-like structures by TFE is known to occur for helices, but, to ourknowledge, this is the first time the stabilization of a native-likebeta-hairpin structure by TFE is reported. Since long-range tertiaryinteractions are absent in the isolated fragment, our results support theidea that, in addition to helices, beta-hairpins may play an active rolein directing the protein folding process.