Structural Basis for alpha 1 Versus alpha 2 Isoform-distinct Behavior of the Na,K-ATPase*

Laura SegallDagger , Zahid Z. Javaid§, Stephanie L. Carl§, Lois K. Lane§, and Rhoda BlosteinDagger

From the Dagger  Department of Biochemistry, McGill University, Montreal, Quebec H3G 1A4, Canada and § Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

Received for publication, November 14, 2002, and in revised form, January 14, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We showed earlier that the kinetic behavior of the alpha 2 isoform of the Na,K-ATPase differs from the ubiquitous alpha 1 isoform primarily by a shift in the steady-state E1/E2 equilibrium of alpha 2 in favor of E1 form(s). The aim of the present study was to identify regions of the alpha  chain that confer the alpha 1/alpha 2 distinct behavior using a mutagenesis and chimera approach. Criteria to assess shifts in conformational equilibrium included (i) K+ sensitivity of Na-ATPase measured at micromolar ATP, under which condition E2(K+) right-arrow E1 + K+ becomes rate-limiting, (ii) changes in K'ATP for low affinity ATP binding, (iii) vanadate sensitivity of Na,K-ATPase activity, and (iv) the rate of the partial reaction E1P right-arrow E2P. We first confirmed that interactions between the cytoplasmic domains of alpha 2 that modulate conformational shifts are fundamentally similar to those of alpha 1, suggesting that the predilection of alpha 2 for E1 state(s) is due to differences in primary structure of the two isoforms. Kinetic behavior of the alpha 1/alpha 2 chimeras indicates that the difference in E1/E2 poise of the two isoforms cannot be accounted for by their notably distinct N termini, but rather by the front segment extending from the cytoplasmic N terminus to the C-terminal end of the extracellular loop between transmembranes 3 and 4, with a lesser contribution of the alpha 1/alpha 2 divergent portion within the M4-M5 loop near the ATP binding domain. In addition, we show that the E1 shift of alpha 2 results primarily from differences in the conformational transition of the dephosphoenzyme, (E2(K+) right-arrow E1 + K+), rather than phosphoenzyme (E1P right-arrow E2P).

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Na,K-ATPase or sodium pump is an integral membrane protein complex found in the plasma membrane of virtually all animal cells. It catalyzes the exchange of three intracellular Na+ ions for two extracellular K+ ions using the energy of hydrolysis of one molecule of ATP. Consequently, the sodium pump plays an essential role in the maintenance of the electrochemical alkali cation gradients, providing the driving force for the transport of various nutrients into the cell. The Na,K-ATPase is a member of the family of P-type ATPases, which during the course of their catalytic cycle undergo phosphorylation and dephosphorylation of a conserved aspartate residue located in the large catalytic loop between transmembrane segments 4 and 5 of the catalytic alpha  subunit. During the catalytic cycle both dephospho- and phosphoenzymes undergo conformational transitions commonly referred to as E1 left-right-arrow E2 and E1P left-right-arrow E2P, respectively. In addition to the large catalytic alpha  subunit, the Na,K-ATPase comprises a smaller, highly glycosylated beta  subunit that is important for the proper folding of alpha  and its insertion into the plasma membrane. At present, four isoforms of alpha  and three isoforms of beta  have been described, and these are distributed in a tissue- and developmentally dependent manner.

The alpha 2 isoform is located primarily in skeletal muscle and in brain, predominantly in glial cells. Our earlier studies indicated that it differs from the ubiquitous alpha 1 subunit primarily in the steady-state E1/E2 equilibrium. Thus, compared with alpha 1, the E1/E2 poise of alpha 2 is shifted toward E1. This shift is reminiscent of the changes in alpha 1 effected by deleting 32 residues from its N terminus (mutant alpha 1M32) which corresponds to the E1-shifted trypsinized kidney enzyme first described by Jorgensen (1). Except for a modest (approx 1.5-fold) increase in K'Na (12), alpha 2 resembles alpha 1 with respect to apparent affinity for extracellular K+ when ouabain-sensitive K+ influx is assayed under physiological conditions of ATP concentration. However, marked differences between alpha 2 and alpha 1 become apparent when, at micromolar ATP, the E2(K+) right-arrowright-arrow E1 conversion is rate-limiting. Under these conditions, alpha 2* 1 resembles closely the alpha 1M32 mutant. We showed previously (2, 3) that, compared with alpha 1, both alpha 2* and alpha 1M32 have faster rates of K+ deocclusion as seen in K+ stimulation rather than alpha 1-like inhibition of Na-ATPase activity at 1 µM ATP, with a concomitantly lower K'ATP for low affinity ATP binding and decreased (50%) catalytic turnover.

One region of marked primary sequence diversity among the otherwise homologous alpha 1, alpha 2*, and alpha 3* isoforms is the N terminus. In previous studies we compared the kinetics of alpha 1/alpha 2 chimeras in which the first 32 residues were interchanged. The results showed that although removal of 32 residues from the N terminus of alpha 1 yields an enzyme with alpha 2*-like kinetics as mentioned above, substitution of the first 32 residues of alpha 1 with those of alpha 2* is without effect. Similarly, substituting the analogous N-terminal sequence of alpha 1 into that of alpha 2* does not dramatically alter the kinetics of alpha 2. It was therefore suggested that either removal of the N terminus, as in the case of alpha 1M32, or alteration of its primary or secondary structure, as in the case of alpha 2*, likely results in a weakening of intramolecular interactions between the N terminus and some other regions of the alpha  protein (2).

The experiments described in the present study were designed to extend the chimera approach to additional domains of isoform diversity to pinpoint regions that confer the alpha 1/alpha 2 distinct behavior. As a prelude to this analysis, we first show that cytoplasmic interactions that underlie the E1/E2 conformational transitions seen with alpha 1 (3, 4) are notably similar in alpha 2*. The subsequent analysis of alpha 1/alpha 2 chimeras shows that the N-terminal segment encompassing the cytoplasmic N terminus and the first (M2-M3) cytoplasmic loop of alpha 2*, the so-called "Actuator" or A domain (5) and, to a lesser extent, the most divergent portion of the nucleotide binding or N domain within the large M4-M5 loop, are responsible for the E1 shift of alpha 2*.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mutagenesis, Transfection, and Cell Culture-- All of the mutant and chimeric alpha 's used in this study were derived from the ouabain-insensitive rat alpha 1 and alpha 2* cDNAs previously described by Jewell and Lingrel (6). The E231K mutation was introduced into a rat alpha 2* cDNA that had been excised from pRcCMV with HindIII(81, 3282) using PCR amplification with synthetic nucleotides and the TaqPlus polymerase reaction kit (Stratagene). The reaction mixtures were eluted from QIAquick spin columns (Qiagen) and digested with DpnI, EcoRI, and SalI. The EcoRI(780)-SalI(1202) alpha 2* fragment was gel-purified and ligated into a modified pIBIalpha 2* shuttle vector in place of the wild type EcoRI-SalI fragment. Clones containing the E231K mutation (G800 right-arrow A) were identified by the presence of adenosine at nucleotide 800, and the complete sequence of the substituted fragment was verified before excising the full-length alpha 2E231K cDNA from the shuttle vector and ligating it into pRcCMV (Invitrogen). Orientation of the alpha 2E231K cDNA in pRcCMV was determined by restriction enzyme analysis.

The alpha 2M30 mutant was constructed by introducing a 30-residue deletion into the 5' HindIII(66)-EcoRI(780) restriction fragment cassette of the rat alpha 2* cDNA as described previously (2). The mutant cassette was then ligated into the rat alpha 2* cDNA in place of the wild type HindIII-EcoRI cassette. The full-length mutant cDNA was released from the shuttle vector by digestion with HindIII(66, 3284) and ligated into the expression plasmid pCDNA3.1 (Invitrogen), and orientation of the cDNA was determined by restriction analysis.

Chimeras alpha 2-(1-309)/alpha 1 and alpha 1-(1-311)/alpha 2 were prepared by exchanging 5' HindIII-BssHII fragments of the two cDNAs. The HindIII site of each cDNA is in the 5'-untranslated region, and the BssHII site splits the codons for Ala-347 in alpha 1 and the corresponding Ala-345 in alpha 2. It should be noted that the two isoforms have identical sequences in the region between residues 311 and 346 of alpha 1. The full-length cDNAs for the chimeric proteins were then excised from the shuttle vectors and cloned into pRcCMV as above.

The N-terminal chimera, alpha 2-(1-63)/alpha 1, denoted as alpha 2nt/alpha 1, was prepared using the oligonucleotide-directed technique of Kunkel (7). Starting with the 5' SacI (230)-SalI(875) cassette of alpha 1-(1-32)alpha 2 (3), nucleotide mutations encoding the alpha 2 amino acid sequence from residue 33 through 63 were introduced. This mutant 5' cassette was sequenced and ligated into an alpha 1 shuttle vector in place of the wild type cassette, and the full-length chimeric cDNA was transferred into pRcCMV. Because residues 64-94 of rat alpha 2 are naturally identical to residues 66-96 of alpha 1, the entire N-terminal cytoplasmic tail of the alpha 2nt/alpha 1 protein has the alpha 2 sequence.

The chimera in which the isoform divergent portion within the M4-M5 loop, alpha 2-(427-562)/alpha 1, denoted as alpha 2L/alpha 1, was prepared using the Kunkel (7) technique to introduce the alpha 2 sequence into five short segments of the H5-H6 cytoplasmic loop of rat alpha 1. Three of the substituted segments are encoded by nucleotides in the SalI(875)-BamHI(1776) cassette: I, 429-442 alpha 1 replaced by 427-440 alpha 2; II, 457-473 alpha 1 replaced by 455-471 alpha 2; III, 489-499 alpha 1 replaced by 487-496 alpha 2. The other two segments are in the BamHI(1777)-KpnI(2290) cassette: IV, 515-530 alpha 1 replaced by 512-527 alpha 2; and V, 552-565 alpha 1 replaced by 549-562 alpha 2. After sequence verification, the mutated cassettes were ligated into an alpha 1 shuttle vector in place of the wild type cassettes, and the alpha 2L/alpha 1 cDNA was then transferred to pRcCMV. The mutated 5' SacI-SalI cassette used above to prepare alpha 2nt/alpha 1 was also substituted into the alpha 2L/alpha 1 cDNA in place of the wild type cassette to produce alpha 2(nt+L)/alpha 1.

HeLa cells were transfected with the pRcCMV-alpha and pCDNA-alpha constructs using either the calcium phosphate method (8) or the LipofectAMINE technique (LipofectAMINE, Invitrogen), and cells expressing the relatively ouabain-resistant rat alpha  enzymes were selected as previously described (6, 9). HeLa cells expressing the mutant alpha  enzymes were amplified in Dulbecco's modified Eagle's medium plus 10% newborn calf serum, 100 units/mg penicillin G, 100 µg/ml streptomycin, and 1 µM ouabain as described previously (2).

Membrane Preparation-- NaI-treated microsomal membranes were prepared from the mutant cells as described earlier (6, 9). Protein content was determined with a detergent-modified Lowry assay (10).

Tryptic Cleavage-- Digestion was carried out at a trypsin/enzyme ratio of 1200 units trypsin/mg of membranes (L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin, Type II, Sigma) in a buffer comprising 100 mM NaCl, 0.2 mM MgSO4, and 20 mM Tris glycylglycine (pH 7.4) at a final enzyme concentration of 35 µg/ml. Digestion was terminated by adding 10-fold excess (units) soybean trypsin inhibitor (Type I-S, Sigma) in a buffer containing 50 mM choline chloride, 0.2 mM MgSO4, and 20 mM Tris glycylglycine (pH 7.4).

Enzyme Assays-- Na,K-ATPase activity was measured as the release of 32Pi from [gamma -32P]ATP as previously described (11). Briefly and unless indicated otherwise, the membranes were preincubated for 10 min at 37 °C with all reactants added except [gamma -32P]ATP. The reaction was initiated by the addition of [gamma -32P]ATP. Final concentrations for Na,K-ATPase activity measurements were 100 mM NaCl, 10 mM KCl, 3 mM MgSO4, 20 mM histidine (pH 7.4), 5 mM EGTA (pH 7.4), and 5 µM ouabain (Sigma). 5 mM ouabain was used to determine base-line hydrolysis activity. As in earlier studies and unless indicated otherwise, assays of Na,K-ATPase activity were carried out using 1 mM ATP to maintain close to saturating ATP concentration and maximize sensitivity of assays of the relatively low activity cultured cells (cf. Refs. 3, 9, and 12). Na-ATPase activity was measured at 1 µM ATP as described previously (2), with varying amounts of added KCl and choline chloride to maintain constant chloride (40 mM) concentration. Base-line activity was determined with 40 mM KCl replacing NaCl. For studies of vanadate sensitivity, inorganic orthovanadate (Fisher) solutions were prepared before the experiment and added with the [gamma -32P]ATP solution to initiate the reaction. Na,K-ATPase activities obtained at various vanadate concentrations and expressed as the percentage of that obtained in the absence of vanadate were analyzed by fitting the data to a one-compartment model using a nonlinear least-square analysis of a general logistic function, as described elsewhere (13). Curve fitting was carried out using the Kaleidagraph computer program (Synergy). Each experiment was carried out at least three times, with one or more chimeras analyzed concurrently with the alpha 2* and alpha 1 isoforms.

Rate of E1P right-arrow E1P-- After formation of E1P in the presence of high chloride concentration (14), the rate of E1P right-arrow E2P was determined by measuring the rate of disappearance of total phosphoenzyme after rapid dilution of the salt (to allow "normal" relaxation of E1P right-arrow E2P) plus the addition of KCl to catalyze rapid hydrolysis of E2P (14, 15). Accordingly, the enzyme was first phosphorylated in medium containing 600 mM NaCl to stabilize E1P, 1 mM MgCl2, 1 mM EGTA, and 20 mM Tris-HCl (pH 7.4) with 1 µM [gamma -32P]ATP for 30 s at 0 °C to obtain maximal phosphoenzyme. Dephosphorylation was then initiated by 6-fold dilution with a "chase" medium containing final concentrations of 20 mM KCl, 10 µM unlabeled ATP, 1 mM EGTA, and 20 mM Tris-HCl (pH 7.4), which simultaneously lowered the NaCl concentration to 100 mM. Samples were taken for measurement of [32P]E for periods up to 30 s. Background phosphoenzyme levels were obtained by allowing the chase to continue for 60 s. The data were fitted to a first-order decay model using the Kaleidagraph nonlinear fitting program (Synergy). At least two different membrane preparations obtained from at least two different clones were assayed. The data presented are representative of at least three independent experiments. Each value shown is the mean ± S.D. of triplicate determinations.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cytoplasmic Interactions of alpha 2*-- Our earlier studies showed that the steady-state E1/E2 conformational equilibrium of alpha 2*, compared with that of the ubiquitous alpha 1 isoform, is poised toward E1. In fact, the kinetic behavior of alpha 2* is generally similar to that of mutants of alpha 1 in which the poise is shifted toward E1. These mutations include a 32-residue deletion of the cytoplasmic N terminus (mutant alpha 1M32) and a Glu-233 right-arrow Lys replacement (mutant alpha 1E233K) in the first cytoplasmic loop. Furthermore, the combination of these two mutations of the so-called Actuator domain (mutant alpha 1M32E233K) in alpha 1 results in a remarkably synergistic shift in poise toward E1 state(s). The findings were interpreted to indicate that interactions between these regions of the Actuator domain (5) and the catalytic loop are critical for conformational coupling of the Na,K-ATPase (4).

Experiments carried out in the present study to address the question of whether cytoplasmic interactions of alpha 2* are analogous to those of alpha 1 are summarized in Fig. 1 and Table I. Expression of alpha 2M30 and alpha 2E231K in HeLa cells yielded functional enzymes capable of supporting cell growth in 1 µM ouabain. Cells transfected with the double mutant alpha 2M30E231K, analogous to alpha 1M32E233K (4), failed to grow even in elevated K+ (cf. Ref. 16). Therefore, a comparable "mutation" was prepared by enzymatically cleaving the N terminus of alpha 2E231K in the E1(Na+) conformation with trypsin (cf. Ref. 1). The single mutants and the cleaved alpha 2E231K (alpha 2E231K-Tryp) were then assessed for shifts in the E1/E2 equilibrium using the following criteria: (i) the effect of K+ on Na-ATPase activity as a measure of E2(K+) right-arrow E1 (see Ref. 2), (ii) K'ATP for low affinity binding to E2(K+), K'ATP(L), and (iii) sensitivity of Na,K-ATPase activity to inhibition by inorganic orthovanadate.


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of K+ on Na-ATPase activity of alpha 1 and alpha 2* cytoplasmic mutants. ATP hydrolysis was assayed in the presence of 1 µM ATP, 20 mM NaCl, and various concentrations of KCl as described under "Experimental Procedures." Data are presented as percent of Na-ATPase activity measured in the absence of added KCl and in the presence of 1 mM KCl. Results shown are the average of at least three separate experiments ± S.D.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Kinetic analysis of cytoplasmic mutants of alpha 1 and alpha 2*
ND, not determined.

Thus, at micromolar ATP concentrations sufficient to saturate only the high affinity phosphorylation site the response of Na-ATPase to K+ is a sensitive means to characterize mutant-specific differences in the K+-deocclusion pathway of the reaction cycle [E2(K+) right-arrowright-arrow E1 + K+] that becomes rate-limiting under these conditions (2, 17). As shown previously and summarized in Table I (upper panel), a low concentration of K+ (1 mM KCl) inhibits the Na-ATPase activity of alpha 1 but stimulates that of alpha 1M32 and alpha 1E233K with a further and notably synergistic stimulation of the double mutant alpha 1M32E233K. The pattern for alpha 2* cytoplasmic mutants is remarkably similar (see Fig. 1 and the lower panel of Table I). With respect to the alpha 2 mutants, K+ stimulates the Na-ATPase activity of alpha 2M30 and alpha 2E231K, ~300 and 900%, respectively, and that of alpha 2E231K-Tryp, more than 2000%. In fact, the apparent stimulation may be an underestimation of the true stimulation since trypsinolysis of alpha 2E231K undoubtedly results in a heterogeneous pool of enzyme species that includes untrypsinized enzyme having inherently lower sensitivity to K+ activation. It should be mentioned that trypsinization of alpha 2 in the presence of Na+ increased K+ activation such that alpha 2-Tryp resembled alpha 2M30 (data not shown; cf. tryptic cleavage of alpha 1 described in Ref. 18), which is not surprising since the region encompassing the tryptic cleavage site (T2; see Ref. 1) is conserved between alpha 1 and alpha 2. These results suggest that alpha 2M30, alpha 2E231K, and alpha 2E231K-Tryp all shift the E1/E2 equilibrium of alpha 2* progressively further toward E1.

As shown in Table I, alpha 2M30, alpha 2E231K, and alpha 2E231K-Tryp also exhibit increased affinities for low affinity ATP binding relative to alpha 2*, with a synergistic effect of the N-terminal deletion and the E231K mutation in the beta -strand region of the M2-M3 loop. It should be noted that, as with the K+ stimulation of Na-ATPase activity shown above, the K'ATP(L) of alpha 2E231K-Tryp is an overestimation of the actual value of the double mutant due to the presence of untrypsinized enzyme.

To gain further insight into the effects of these mutations on conformational equilibrium, we investigated the sensitivity of the Na,K-ATPase activity of the mutants to inhibition by vanadate. Inorganic orthovanadate is a transition state analog of inorganic phosphate that binds to P-type ATPases in the E2 conformation during steady-state catalysis. Consequently, sensitivity of an enzyme to inhibition by vanadate is a measure of the proportion of enzyme in the E2 state (19). As shown by the representative experiment in Fig. 2 and summarized in Table I, alpha 2M30 and alpha 2E231K are both less sensitive to vanadate inhibition than alpha 2*, suggesting that these mutations further shift the E1/E2 equilibrium of alpha 2* toward E1. We were unable to determine accurately the IC50 for vanadate inhibition of alpha 2E231K-Tryp because the residual Na,K-ATPase activity was too low relative to the background (approx 15 nmol/mg·min).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   Vanadate sensitivity of Na,K-ATPase activity of alpha 2* cytoplasmic mutants. ATP hydrolysis at varying vanadate concentrations was determined with 100 mM NaCl, 10 mM KCl, and 1 mM ATP as described under "Experimental Procedures." Data are presented as percent Na,K-ATPase (control) measured in the absence of vanadate. Results shown are from a representative experiment. Each value is the mean ± S.D. of triplicate determinations. Symbols are: open circle , alpha 1; , alpha 2M30; diamond , alpha 2E231K. 100% maximal activities are, 176.0 ± 3.3, 162.7 ± 5.1, and 127.5 ± 8.3 for alpha 1, alpha 2M30, and alpha 2E231K, respectively.

Chimeras of the alpha 1 and alpha 2* Isoforms-- The above analysis of cytoplasmic mutations of the alpha 2* isoform of the Na,K-ATPase support the notion that interactions between the cytoplasmic domains of alpha 2* that modulate conformational shifts are fundamentally similar to those of alpha 1. The analysis also suggests that it is the alpha 2-specific regions of the A domain and/or the catalytic domain with which A interacts that underlie the predilection of this isoform for the E1 state(s). Accordingly, an alpha 1/alpha 2 chimera approach was used to address this issue.

From a comparison of the primary structure of the cytoplasmic regions of alpha 1 and alpha 2* (Fig. 3B), it is important to note that the beta  strand region in the M2-M3 loop of alpha 1 encompassing Glu-233 is highly homologous to that of alpha 2*. In contrast, the two isoforms differ significantly in the primary sequence of their N termini and in the sequence encompassing the ATP binding site in the M4-M5 cytoplasmic loop. Therefore, chimeras were constructed in which the entire N terminus (residues 1-65) and the divergent portion of the nucleotide binding (N) domain in the large M4-M5 loop (residues 429-565) of alpha 1 were substituted with the analogous residues of alpha 2*. It should be noted that our original intent was to investigate 5 individual divergent regions contained within residues 429-565 (see "Experimental Procedures," cassettes I-V) on the E1/E2 equilibrium of alpha 1. Because the individual domains showed no effect on the K+ inhibition of Na-ATPase of alpha 1 relevant to the K+ deocclusion pathway,2 we constructed an alpha 1/alpha 2 chimera where all 5 cassettes of alpha 2* were substituted into alpha 1. Two additional chimeras were constructed. In these chimeras, the first 311 residues of alpha 1, which encompass the entire A domain, are interchanged between alpha 1 and alpha 2* because this region comprises the portion of the cytoplasmic domain that undergoes large rotational displacements during E1/E2 transitions (5, 20). A schematic representation and the designation of the chimeras are illustrated in Fig. 3A.


View larger version (52K):
[in this window]
[in a new window]
 
Fig. 3.   Chimeras of alpha 1 and alpha 2. A, schematic representation of alpha 1/alpha 2 chimeras. alpha 1/alpha 2 chimeras were constructed as described under "Experimental Procedures" whereby the N terminus (alpha 2nt/alpha 1, residues 1-65) and a portion of the catalytic M4/M5 loop of alpha 1 (alpha 2L/alpha 1, residues 429-565) were substituted with the analogous residues of alpha 2* either individually or together (alpha 2(nt+L)/alpha 1). Two additional chimeras were constructed in which the region encompassing the entire Actuator domain (residues 1-311 in alpha 1 and the analogous region of alpha 2*) was interchanged (alpha 1-(1-311)/alpha 2 and alpha 2-(1-309)/alpha 1). B, sequence alignment of alpha 1 and alpha 2. The sequences for the regions described in A of the rat alpha 1 and alpha 2* were aligned using ClustalW, namely (i) the N-terminal segment and (ii) the L region of the N domain. Transmembrane segments are shaded.

To assess the effect of these different regions on the E1/E2 poise of alpha 1, we first investigated the effect of K+ on Na-ATPase activity measured at micromolar ATP for each of the chimeras as described above. As shown in Fig. 4, the Na-ATPase activities of wild type alpha 1 and chimeras alpha 2nt/alpha 1,3 alpha 2L/alpha 1, and alpha 2(nt+L)/alpha 1 are all inhibited by K+. In contrast, wild type alpha 2* and alpha 2-(1-309)/alpha 1 are stimulated by K+ at least up to 1 mM, consistent with a faster E2(K+) right-arrow E1 transition and, hence, with a preponderance of the E1 conformation. It is noteworthy that although the alpha 2nt/alpha 1 construct encompasses the most divergent portion of the primary sequence, it is only upon replacement of the homologous N-terminal segment (residues 1-311) of alpha 1 with that of alpha 2*, chimera alpha 2-(1-309)/alpha 1, that the shift toward E1 is observed.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of K+ on Na-ATPase activity of alpha 1, alpha 2*, and chimeras. Assays were carried out as in Fig. 1. Data are presented as percent of Na-ATPase activity (control) measured in the absence of added KCl. Results shown are from a representative experiment. Each value is the mean ± S.D. of triplicate determinations. Symbols are: open circle , alpha 1; , alpha 2*; diamond , alpha 1-(1-311)/alpha 2; ×, alpha 2-(1-309)/alpha 1; black-square, alpha 2L/alpha 1; triangle , alpha 2nt/alpha 1; , alpha 2(nt+L)/alpha 1.

Fig. 5 shows the determination of K'ATP(L) using the Lineweaver-Burk transformation of the simple Michaelis-Menten analysis of Na,K-ATPase activity as a function of ATP concentration, with values presented in the inset. Only the alpha 2-(1-309)/alpha 1 chimera, like wild type alpha 2*, showed an increased affinity for ATP. Thus, the K+ stimulation of alpha 2-(1-309)/alpha 1 described in Fig. 4 correlates with its increased apparent affinity for ATP, consistent with a shift in the E1/E2 poise in favor of E1. Chimeras alpha 2L/alpha 1 and alpha 2(nt+L)/alpha 1 show no difference in K'ATP(L) relative to alpha 1.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5.   Lineweaver-Burk plot of Na,K-ATPase activity as a function of ATP concentration. ATP hydrolysis was assayed in the presence of 100 mM NaCl, 10 mM KCl, 3 mM MgSO4, and varying ATP concentrations as described under "Experimental Procedures" and normalized to 100% VMAX. Each value is the mean ± S.D. of triplicate determinations. Results are taken from a representative experiment, with K'ATP values show in the inset. Symbols are as in Fig. 4.

To further examine the contribution of the various domains to the poise of E1/E2, we used vanadate as a conformational probe for E2 forms as described above. In one set of experiments, chimeras alpha 1-(1-311)/alpha 2 and alpha 2-(1-309)/alpha 1 were analyzed concurrently with alpha 1 and alpha 2* (Fig. 6A), and in another, the alpha 2L/alpha 1 chimera was compared with alpha 1 and alpha 2* (Fig. 6B). As shown previously (21), the Na,K-ATPase activity of alpha 2* is ~25-fold less sensitive to vanadate than alpha 1 (Fig. 6A). Interestingly, the alpha 1-(1-311)/alpha 2 enzyme is 3.7-fold less sensitive to vanadate than alpha 1 and resembles alpha 1 with respect to K+ inhibition of Na-ATPase and K'ATP(L), whereas alpha 2-(1-309)/alpha 1 is 8.5-fold less vanadate-sensitive than alpha 1 and resembles alpha 2* with respect to K+ activation of Na-ATPase and K'ATP(L). A modest shift in conformational poise is also indicated by the behavior of the alpha 2L/alpha 1 chimera. This replacement of the divergent region of the N domain of alpha 1 by that of alpha 2 reduces the vanadate sensitivity of alpha 1 by a factor of 2.3. In other experiments (not shown) a similar approx 2-fold shift was effected by including the loop insertion of alpha 2* with the alpha 2* N terminus, i.e. a 2-fold higher IC50 for vanadate seen for alpha 2nt/alpha 1 compared with alpha 1, which was further doubled, resulting in a 4-fold higher IC50 compared with alpha 1 for alpha 2(nt+L)/alpha 1. Taken together these findings imply a major contribution of the N-terminal segment encompassing residues 1-309 and a smaller contribution of the L region of alpha 2* in effecting shifts away from E2.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 6.   Vanadate sensitivity of Na,K-ATPase activity of alpha 1, alpha 2*, and chimeras. Assays were carried out as in Fig. 2. One series of experiments was carried out with chimeras alpha 1-(1-311)/alpha 2 and alpha 2-(1-309)/alpha 1 assayed concurrently with alpha 1 and alpha 2*, and a representative experiment is shown in panel A. In a second series, chimera alpha 2L/alpha 1 was assayed concurrently with alpha 1 and alpha 2*, and a representative experiment is shown in Panel B. Symbols are as in Fig. 4.

It has been observed that the alpha 2* isoform and the 32-residue deletion mutant of alpha 1, alpha 1M32, are both enzymes with their E1/E2 equilibrium shifted toward E1 forms. Both enzymes show similar K+ sensitivities of Na-ATPase activity at low ATP, K'ATP(L) values, catalytic turnovers, and K+ deocclusion rates. One major difference between the two enzymes is their sensitivity to inhibition by vanadate. Thus, alpha 2* has a 25-fold lower IC50 than alpha 1M32. Because vanadate sensitivity reflects the steady-state levels of E2, we investigated differences not only in the E2(K+) right-arrow E1 transition rate but also the E1P right-arrow E2P transition rate. As shown previously (22) and represented in Fig. 7, alpha 1M32 has a 5-fold slower conversion of E1P to E2P than alpha 1, consistent with a preference for the E1P form. This does not hold true for alpha 2*; its E1P right-arrow E2P transition rate is only slightly slower than that of alpha 1, providing an explanation for its higher sensitivity to vanadate compared with alpha 1M32. The implication of vanadate sensitivity as a measure of the E1/E2 poise during steady-state catalysis is discussed further below.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 7.   Rate of formation of E2P from E1P. The rate of E1P right-arrow E2P was measured indirectly at 0 °C as described under "Experimental Procedures." The results are taken from a representative experiment with mean ± S.D. of triplicate determinations. Symbols are open circle , alpha 1; , alpha 2*; black-diamond , alpha 2-(1-309)/alpha 1; ×, alpha 1M32.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our earlier studies showed a notable similarity between the alpha 2* isoform of the Na,K-ATPase and the "E1-shifted" mutants of alpha 1, particularly the deletion mutant alpha 1M32. Nevertheless, the behavior of alpha 1/alpha 2 chimeras in which the first 32 residues of the N termini were interchanged showed that the kinetic difference between alpha 1 and alpha 2* could not be explained by their distinct N-terminal 1-32 residue per se. More likely, those findings indicate that the difference is due to the interaction of the N terminus with another, isoform-distinct region(s) of the enzyme.

As a preliminary step toward defining the structural basis for alpha 1/alpha 2 differences, we have used a mutagenesis approach to obtain evidence for cytoplasmic interactions between the N terminus, the M2-M3 loop, and large M4-M5 loop of alpha 2*, as noted previously for alpha 1 (3, 4). As shown in Table I (lower panel), a kinetic analysis of cytoplasmic mutants of alpha 2*, namely alpha 2M30 and alpha 2E231K, show that both mutations effect shifts in the E1/E2 poise of alpha 2* analogous to those seen for alpha 1, although even further toward E1. HeLa cells transfected with the double mutant, alpha 2M30E231K, analogous to alpha 1M32E233K, failed to grow. Noting that the catalytic turnover of alpha 1M32E233K is <= 500 min-1 (4), it is likely that the catalytic turnover of alpha 2M30E231K is much too low to support HeLa cell growth in 1 µM ouabain. Consequently, the N terminus of alpha 2E231K was enzymatically cleaved by trypsinolysis of membranes isolated from the alpha 2E231K-transfected cells as originally described by Jorgensen (1). Like alpha 1M32E233K, alpha 2E231K-Tryp showed a strong, synergistic effect on E1 left-right-arrow E2, shifting it even further in favor of E1 forms. Therefore, we conclude that the interaction of the cytoplasmic domains of alpha 2* that modulate conformational shifts are fundamentally similar to those of alpha 1.

As already mentioned, the primary structure of the beta -strand region in the M2-M3 loop encompassing Glu-231 of alpha 2* is highly homologous to that of alpha 1 containing Glu-233. In contrast, the primary structures of the two isoforms differ significantly in their N termini (domain nt, 56% identity)4 and a region within the N domain of the M4-M5 loop and referred to here as the L region (61% identity). It is noteworthy that for the remainder, the sequence identity is very high, namely 86% between the end of the nucleotide and the beginning of the L region (residues 429-565; see Fig. 3B) and 92% from the C-terminal end of the L region to the C terminus of the protein. Therefore, alpha 1/alpha 2 chimeras were constructed in which domains of divergent primary sequence were interchanged. Thus, the N terminus (nucleotide residues 1-65) and L region (residues 429-565) of alpha 1 were substituted with the analogous regions of alpha 2*. This was done either individually (alpha 2nt/alpha 1 and alpha 2L/alpha 1, respectively) or in combination (alpha 2(nt+L)/alpha 1) (see Fig. 3A for a schematic representation). In addition to the above, chimeras encompassing the entire Actuator domain, i.e. alpha 1-(1-311)/alpha 2 and alpha 2-(1-309)/alpha 1), were analyzed. As shown in recent structural studies, this domain undergoes large rotational motions (estimated at 110° in the sarcoplasmic reticulum Ca-ATPase) in the course of the conformational transitions (20). The present results show that although a switch of the entire N terminus is without effect, inclusion of the entire isoform-divergent N-terminal segment up to residue 309 of alpha 2* is capable of conferring alpha 2*-like kinetics to alpha 1. This is apparent from the K+ activation of Na-ATPase activity at low ATP when E2(K+) right-arrow E1 is rate-limiting as well as a decrease in K'ATP(L). However, this N-terminal segment of alpha 2* only partially decreases the vanadate sensitivity of alpha 1 (approx 8.5-fold compared with 20-25-fold for alpha 2* relative to alpha 1). In addition, however, the L region of alpha 2* confers a 2.3-fold decrease in vanadate sensitivity to alpha 1, such that synergistic effects of the two domains, residues 1-309 in the N-terminal segment and residues 427-562 in the N domain, can account for the alpha 2* versus alpha 1 differences.

Although it is attractive to hypothesize that the E1 shift of alpha 2-(1-309)/alpha 1 is due to the A domain, one cannot exclude the possible contribution of amino acid replacements in other regions, namely M1, M2, and M3 and the extracellular M1-M2 and M3-M4 loops. Of these regions, it is noteworthy that Coppi et al. (31) showed that an alpha 2-(1-129)/alpha 1 chimera is not E1-shifted because, like alpha 1, its Na-ATPase activity is inhibited by K+ at low ATP concentration (31). This result provides a basis for eliminating M1 and the M1-M2 loop as candidates for effecting the E1 shift. For the rest, there are four replacements, namely Ser-132 right-arrow A in M2, His-288 right-arrow Gln in M3, and Glu-309 right-arrow Gly and Thr-311 right-arrow Ser in the M3-M4 loop. Experiments are currently under way to determine whether and/or which ones of these replacements are important or whether it is the A domain per se that confers the alpha 2-like E1 shift seen with the alpha 2-(1-309)/alpha 1 mutant.

It is instructive to consider likely alpha 1/alpha 2 differences in the interactions of the A domain with the N and P domains in the E1 and E2 conformations as well as interactions within regions of the A domain. With the Na,K-ATPase, metal-catalyzed oxidative cleavage studies of domain interactions reveal that in the E1 conformation, the N domain docks onto the phosphorylation (P) domain, and A moves apart; in E2, A docks onto P, and N is displaced (32), consistent with putative domain interactions deduced from crystal structures of sarco(endo)plasmic reticulum calcium ATPase in E1 and E2 states (5, 20, 33). Concerted effects of alpha 1 versus alpha 2 distinct residues within the N-terminal segment encompassing domain A and the L region within domain N can be explained by the recent model for regulation of the aforementioned domain interactions regulated by the N terminus (22). Thus, assuming that the secondary structure of the alpha 2* N terminus, like that of alpha 1, has the propensity to form three short helices (H1, H2, and H3), intramolecular interactions between helices 1 and 2 of the N terminus allow the M2-M3 loop in A and the N domain to come together in the E2 conformation. In the E1 conformation, H2 interacts with the M2-M3 loop to keep it apart from N. Thus, isoform diversity in the N terminus impacts intramolecular interactions of H2 with M2-M3 within the A domain. The minor E1 shift effected by the swap of the isoform divergent portion of the N domain (L swap) probably indicates interaction between N and P in the E2P conformation mediated by the M2-M3 loop. Accordingly, the following are likely interactions deduced from the proximity (<= 3Å) of backbone carbonyl groups of sarco(endo)plasmic reticulum calcium ATPase. (i) The N terminus may interact with the region near Asp-203 of the sarco(endo)plasmic reticulum calcium ATPase M2-M3 loop (Glu-233 of alpha 1) in the E1 (5) but not in E2P (vanadate-trapped intermediate) (33) or E2 (thapsigargin-bound intermediate) (20) conformations. (ii) The region near Asp-203 of the sarco(endo)plasmic reticulum calcium ATPase M2-M3 loop does not interact with either the N or P domains in E1 but could interact with both N and P in E2P and with only N in E2; there are no nearby sites of interaction between the N terminus and either the N or P domains, consistent with an intermediary role of the M2-M3 loop of the A domain in E1 left-right-arrow E2 conformational changes (22).

The present kinetic analysis involves evaluation of the overall reaction cycle under different conditions of rate limitation or ligand perturbations as well as certain partial reactions relevant to conformation transitions. The usefulness of this approach is indicated by the comparison of alpha 2* and alpha 1M32 as summarized in Table II. Compared with alpha 1, alpha 1M32 but not alpha 2*, exhibits a substantial slowing of the E1P right-arrowright-arrow E2P conversion, providing an explanation for its much larger decrease in sensitivity to vanadate compared with alpha 2*, i.e. IC50 values of alpha 1M32 and alpha 2 are decreased ~500- and 20-fold, respectively, compared with alpha 1. Accordingly, the E1 shift in E1/E2 poise of alpha 2* is due to a shift in dephosphoenzyme [E2(K+) right-arrow E1] but not phosphoenzyme [E1P right-arrow E2P]. This result indicates the "nonequivalence" of the two conformational transitions and also highlights a limitation of the sole use of vanadate as a probe of conformation since the steady-state proportion of enzyme in the E2 state reflects the rates of transition between both dephospho- and phosphoenzyme states.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Criteria for assessing E1/E2 poise of alpha 1, alpha 1M32, and alpha 2*

This report deals only with the structure/function analysis of isoform-specific kinetic behavior. Isoform-specific interactions of the pump with other proteins such as cytosolic second messengers may be important for adapting sodium pump function to cell-specific requirements as suggested by Blanco et al. (23). Thus, targets for protein kinase C phosphorylation present in the N terminus of alpha 1 are absent in alpha 2. The significance of this difference is underscored by the observation that in transfected opossum kidney cells (24) protein kinase Cbeta -mediated phosphorylation of alpha 1 at these residues promotes its translocation to the plasma membrane. Similarly, in alpha 1 but not alpha 2, the presence of Tyr-5 within a consensus sequence for phosphorylation by tyrosine kinases of the Src family has been implicated in the insulin stimulation of pump activity in the proximal convoluted tubules of the rat kidney (25). Furthermore, although insulin acts via tyrosine kinases in rat proximal convoluted tubules to stimulate pump activity by increasing the apparent Na+ affinity (26) in skeletal muscle, it promotes translocation of alpha 2 to the plasma membrane (27) via the action of protein kinase C (28). There is also direct evidence for a role of the isoform-specific primary sequence within the M4-M5 loop domain, in regulation of the pump by second messengers. In agreement with our findings, Pierre et al. (29) failed to detect an effect of the introduction of an alpha 2* distinct region of the large catalytic loop (residues 489-499, contained within the L region) on the E1/E2 conformational equilibrium of alpha 1. However, a role for this domain in the isoform-specific response of transfected opossum kidney cells to protein kinase C activation was observed in the differential response of the isoforms to hormones via the action of second messengers. Taken together, the foregoing studies suggest that the primary sequence diversity may in part be relevant to isoform-specific pump regulation, separate from a role in isoform-distinct pump kinetics.

In conclusion, we have shown that the E1 shift in the conformational equilibrium of alpha 2 can be largely accounted for by the N-terminal third of the alpha  subunit that comprises mainly the A domain, with a small contribution of the isoform-specific sequence of the N domain within the M4-M5 loop. In addition, despite the kinetic similarities of the alpha 2 isoform with cytoplasmic mutants of alpha 1, such as alpha 1M32, the E1 shift of alpha 2 results primarily from differences in the dephosphoenzyme conformational transition, i.e. E2(K+) right-arrow E1, with its E1P right-arrow E2P transition rate similar to that of alpha 1.

    ACKNOWLEDGEMENTS

We thank Drs. E. Jewell and J. B. Lingrel for the rat alpha 2* transfected cells.

    FOOTNOTES

* This work was supported by Canadian Institutes of Health Research Grant MT-3876, an operating grant from the Quebec Heart and Stroke Foundation (to R. B.), National Institutes of Health Grant HL 49204 (to L. K.), and a predoctoral fellowship from the Heart and Stroke Foundation of Canada (to L. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Montreal General Hospital Research Institute, 1650 Cedar Ave., Montreal, Quebec H3G 1A4, Canada. Tel.: 514-934-1934 (ext. 44501); Fax: 514-934-8332; E-mail: rhoda.blostein@mcgill.ca.

Published, JBC Papers in Press, January 14, 2003, DOI 10.1074/jbc.M211636200

1 alpha 2* denotes the rat alpha 2 enzyme rendered relatively ouabain-resistant to permit its distinction from the endogenous, ouabain-sensitive HeLa enzyme (6). The asterisk is not shown for the alpha 2 mutants.

2 L. Segall, L. K. Lane, and R. Blostein, unpublished information.

3 Chimera abbreviations: alpha 2nt/alpha 1, alpha 2-(1-63)/alpha 1; alpha 2L/alpha 1, alpha 2-(427-562)/alpha 1; alpha 2(nt+L)/alpha 1, alpha 2-(1-63, 427-562)/alpha 1.

4 As determined by BLAST, NCBI.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Jorgensen, P. L. (1975) Biochim. Biophys. Acta 401, 399-415[Medline] [Order article via Infotrieve]
2. Daly, S. E., Lane, L. K., and Blostein, R. (1994) J. Biol. Chem. 269, 23944-23948[Abstract/Free Full Text]
3. Daly, S. E., Lane, L. K., and Blostein, R. (1996) J. Biol. Chem. 271, 23683-23689[Abstract/Free Full Text]
4. Boxenbaum, N., Daly, S. E., Javaid, Z. Z., Lane, L. K., and Blostein, R. (1998) J. Biol. Chem. 273, 23086-23092[Abstract/Free Full Text]
5. Toyoshima, C., Nakasako, M., Nomura, H., and Ogawa, H. (2000) Nature 405, 647-655[CrossRef][Medline] [Order article via Infotrieve]
6. Jewell, E. A., and Lingrel, J. B. (1991) J. Biol. Chem. 266, 16925-16930[Abstract/Free Full Text]
7. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 488-492[Abstract]
8. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745-2752[Medline] [Order article via Infotrieve]
9. Lane, L. K., Feldmann, J. M., Flarsheim, C. E., and Rybczynski, C. L. (1993) J. Biol. Chem. 268, 17930-17934[Abstract/Free Full Text]
10. Markwell, M. A., Haas, S. M., Tolbert, N. E., and Bieber, L. L. (1981) Methods Enzymol. 72, 296-303[Medline] [Order article via Infotrieve]
11. Blostein, R. (1988) Methods Enzymol. 156, 171-178[Medline] [Order article via Infotrieve]
12. Munzer, J. S., Daly, S. E., Jewell-Motz, E. A., Lingrel, J. B., and Blostein, R. (1994) J. Biol. Chem. 269, 16668-16676[Abstract/Free Full Text]
13. DeLean, A., Munson, P. J., and Rodbard, D. (1978) Am. J. Physiol. 235, E97-E102[Medline] [Order article via Infotrieve]
14. Klodos, I., Post, R. L., and Forbush, B., III (1994) J. Biol. Chem. 269, 1734-1743[Abstract/Free Full Text]
15. Vilsen, B. (1997) Biochemistry 36, 13312-13324[CrossRef][Medline] [Order article via Infotrieve]
16. Arguello, J. M., and Lingrel, J. B. (1995) J. Biol. Chem. 270, 22764-22771[Abstract/Free Full Text]
17. Post, R. L., Hegyvary, C., and Kume, S. (1972) J. Biol. Chem. 247, 6530-6540[Abstract/Free Full Text]
18. Wierzbicki, W., and Blostein, R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 70-74[Abstract]
19. Cantley, L. C., Jr., Cantley, L. G., and Josephson, L. (1978) J. Biol. Chem. 253, 7361-7368[Medline] [Order article via Infotrieve]
20. Toyoshima, C., and Nomura, H. (2002) Nature 418, 605-611[CrossRef][Medline] [Order article via Infotrieve]
21. Segall, L., Daly, S. E., and Blostein, R. (2001) J. Biol. Chem. 276, 31535-31541[Abstract/Free Full Text]
22. Segall, L., Lane, L. K., and Blostein, R. (2002) J. Biol. Chem. 277, 35202-35209[Abstract/Free Full Text]
23. Blanco, G., Sanchez, G., and Mercer, R. W. (1998) Arch. Biochem. Biophys. 359, 139-150[CrossRef][Medline] [Order article via Infotrieve]
24. Efendiev, R., Bertorello, A. M., Pressley, T. A., Rousselot, M., Feraille, E., and Pedemonte, C. H. (2000) Biochemistry 39, 9884-9892[CrossRef][Medline] [Order article via Infotrieve]
25. Feraille, E., Carranza, M. L., Gonin, S., Beguin, P., Pedemonte, C., Rousselot, M., Caverzasio, J., Geering, K., Martin, P. Y., and Favre, H. (1999) Mol. Biol. Cell 10, 2847-2859[Abstract/Free Full Text]
26. Feraille, E., Carranza, M. L., Rousselot, M., and Favre, H. (1994) Am. J. Physiol. 267, F55-F62[Medline] [Order article via Infotrieve]
27. Lavoie, L., Roy, D., Ramlal, T., Dombrowski, L., Martn-Vasallo, P., Marette, A., Carpentier, J. L., and Klip, A. (1996) Am. J. Physiol. 270, C1421-C1429[Medline] [Order article via Infotrieve]
28. Sampson, S. R., Brodie, C., and Alboim, S. V. (1994) Am. J. Physiol. 266, C751-C758[Medline] [Order article via Infotrieve]
29. Pierre, S. V., Duran, M.-J., Carr, D. L., and Pressley, T. A. (2002) Am. J. Physiol. 283, F1066-F1074
30. Daly, S. E., Blostein, R., and Lane, L. K. (1997) J. Biol. Chem. 272, 6341-6347[Abstract/Free Full Text]
31. Coppi, M. V., Compton, L. A., and Guidotti, G. (1999) Biochemistry 38, 2494-2505[CrossRef][Medline] [Order article via Infotrieve]
32. Patchornik, G., Munson, K., Goldshleger, R., Shainskaya, A., Sachs, G., and Karlish, S. J. D. (2002) Biochemistry 41, 11740-11749[CrossRef][Medline] [Order article via Infotrieve]
33. Xu, C., Rice, W. J., HE, W., and Stokes, D. L. (2002) J. Mol. Biol. 316, 201-211[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.