Molecular and Cellular Biochemistry 114: 49-56, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Probing the structure of the Neurospora crassa plasma membrane H+-ATPase Gene A. Scarborough Department of Pharmacology University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

Abstract The structure of the Neurospora crassa plasma membrane H+-ATPase has been investigated using a variety of chemical and physicochemical techniques. The transmembrane topography of the H+-ATPase has been elucidated by a direct, protein chemical approach. Reconstituted proteoliposomes containing purified H+-ATPase molecules oriented predominantly with their cytoplasmic surface facing outward were treated with trypsin, and the numerous peptides released were purified by HPLC and subjected to amino acid sequence analysis. In this way, seventeen released peptides were unequivocally identified as located on the cytoplasmic side of the membrane, and numerous intervening segments could be inferred to be cytoplasmically located by virtue of the fact that they are too short to cross the membrane and return between sequences established to be cytoplasmically located. Additionally, three large membrane-embedded segments of the H+-ATPase were isolated using our recently developed methods for purifying hydrophobic peptides, and identified by amino acid sequence analysis. This information established the topographical location of virtually all of the 919 residues in the H+-ATPase molecule, allowing the formulation of a reasonably detailed model for the transmembrane topography of the H+-ATPase polypeptide chain. Separate studies of the cysteine chemistry of the H+-ATPase have demonstrated the existence of a single disulfide bridge in the molecule, linking the NH2- and COOH-terminal membrane-embedded domains. And, analyses of the circular dichroism and infrared spectra of the purified H+-ATPase have elucidated the secondary structure composition of the molecule. A first-generation model for the tertiary structure of the H+-ATPase based on this information and other considerations is presented. (Mol Cell Biochem 114: 49-56, 1992)

Key words: membrane transport, H+-ATPase, P-type ATPase, molecular structure, folding model

Introduction Ever since the turn of the century, when the existence of a cell plasma membrane, or 'lipoid layer' began to be appreciated [1], a major question in biology has been the means by which polar compounds are made able to penetrate this hydrophobic barrier in the process known as membrane transport. Over the last several decades, numerous different transport systems have

been established for probing the mechanisms of membrane transport, and a great deal of experimental information about these various systems has accumulated. Nevertheless, our understanding of this fascinating process remains primitive, at best. The field of enzymic catalysis has shown us clearly that in order to understand how membrane transport proteins work, it is

Address for offprints: G.A. Scarborough, CB #7365 F.L.O.B., Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

50 going to be necessary to understand, in detail, their molecular structures. In this article, progress made in this laboratory toward understanding the molecular structure of one membrane transport protein, the proton-translocating ATPase from the plasma membrane of Neurospora, is described. From the information we have gained about this transporter, and additional considerations regarding protein structure in general, a feasible working model for the structure of the H +ATPase is proposed.

Results and discussion Previous studies Our original studies with the H+-ATPase were carried out using plasma membrane vesicles isolated by the concanavalin A method [2]. The hydrolytic moiety of the ATPase was identified as a polypeptide with a molecular mass of about 100 kDa [3], and the physiological role of the ATPase as an electrogenic proton pump was firmly established [4, 5]. It was also shown that the catalytic mechanism of the H+-ATPase involves transient phosphorylation and dephosphorylation of the beta-carboxyl group of an aspartic acid residue in the 100 kDa polypeptide chain [3, 6], establishing the kinship of this ATPase with animal cell counterparts such as the Na+/K + - , H+/K + - , and Ca++-ATPases of plasma membranes, and the Ca++-ATPase of muscle sarcoplasmic reticulum. The more recent availability of amino acid sequence information deduced from the corresponding gene sequences has borne this out [7, 8] and in addition has included a great many other ATPases in this rather large family of transport enzymes, now conveniently referred to as the P-type ATPases [9]. ATPase conformational changes correlating with the attainment of the transition states of the H+-ATPase phosphorylation and dephosphorylation reactions during the catalytic cycle have been demonstrated as well [10]. Procedures have also been developed for solubilizing and purifying active H+-ATPase to near homogeneity in 100 milligram quantities [11, 12], and for reconstituting the purified molecule in fully functional form into artificial phospholipid vesicles [13]. And, using this reconstituted system, we have been able to demonstrate that a single copy of the 100 kDa hydrolytic moiety alone is capable of efficient ATP-hydrolysisdriven proton translocation [13, 14]. With this important groundwork laid, our efforts to understand the

structure and mechanism of the Neurospora plasma membrane proton pump have been reduced to considerations of the events occurring in a single copy of the 100 kDa polypeptide chain.

Transmembrane topography of the H+-ATPase In addition to providing information as to the subunit composition and minimal functional unit of the H ÷ATPase, reconstituted ATPase proteoliposomes proved to be quite useful for probing the transmembrane topography of the molecule. On the basis of susceptibility to degradation by trypsin and protection against this degradation by the ATPase ligarlds, MgATP and vanadate, it was shown that 85-90% of the ATPase molecules in the proteoliposomes are oriented with their cytoplasmic surface facing outward, whereas the remaining 10-15% are present with the opposite orientation and are totally resistant to tryptic cleavage [15, 16]. It was also shown [15] that tryptic cleavage in the presence of ligands produces ca. 97, 95, and 88kDa degradation products similar to those shown by Mandala and Slayman [17] to result from cleavage of the ATPase at lys24, lys36, and arg 7~, indicating that these Nterminal sites are present on the cytoplasmic side of the membrane. Tryptic cleavage in the absence of ligands leads to the release of numerous H+-ATPase peptides from the proteoliposomes, and in our initial investigation of these peptides [15], one was purified by HPLC and shown by N-terminal amino acid sequence analysis to comprise residues 901-911 at the extreme C-terminus of the molecule. These results established the location of the N- and C-termini of the ATPase molecule on the cytoplasmic side of the membrane. The same conclusion has been reached by Mandala and Slayman [18]. Following up on our first investigation, fourteen additional peptides released from the liposomes were purified by HPLC and identified by N-terminal sequence analysis [16]. The results obtained established residues 70-100, 186-219,238-256, 441.460,471-512, 545-559, and 567-663 to be located on the cytoplasmic side of the membrane. Moreover, this information allowed the identification of several additional flanking sequences, including residues 29-32, 41-69, 220-237, 461-470, 513-544, 560-566, and 916-920, as also likely to be cytoplasmically located, since they are too short to cross the membrane and return. These results collectively indicate that residues 21-100, 186256, 441-663, and 897-920 are located on the cytoplasmic side of the mem-

51 brane. Additional experiments have also localized residues 359-440 on the cytoplasmic side of the membrane (manuscript submitted). After the development of methodology for manipulating the hydrophobic segments of the H+-ATPase [19, 20], these studies were extended to define the parts of the ATPase molecule remaining associated with the liposomes after removal of the released peptides [21]. The liposome-bound peptides were fractionated by Sephadex LH60 chromatography in chloroform-methanol-trifluoroacetic acid and the resulting eluate was analyzed by an SDS-PAGE procedure specifically developed for analyzing hydrophobic peptides [20]. Three major peptides with approximate Mr's of 7, 7.5, and 21 kDa were identified by N-terminal sequence analysis as H+-ATPase peptides beginning at residues 100,272, and 660, respectively. On the basis of their size, these peptides probably end near residues 173,355, and 891, respectively. These peptides were also labeled from the liposomal membrane interior by the lipophilic photolabeling reagent, [125I]-trifluoromethyliodophenyldiazifine, whereas other parts of the molecule were not. It was therefore concluded that these three peptides constitute the great majority of the membrane-embedded region of the H+-ATPase molecule. Additional considerations from these studies localized residues 174-185 and 257-271 on the cytoplasmic side of the membrane. The results of all of these topography studies are summarized in Fig. 1. The filled circles indicate residues of the H+-ATPase shown directly to be located on the cytoplasmic side of the membrane by purification and amino acid sequence analysis. The open circles indicate residues deduced to be cytoplasmically located by virtue of their positions between residues directly shown to be cytoplasmically located. The hatched circles indicate residues in membrane-embedded segments. The lines in the sequence indicate minor regions with locations as yet not established. Thus, the topographical locations of nearly all of the 919 residues in the molecule have been established. It should be emphasized that the exact points of entry and exit of the polypeptide chain into and out of the membrane are not implied in the model.

Chemical state of the cysteine residues in the H+-ATPase molecule As indicated by the gene sequence, there are eight cysteine residues in the H+-ATPase molecule [7, 8]. To

determine the chemical state of these cysteine residues, direct chemical studies with established cysteine and cystine reagents were carried out [22]. Titrations with cysteine and cystine reagents indicated the presence of six free cysteines and one disulfide bridge in the molecule. Quantitative carboxymethylation experiments with radioactive iodoacetate under reducing and nonreducing conditions confirmed this conclusion. ATPase carboxymethylated under both conditions was then cleaved with trypsin and the digests resolved into hydrophilic and hydrophobic fractions [19]. Five of the six labeled free cysteine peptides partitioned into the hydrophilic fraction and were purified and established to contain cys 376, cys 4°9, cys 472, cys 532, and cys 545, The labeled free cysteine in the hydrophobic fractions was identified as either cys 84° or cys s69, which in turn identified the other as one of the disulfide bridge cysteines. The other disulfide bridge cysteine was identified as cys14s by purification and N-terminal amino acid sequencing of an additional peptide labeled in the reduced enzyme. Thus, the disulfide bridge in the H +ATPase molecule is between cys148 and either cys 84° o r cys 869.

Secondary structure of the H+-ATPase molecule As isolated by the large-scale isolation procedure mentioned above [11, 12], the purified H+-ATPase is a hexamer of 100kDa monomers [23]. Curiously, after purification of the ATPase in the presence of detergents, the detergents can be removed by molecular sieve chromatography with essentially no effect on the quaternary structure or stability of the hexamers [23, 24]. The resulting preparation contains ATPase hexamers with only 5-10% non-ATPase protein, approximately 12 moles of tightly-bound lysolecithin/mole of ATPase monomer, and little or no plasma membrane phospholipid [24]. Importantly, the H+-ATPase monomers in the hexamers are fully functional, indicating that the functional properties of the soluble hexamers are relevant to those of the enzyme in its membranebound state [24]. This form of the ATPase was quite useful for studies of the secondary structure of the H +ATPase by circular dichroism, since such preparations are virtually free of light scattering and other artifacts that commonly hinder optical studies of membranebound proteins or even detergent-solubilized preparations. Thus it was possible to determine the circular dichroism spectrum of the H+-ATPase from 184 to

52 260 nm, from which the secondary structure composition of the ATPase was estimated by the singular value decomposition procedure of Hennessey and Johnson [25]. The results indicated that the H+-ATPase contains approximately 36% helix, 12% antiparallel beta-sheet, 8% parallel beta-sheet, 11% beta-turn, and 26% irregular structure [24]. Importantly, no detectable changes in the circular dichroism spectrum of the ATPase were found to occur in the presence of ATPase ligands known to induce enzyme conformational changes resembling those that occur during the ATP hydrolytic cycle, indicating that substantial changes in the secondary structure of the ATPase are probably not involved in the transport mechanism. Finally, the circular dichroism spectrum of the H+-ATPase was compared to the corresponding spectra of the Na+/K +- and Ca++-translocat ing ATPases and shown to be quite similar, indicating in yet another way that the P-types ATPases possess considerable structural similarity. In another study, the H+-ATPase in reconstituted proteoliposomes was analyzed by infrared attenuated total reflection spectroscopy, and the secondary structure elements of the molecule were determined by Fourier self-deconvolution [26]. Essentially identical secondary structure estimates for the ATPase were obtained by this entirely different approach, suggesting quite strongly that these secondary structure estimates are reasonably accurate. Thus, models for the structure of the H+-ATPase must take this information into account.

A model for the teriary structure of the H+-ATPase With this information, it is possible to begin to consider how the H+-ATPase polypeptide chain might fold into its functional three-dimensional structure. First, regarding the number of membrane-spanning stretches, the experimental data indicate only that each of the three membrane-embedded peptides must have an even number of and at least two such stretches. However, hydropathy analysis by the method of Mohana Rao and Argos [27] suggests that the second membrane-embedded segment beginning at residue 272 could have four membrane-spanning stretches, and that the third segment beginning at residue 660 could have as many as six [19]. Thus, as indicated in Fig. 1, our working model for the membrane-embedded region of the ATPase has twelve membrane-spanning stetches. If so, it would add the H+-ATPase to the large and rapidly

growing family of transport molecules with twelve posited membrane-spanning stretches [28-31]. Although most models for the P-type ATPase do not propose this many membrane-spanning stretches, this could be a reflection of the rules for hydropathy analysis, which reject polar residues from membrane-embedded regions. Since the membrane-embedded regions of the P-type ATPases must contain the path for ion transit through the membrane, which could be quite polar, it may be that strict hydropathy analyses underestimate the actual number of membrane-embedded stretches. Except for the two additional membrane-spanning stretches in the second membrane-embedded segment, the proposed membrane-embedded stretches indicated in Fig. 1 are similar to those proposed for the closely related Ca++-ATPase [32]. In fact, the overall topography proposed in Fig. 1 is quite similar to the Ca ++ATPase model, lending additional credibility to each. While there may be exceptions [33], it is generally agreed that the membrane-embedded stretches in integral membrane proteins are probably helices more or less parallel to the membrane normal [34]. This is clearly the case for the two integral membrane proteins of known structure, the photosynthetic reaction center [35] and bacteriorhodopsin [36]. For these reasons, it is reasonable to assume at least tentatively that the transmembrane stretches of the Neurospora H+-ATPase are also folded this way. Four of the proposed helices in the N-terminal segment are connected by short loops, so it is likely that these helices are close to each other in the folded molecule. This is also the case for at least four of the six proposed helices in the C-terminal membraneembedded region. Moreover, as described above, there is a disulfide bridge in the H+-ATPase molecule between cys 14s in the N-terminal membrane-embedded region and either cyss4° o r c y s 869 in the C-terminal membrane-embedded region [22]. It is therefore likely that many, if not all, of the N- and C-terminal transmembrane helices are clustered together in the folded molecule. Thus, the N-terminal group of membrane-embedded helices in the two-dimensional model of Fig. 1 can be folded over into juxtaposition with the C-terminal group. How the molecule might look after this is shown in Fig. 2. The Z-shaped line denotes one of the two possible disulfide bridge configurations. Although this model is speculative, several features are worth noting. First, the transmembrane helices (cylinders) have been grouped into three sets of antiparallel four-helix bundles. In view of the documented marked stability of the antiparallel four-helix bundle [37-41],

53 84O

t48

OUT

272

33

~

~

•

I

X~

~J~ ; ~

~

IN

66O

Fig. 1. Model for the transmembrane topography of the H+-ATPase. OUT and IN indicate points of reference outside and inside an intact cell, respectively. See text for additional details.

enhanced for helices closely spaced in a linear sequence or held together by a disulfide bridge, and further enhanced in an hydrophobic environment such as the interior of a lipid bilayer [37, 41], an antiparallel fourhelix bundle configuration for the transmembrane helices is quite feasible. In support of this suggestion, antiparallel helix alignments and four-helix bundle configurations dominate the structures of the membraneembedded helix sectors of both the photosynthetic reaction center [42] and bacteriorhodopsin [36]. Moreover, since four-helix bundles have a marked tendency to

interact with each other in highly symmetrical ways [37], the proposed bundles are suggested to be present in the molecule as a symmetrical ring, as shown. Many isomers of this structure with different interhelix contacts are equally possible; the helix numbers indicated in the figure are included only to facilitate tracking the path of the polypeptide chain. Interestingly, an helix arrangement similar to this, but without four-helix bundles, was recently suggested by Maloney [28] on the basis of an entirely different line of reasoning. The helix arrangement proposed in Fig. 2 is also similar to one of

54 cytoplasmic portion of the H+-ATPase polypeptide chain in the model of Fig. 2 has been drawn to suggest this situation. The proposed interdomain cleft is indicated by the arrow. Alternatively, the active-site cleft may be at the side of the molecule near the surface of the membrane, as can be imagined from inspection of the structures proposed by Taylor et al. [54] and Stokes and Green [55] for the Ca++-ATPase. Thus, from the information we have generated from a variety of chemical and physicochemical approaches, and considerations of known modes of protein folding, it has been possible to construct a first generation model for the structure of the Neurospora crassa plasma membrane H+-ATPase that has a reasonable chance of being approximately correct. Future work will be aimed at testing these notions experimentally. N

Acknowledgement Supported by USPHS NIH grant GM24784.

References

Fig. 2. Working model for the tertiary structure of the H+-ATPase. See text for details. Reproduced from 'Molecular Aspects of Transport Proteins', Elsevier Science Publishers B.V., Amsterdam.

the arrangements proposed by Green [43] for the Ca ++ATPase, if two of the outer helices are removed from the model, as would be required if the H+-ATPase has only ten helices. Second, our studies of the secondary structure of the H+-ATPase indicate that about 36% of the polypeptide chain is present in a helical configuration [24, 26]. If the membrane-embedded sector of the molecule is helical as shown, only 90 or so additional residues are present in a helical configuration. Thus, the majority of the cytoplasmic portion of the molecule must comprise beta-sheet, beta-turn, and other structure, in contrast to certain predicted models for the cytoplasmic portions of the Ca ++- and Na+/K+-ATPases [44, 45]. And finally, since essentially all enzymes [46], particularly phosphoryl-transfer enzymes [47-53], have structures with at least two, separate structural domains, or lobes, separated by a usually deep, active-site cleft, the

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