Structures of P-Type and F-Type Ion Pumps David L Stokes and Robert K Nakamoto

Structures of P-type and F-type ion pumps David L Stokes and Robert K Nakamoto

University of Virginia, Charlottesville, USA

P-type ion pumps are large, integral membrane proteins, whereas the F-type ATP synthase is an enormous multiple-subunit complex. Both have been the focal points of intensive physiological and mechanistic studies for many years; however, critical information about three-dimensional structure has been lacking. During the past year, new crystal forms of both P-type and F-type ATPases were reported. Although the resulting structures are at medium to low resolution, they provide novel structural and functional information, as well as prospects for higher resolution structures in the foreseeable future.

Current Opinion in Structural Biology 1994, 4:197-203

Introduction termined for both of these molecules, but better specimen preparation of CaZ+-ATPase improved the resolu- Pumping ions across biological membranes represents ° tion, and a new, more stable crystal form helped the a crucial activity of cells. The resulting ion gradients are determination of the Na+/K+-ATPase structure. In ad- used for a wide variety of purposes, including such es- dition, X-ray diffraction of non-crystalline CaZ+-ATPase sential functions as intracellular signaling, transport of has investigated the structural effects of Ca 2+ binding. nutrients and synthesis of ATP. Given the huge dive> sity in the function of these proteins, we have chosen to limit the review to the recent structural progress on Structure of Ca2÷-ATPase by electron microscopy and P-type and F-type ion pumps. X-ray diffraction P-type ion pumps are named for the covalent phos- In the case of Ca2+-ATPase, vanadate has long been phoenzyme formed as part of the reaction cycles of, known to induce crystallization, with three-dimen- for example, Ca2+-ATPase, Na+/K+-ATPase, or H+/K +- sional reconstructions by electron microscopy having ATPase; F-type pumps are named from the original previously been reported at 25,~ resolution [1-3]. For FoF 1 nomenclature of the mitochondrial ATP synthase, the new reconstruction, Toyoshima et al. [4*'] used which also includes ATP synthases from chloroplasts frozen-hydrated specimens that better preserved not and bacteria. We have omitted the third major family, only the crystalline order, but also the cylindrical shape V-type pumps from vacuoles and acidic organelles, as of the long helical tubes obtained from the native mem- little structural information has yet been derived from brane. Using helical reconstruction methods, a com- this family. plete three-dimensional dataset was recovered, with- out having to tilt the specimen. The resulting reconstruction (Fig. 1) had a resolution of 14 A, which was sufficient to identify protein domains both inside and outside the bilayer. Some tentative assignments were P-type ion pumps made for transmembrane densities based on a popular folding model containing 10 transmembrane helices [5]. This family of ion pumps functions to establish and Furthermore, a cleft identified in the cytoplasmic head maintain ion gradients across membranesl All mem- was proposed as a potential ATP-binding site. In fu- bers have an ~t-subunit of -100kDa that hydrolyzes ture work, a structure at slightly higher resolution (bet- ATP and transports ions. Several members also have ter than 10A) should reveal some secondary structure. a ~-subunit that influences their targeting to particu- This could be obtained, either by more extensive pro- lar cell membranes and that may, or may not, con- cessing of a larger number of tube images, as was done tribute to ion transport. To date, no crystals suitable for the acetylcholine receptor [6], or by reconstruction for x-ray diffraction have been reported , but two from a different crystal form. In fact, a different crystal new structures were recently determined by electron form, which consists of an ordered stack of bilayers, is microscopy: one for Ca2+-ATPase from sarcoplasmic currently being studied at higher resolution (-6-&) [7]. reticulum, and one for Na+/K+-ATPase from kidney. This second Ca2+-ATPase crystal form may also reveal Three-dimensional structures have previously been de- conformational differences between reaction interme-

Abbreviations DCCD--dicyclohexylcarbodiimide; GST glutathione-S-transferaseNOE--nuclear Overhauser effect.

diates, as the crystallization conditions trap the enzyme Structure of Na+/K+-ATPase by electron microscopy in a different reaction state. A relatively new crystal form of Na+/K+-ATPase was negatively stained and used for traditional reconstruction by electron microscopy from multiple tilts [9"]. Unlike previous crystals grown after treatment with phospholipase A2, or vanadate, Co(NH3)4ATP was used to trap a specific reaction intermediate, thus in- ducing the growth of tetrameric crystals [10]. These crystals have more reproducible unit cell parameters than those previously used; however, crystalline regions occur in a minority of membrane patches, are still relatively small (<0.2 ~trn) and are not usually coherent across a given patch. Perhaps because of these problems, only three small crystals were used for the reconstruction, and resolution was therefore limited to 25 A, which is similar to previously reported reconstructions of Na+/K+-ATPase [11-14]. Nevertheless, the four-fold crystal symmetry appears to have helped this newest reconstruction, which clearly shows the expected pear- shaped cytoplasmic head. This observation, together with a proposed location for the membrane based on a region of reduced contrast, provided plausible assignments for cytoplasmic, intramembranous and extracellular portions. These assignments should, however, be regarded with some caution given the unpredictable contrast of negative stain within a bilayer. In this regard, frozen-hydrated specimens would be well worth studying, especially if larger, coherent crystalline areas could be obtained. Another new crystal form of Na+/K+-ATPase was recently reported [15"] which may alleviate some of the problems associated with previous crystals. In particular, the authors claimed that a high percentage of membrane fragments contained single crystalline domains, and diffraction to N 20 A resolution was obtained both from negatively stained and frozen-hydrated specimens. Although their use of citrate buffer at pH 4.8 for crystallization is non-physiological, ATPase activity decreased only slightly during crystallization. One potential drawback of this crystal form is the pairing of membranes, which appears to be necessary for crys- Fig. 1. Balsa wood model of the Ca2+-ATPase at 14,~ resolution. tallization, perhaps because lattice stability requires the Dark grey regions represent intramembranous protein. The large additional set of intermolecular contacts. However, as cytoplasmic domain is at the top of the model, while the small long as stacking is well ordered and molecular pack- luminal domain is at the bottom. The volume of the model is ap- ing is reproducible from one crystal to the next, this proximately 75% of theexpected volume of Ca2+-ATPase and the scale bar represents 20 A. (Adapted from Toyoshima et al. [4"].) crystal form should be amenable to three-dimensional reconstruction from multiple tilts.

X-ray diffraction of membrane pellets has been used by Blasie and colleagues to determine a cylindrically av- Modeling the protein fold eraged profile of Ca2+-ATPase across the bilayer. Their A great deal of energy has been spent trying to predict most recent result [8"] comes after release of caged the folding of the pump polypeptides based on their Ca 2+ and shows small changes that are attributed to amino acid sequences. A primary goal has been to Ca 2+ binding at three locations: within the bilayer at determine the number of transmembrane crossings putative Ca 2+ transport sites, on the cytoplasmic head, (commonly thought of as helices) and their location and on the luminal surface of the bilayer. These Ca 2+ in the structure. Currently, arguments are commonly sites correlate to sites of lanthanide binding (analogues heard for models with either eight, or ten transmem- of Ca 2+) but the conformation of the protein itself also brane helices (both amino and carboxyl termini are changes upon Ca 2+ binding, which may account for at known to be in the cytoplasm). This issue has been least some of the differences observed. addressed by a variety of methods (chemical labeling, Structures of P-type and F-type ion pumps Stokes and Nakamoto 199 antibody binding, proteolytic cleavage) on Ca2+-AT- cies between the two structures, which may be traced Pase, Na+/K+-ATPase, H+/K+-ATPase, and H+-ATPase. to the contribution of the minor subunits. Although virtually everyone agrees on the existence Amzel and Pedersen [25] observed that crystals of the and location of the first four transmembrane crossings, rat liver mitochondrial Fa belonged to space group R32, the remaining four to six crossings remain highly con- and that the complex sat on a crystallographic three- troversial because there is little conserved sequence in . fold axis. Hence, each asymmetric unit contained only this region and each protein nmst be considered indi- one third of the F 1 complex, and the resulting struc- vidually. Clearly, any attempt to model protein folding ture necessarily revealed a highly symmetric particle requires that the number of crossings be established. in which each ~-[3 pair was identical to the other Despite this critical uncertainty, the packing of helices two. This crystal packing implies that the g-, 8- and has been modeled using a new algorithm originally de- e-subunits, which appear only once in each Ft complex veloped for the seven-helix receptors (e.g. rhodopsin and do not have internal three-fold symmetry, did not [16]). This method of analysis involves identification of participate in packing contacts and were thus free to the conserved and variable faces of helices, its ultimate occupy random positions about the three-fold axis. goal being to arrange them so that variable faces con- As a result, their contributions to the structure were tact the lipid and conserved faces interact with other smeared around this three-fold axis. Despite this de- transmembrane helices. In fact, when this analysis is ficiency, the structure was refined and the authors performed on a group containing both Ca2+-ATPase claim to have traced the chain for 900 of the nearly and Na+/K+-ATPase [17"], many of the putative trans- 1000 amino acids of the co- and [3-subunits, yielding an membrane helices (assumed to be 10) show such con- R factor of 37%. served and variable faces. This analysis could provide In contrast, the structure of bovine heart mitochondrial important structural constraints for packing models of. F1 [23"] was very asymmetric with regard to the three- transmembrane helices, such as that recently presented fold axis, even though the bovine heart mitochondrial by Inesi and colleagues [18"]. F 1 is essentially the same as the rat complex (ct and 13 sequence identities are 98% and 95%, respectively). In these orthorhombic crystals, the asymmetric unit contained the entire F 1 complex and three-fold symmetry was not mandated by crystal packing. Distinctly asym- F-type ion pumps (ATP synthase) metric features in the resulting structure (Fig. 2a) were the 40A 'stem' that extended from the bottom of the The function of this family of ion pumps is to synthe- complex, a 'pit' next to this stem, and a 15A de- size ATP from existing proton gradients. In contrast to pression with a trough at the top of the complex. In the single catalytic subunit of the P-type ATPases, the addition, a long internal rod extended 90A along the FoF1 ATP synthase contains at least eight different kinds pseudo three-fold axis from the stern to the top of the of subunits -- five in the soluble F1, and three or more complex (Fig. 2b). This rod, which is likely to be an in the membrane-bound F o. The organization of these ~t-helix, may be part of the 8- or ~,-subunits, as both subunits has been visualized by electron microscopy are predicted to have high 0t-helical contents. Other of non-crystalline specimens (reviewed in [19",20]). In features appear to be related by three- or six-fold sym- addition, the F 1 sector, which is easily dissociated from metry and thus are likely to belong to the homologous the membrane, has been crystallized and analyzed by ct- and [3-subunits. No attempts were made to delineate X-ray diffraction. The architecture of the Fo sector is individual subunits. still mysterious; however, the fine structure of the proteolipid has been examined by NMR. The discrepancy in the molecular symmetries from the two crystal forms must reflect the structural effects of the y-, 8-, and e-subunits on the crystal contacts, which in turn must be due to differences in the crystallization Structure of the F 1 complex by X-ray crystallography conditions. Whereas the rat liver enzyme was crystal- Although the F 1 complex can be dissociated from the lized from a mother liquor containing ATP and potas- membranous Fo and treated as a soluble protein, the sium phosphate, the bovine heart crystals were made large size of F 1 is a considerable crystallographic chal- in the presence of AMP-PNP, ADP and Mg 2+. ATP and lenge. The F1 complex consists of three copies each of phosphate seem to prevent the minor subunits from the major subunits ~ and [3, and single copies of the perturbing the three-fold symmetry of the ~t-[3 pairs, minor subunits, y, 8 and e. The total molecular mass whereas AMP-PNP, ADP, and Mg 2+ force asymmetry, is 371 kDa in the case of the bovine heart mitochon- which can only be accommodated if the asymmetric drial F 1 [21]. After years of crystallization studies, an unit contains an entire F 1 molecule. This asymmetry orthorhombic crystal form (P21212 t) of bovine heart could result either from an indirect effect on the ring F 1 was presented last year by Walker's group [22"], and of ~- and [3-subunits, or from direct participation of y-, the corresponding structure was published at 6.5 A res- 8-, or e-subunits in crystal contacts. olution [23"]. Previously, Amzel's group [24] presented The influence of the different nucleotides on the con- a structure from trigonal crystals of rat liver F 1 at 3.6A formation of the complex suggests that the two crystal resolution. Even considering differences in resolution forms may have a functional significance. The differ- and stages of refinement, there were major discrepan- ing patterns of crystallization are reminiscent of results 200 Macromolecular assemblages

( (b)

Fig. 2. Topology and electron density map of the F1 complex at 6.5 A. (a) Topological view of the F1 complex. The stem is believed to be a part of the stalk connecting F1 to.F o and distinguishes the bottom of the complex that is likely to be an s-helix. (b) Cross section of the electron density map. Note the 90 A rod extending from the stem to the top of the complex. Not readily seen in these figures is the pseudo three- or six-fold symmetry of several features. Note that (a) and (b) are not at the same scale. (Reproduced with permission from Abrahams et al. [23"].)

from electron microscopy (summarized in [13"]), which cation, the desired portion was freed by cleavage at show that the minor subunits change conformation in an artificial thrombin site. Previously, Dunn [29] re- response to the addition of nucleotides. These results ported that isolated ~,-subunit remained soluble only are consistent with mechanistic studies, which estab- when in complex with the e-subunit. In a manner lished a kinetic asymmetry amongst the three potential consistent with this observation, a soluble GST-~,-sub- catalytic sites on the [~-subunits [26]. Because the three unit fusion protein was obtained only when expressed c~- and the three [3-subunits, respectively, have identical concurrently with e. On the one hand, a potential prob- sequences, asymmetry must be imposed by the minor lem with this approach may be in obtaining properly subunits. Furthermore, it is likely that the asymmetry folded subunits in the absence of other F 1 subunits. On changes during the catalytic cycle as an integral part the other hand, a potential benefit may be the oppor- of the catalytic and transport mechanism. In a simi- tunity to reach true atomic resolution with individual lar manner to the P-type ATPases, structures derived subunits, which could then be fitted into a lower res- from the two different crystal forms may provide an olution structure of the whole F 1 complex. opportunity to understand the nature of the conformational changes. As yet, the hydrolytic states of the nucleotides bound within the crystals are not known, Structure of the Fo sector so the published structures cannot be related to the cat- As yet, very little experimental evidence is available re- alytic cycle. garding the organization of the F o complex. Recently, Vik and Dao [30"] used the same hypothesis as Bald- win [16] and Green [17" ] (i.e. that variable faces con- Crystallization of single F 1 subunits tact lipid and conserved faces contact other helices) to An alternative method for determining the structure of generate a model by sequence analysis. Vik and Dao the F 1 complex is to crystallize single subunits or sub- [30"].examined closely related sequences of Fo subunits complexes. Indeed, Cox and co-workers [27"] have for hydrophobicity and amino acid variation. Using a crystallized the e-subunit alone, and a y--e complex model of six transmembrane crossings for subunit a, [28"] from Escherichia coB. Instead of extracting sub- one for b and two for c, Fourier analysis of sequence units from FoF1, large amounts of polypeptides were variation was used to identify variable and conserved purified from fusion proteins. Fusions with glutathione- helical faces. The analysis indicated that subunit a has S-transferase (GST) were expressed, and after purifi- two helices with variable faces, suggesting contact be- Structures of P-type and F-type ion pumps Stokes and Nakamoto 201 tween these two helices and the lipid; the remaining resolution, but the large size of the particle will make four helices were predicted entirely to contact other this a slow process. It is not yet clear how the structure helices. From the Fourier analysis, it was also predicted of F o will be solved or how we will come to understand that subunits b and c each had one variable face. On the interactions between F o and F 1. the basis of these predictions and previous results from What is apparent is that our ultimate picture of these mutational studies, an arrangement of subunit a helices pumps will come from combining results from a was proposed. Most of the conserved faces of subunit range of physical techniques. Crystallization of individ- a helices were positioned to interact with each other. In ual subunits from FoF1 [27°,28 "] or of specially con- addition, in this model, a single helix of subunit a con- structed and expressed domains from P-type pumps tacts a cluster of 9-10 c subunits, and two other helices [33] may provide individual structural elements for of subunit a contact the two conserved helices from the fitting into lower resolution structures. Modeling of b subunits. structure based on a variety of constraints may also The only direct structural information on the F o com- be necessary to fill gaps in the structural informa- plex has been obtained by Girven and Fillingame tion. Once structures have been established, we can [31",32"'] using multidimensional NMR to examine the start to address how conformational changes carry out structure of the E. coli proteolipid subunit c. The puri- the process of ion transport. fied polypeptide was studied in chloroform-methanol- water where it retained at least some characteristics of the native protein [i.e. specific dicyclohexylcarbodiimide (DCCD) reactivity of the conserved Asp61 and Acknowledgments decreased DCCD reactivity in the Ile28Thr mutant] and no apparent interactions between subunit molecules. DL Stokes was supported in part by NIH grams AR40997 and Using standard two-dimensional NMR, spin systems of HL48807. 78 of the 79 amino acid side chains were assigned to residue type, and 44 of these were assigned to specific residues in the sequence [31"]. Nuclear Overhauser effects (NOEs) were observed between the ends of the References and recommended reading predicted transmembrane helices suggesting that the protein in solvent was folded as a hairpin, as it was Papers of particular interest, published within the annual period of review, have been highlighted as: believed to fold in the membrane. Further proof of the • of special interest hairpin conformation was obtained by specific modifi- • - of outstanding interest cation of Asp61 with a mixture of a nitroxide analog 1. TAYLORKA, DUX L, MARTONOSI A: Three-Dimensional Re- of DCCD, N-(2,2,6,6-tetramethylpiperidine-l-oxy)-N'- construction of Negatively Stained Crystals of the Ca++-AT- cyclohexylcarbodiimide [32",]. The paramagnetic ni- Pase from Muscle Sarcoplasmic Reticulum. J Mol Btol 1986, troxide broadened many assigned 1H resonances in 187:417-427. both transmembrane helices and provided a means 2. TAYLORKA, HO MH, MARTONOSI A: Image Analysis of the to calculate distance ranges or limits. Using these con- CaX+-ATPase from Sarcoplasmic Reticulum. Ann N Y Acad straints along with inter- and intra-helical NOE deter- Sci 1986, 483:31-43. minations, a hairpin structure with two slightly curved 3. CASTELLANIL, HARDWICKE PM, VIBERT P: Dimer Ribbons in transmembrane helices was proposed. The DCCD-re- the Three-Dimensional Structure of Sarcoplasmic Reticulum. active Asp61 on helix-2 was placed within the bilayer J Mol Biol 1985, 185:579-594. very near the side chains of residues Ala24 and I1e28 4. TOYOSHIMAC, SASABE H, STOKES DL: Three-Dimensional on helix-1. The model was consistent with the effects • , Cryo-Electron Microscopy of the Calcium Ion Pump in the Sarcoplasmic Reticulum Membrane. Nature 1993, of chemical nmtagenic modifications made at these po- 362:469-471. sitions in the complex. The authors studied an old crystal form of CaX÷-ATPase, which is induced by vanadate in its native membrane. They obtained long, cylindrical tubes that were imaged in the frozen-hydrated state and determined the structure by helical reconstruction at 14 .i, resolution. Although secondary structure was not resolved, the domain structure Conclusion within, and on either side of the bilayer was easily visible. Based on a popular folding m(nlel, the authors speculate about the locations of proposed transmembrane helices and of the ATP-binding pocket. Determination of ion pump structures has been an im- mense challenge; however, we can look forward to 5. MACLENNANDH, BRANDL CJ, KORCZAK B, GREEN NM: Amino- Acid Sequence of a Ca 2+ + Mg2+-dependent ATPase from having reasonable structures for both P-type and F- Rabbit Muscle Sarcoplasmic Reticulum, Deduced from its type pumps in the foreseeable future. For the P-type Complementary DNA Sequence. Nature 1985, 316:696-700. pumps, electron microscopy provides good prospects 6. UNW1NN: Nicotinic Acetylcholine Receptor at 92{ Resolu- for the determination of medium resolution structures tion. J Mol Biol 1993, 229:1101-1124. (6 ~_). Furthermore, different enzymatic states of both 7. STOKF~SDL, GREEN NM: Structure of CoATPase: Electron Mi- Ca2+-ATPase and Na+/K+-ATPase have been crystal- croscopy of Frozen-Hydrated Crystals at 6A Resolution in lized which will give insights into conformational Projection. J Mol Biol 1990, 213:529-538. changes required for ion transport. For the F 1 complex, 8. DELONGLJ, BLAISE JK: Effect of Ca 2+ Binding on the Pro- X-ray crystallography may eventually achieve atomic • file Structure of the Sarcoplasmic Reticulum Membrane 202 Macromolecular assemblages

using Time-Resolved X-Ray Diffraction. Biophys J 1993, pumps, and the scores averaged across the diverse group of Ca 2÷- 64:1750-1759 ATPases and Na+/K+-ATPases. The analysis was based on a model Partially oriented membrane pellets of sarcoplasmic reticulum were with 10 tmnsmembrane helices and the faces thus determined will be examined by low-angle X-ray diffraction before, and after photoly- used in future attempts to pack transmembrane helices into a three- sis of caged Ca 2+. Before photolysis, -0.6 Ca 2+ ions per molecule dimensional structure. Many such packing schemes can be generated were bound, whereas -1.6 Ca 2+ ions per molecule were bound after and ranked according to their compliance to the original hypothe- photolysis. The resulting changes to the X-ray pattern were small, sis; other constraints based on experimental evidence can then be but after calculating the cylindrically symmetric profile of Ca2+-AT - employed to choose between the most probable schemes. Pase across the membrane, these changes were mostly attributed to the binding of Ca 2+ at the three distinct sites on the Ca2+-ATPase 18. INESI G, LEWIS D, NIKIC D, HUSSAIN A, KIRTI.EY ME: Long- molecule. Although these sites correlate with those found for lan- • Range Intramolecular Linked Functions in the Calcium thanides, which generate a substantially larger signal, changes in Transport ATPase. Adv Enzym 1992, 65:185-215. Ca2+-ATPase conformation upon Ca 2+ binding probably account for A review of the enzymatic, spectroscopic, structural and mutagenic at least some of the observed differences. studies of Ca2+-ATPase with the aim of understanding the structural basis for energy coupling between the ATP site and the distant ion- 9. SKRIVERE, KAVEUS U, HEBERT H, MAUNSBACH AB: Three-Di- binding sites. • • mensional Structure of Na,K-ATPase Determined from Mem- 19. CAPALDIRA, AGGELER R, GOGOL EP, WILKENS S: Structure of brane Crystals Induced by Cobalt-Tetrammine-ATP. J Str~tct Btol 1992, 108:176-185. • the Escherlchla coil ATP Synthase and Role of the ~ and t~ Subunits in Coupling Catalytic Site and Proton Channeling A relatively new crystal form of Na+/K+-ATPase in the E1 conforma- Functions. 1992, 24:435--439. tion was used for reconstruction from multiple tilts at 25 A resolution. J Btoenerg Biomemb A review which summarizes many electron microscopic and chemi- Only three small negatively stained crystals were used for the re- cal labeling studies concerning the organization of the F 1 complex, as construction, but the four-fold symmetry helped fill in Fourier space, well as the effect of various nucleotides on structure. The paper em- yielding a structure with an expected resemblance to Ca2+-ATPase. The authors make plausible assignments for cytoplasmic, membra- phasizes the conformational changes of the F 1 complex in response to bound ATP, ADP and Pi. The authors suggest that the confor- nous, and extracellular parts of the molecule. Because unit cell pa- mational 'switching' is a key to the coupling mechanism between rameters are stable, a study of this crystal form in the frozen-hydrated catalysis and transport. state should be possible and should help confirm these assignments. 20. PEDERSEN PL, AMZEI. LM: ATP Synthase: Structure, Reaction 10. SKRIVER E, MAUNSBACH AB, HEBERT H, SCHEINER-BOBIS Center, Mechanism, and Regulation of One of Nature's Most G, SCHONER W: Two-Dimensional Crystalline Arrays of Unique Machines. J Biol Chem 1993, 268:9937-9940. Na,K-ATPase with New Subunit Interactions Induced by Cobalt-Tetrammine-ATP. J Ultrastruct Mol Str~tct Res 1989, 21. WAI.KERJE, FEARNLEY IM, GAY NJ, GIBSON BW, NOR'IYIROP 102:189-195. FD, POWELL SJ, RUNSWICK MJ, SARASTE M, TYBULEWICZ VLJ: Primary Structure and Subunit Stoichiometry of FI-ATPase 11. OVCHINNIKOV YA, DEM1N VV, BARNAKOV AN, KUZ1N AP, from Bovine Mitochondria. J Mol Btol 1985, 184:677-701. LUNEV AM, MODYANOV NN, DZANDZHUGAYAN KN: Three- Dimensional Structure of (Na + + K+)-ATPase Revealed by 22. LUTI'ERR, ABRAHAMSJP, VAN RAAIJ iJ, TODD RJ, LUNDQVIST Electron Microscopy of Two-Dimensional Crystals. FEBS Lett • T, BUCHANAN SK, LESLIE A, WALKERJE: Crystallization of F 1- 1985, 190:73-76. ATPase from Bovine Heart Mitochondria. J Mol Bgol 1993, 229:787-790. 12 MOHRAZM, SIMPSON MV, SMITH PR: The Tree-Dimensional This report de~ribes crystallization of F 1 from conditions which Structure of Renal Na,K-ATPase from Electron Microscopy. included AMP-PNP, ADP and Mg 2+. The crystals diffracted to 2.9A J Cell Biol 1987, 105:1-8. and belonged to the space group P21212 t. The unit cell dimensions 13. HEBERTH, SKRIVER E, MAUNSBACH AB: Three-Dimensional were a=285A, b=108A and c=140A and the asymmetric unit was Structure of Renal Na,K-ATPase Determined by Electron Mi- large enough to accommodate the entire F 1 complex. croscopy of Membrane Crystals. Febs Lett 1985, 187:182-186. 23. ABRAHAMSJP, LUTTER R, TODD RJ, VAN RAAIJ MJ, LESLIE AGW, 14. HEBERT H, SKRIVER E, SODERHOLM M, MAUNSBACH AB: • • WALKERJE: Inherent Asymmetry of the Structure of F1-AT- Three-Dimensional Structure of Renal Na,K-ATPase Deter- Pase from Bovine Heart Mitochondria at 6.5,~ Resolution. mined from Two-Dimensional Membrane Crystals of the pl EMBO J 1993, 12:1775-1780. Form. J Ultrastn~ct Mol Struct Res 1988, 100:86-93. This paper presents the first medium resolution (6.5 A) structure for which the entire F l complex is represented in the asymmetric unit. 15. TAHARAY, OHNISHI S, FUJIYOSHI Y, KIMURAY, HAYASHI Y: A Although no attempt is made to delineate the subunits, the structure • • pH Induced Two-Dimensional Crystal of Membrane-bound is remarkable for the significant asymmetry of the complex, which Na+,K+-ATPase of Dog Kidney. F#2BS Lett 1993, 320:17-22. at least in part must be due to the minor subunits, T, 8 and e (see A novel crystal form of Na+/K+-ATPase is described, which the au- Fig. 2). Some of the asymmetric features were a 40A stem at the thors claim is present in 80% of membrane fragments and which has bottom of the complex, which may be a part of the stalk observed larger coherent crystalline areas than those of previous crystal forms. in mitt•graphs of the whole FoF 1 complex, a pit next to the stem, Diffraction around 20A is shown for both negatively stained and and a 15A depression at the top of the complex. Internally, several frozen-hydrated crystals. The crystals consist of membrane pairs, features in three- and six-fold symmetry were observed along with which presents potential problems if the relationship between the a 90 A rod extending along the pseudo three-fold axis. pairs is not strictly identical in all membranes. Assuming this pairing to be consistent, this new crystal form holds promise for future 24. BlANCHEr M, YSER X, HULLIHEN J, PEDERSEN PL, AMZEL LM: three-dimensional reconstruction. Mitochondrial ATP Synthase: Quaternary Structure of the F 1 Moiety at 3.6/~ Determined by X-Ray Diffraction Analysis. J 16. BALDWINJM: The Probable Arrangement of the Helices in Biol Chem 1991, 266:21197-21201. G Protein-Coupled Receptors. EMBO J 1993, 12:1693-1703. 25. AMZEL Li, PEDERSEN PL: Adenosine Triphosphatase from 17. GREEN NM: Conservation Patterns Define Interactions be- Rat Liver Mitochondria: Crystallization and X-Ray Diffrac- * tween Transmembrane Helices of P-Type Ion Pumps. In The tion Studies of the Fl-Component of the Enzyme. J Biol Sodium Pump, Edited by Bamberg E, Schoner W. Darmstadt: Chem 1978, 253:2067-2069. Steinkopff Verlag; 1993. 26. PENEFSKY HS, CROSS RL: Structure and Mechanism of FoF l- Sequence analysis of predicted transmembmne helices from Ca 2+- Type ATP Synthases and ATPases. Adv Enzymol 1991, and Na+/K+-ATPase based on the hypothesis that variable residues 64:173-213. within the membrane will generally face the lipid, whereas the conserved residues will interact with other transmembrane helices. Vari- 27. CODD R, COX GB, GUKS JM, SOLOMON RG, WEBB D: The ability scores were assigned for small groups of very closely related • Expression, Purification and Crystallization of the e Subunit Structures of P-type and F-type ion pumps Stokes and Nakamoto 203

of the F 1 portion of the ATPase of Escherichia coll. J Mol esis as Baldwin [10] and Green [11"] that variable faces contact lipid Btol 1992, 228:306-309. and conserved faces contact other helices. A GST-e fusion protein was overexpressed and the e-subunit moiety 31. GRIVENME, FILLINGAME 1Ll-I: Structure and Folding of Subunit isolated and crystallized. The crystals diffracted to at least 2.9 A with o. c of F1F o ATP Synthase: 1H NMR Resonance Assignments unit cell dimensions Of a=/?=94.9 A, and c=57.1 .~. The much smaller and NOE Analysis Biochemistry 1993, 32:12167-12177. unit cell will be much easier to solve, and at a higher resolution, than A review which presents a structural model of F o subunit c based on the entire F 1 complex. multidimensional NMR studies. Although a complete set of distance 28. Cox GB, CROMER BA, GUSS JM, HARVEY I, JEFFREY PD, constraints was not obtained, NOE's were measured for amino- and • SOLOMONRG, WEBB, DC: Formation in vivo, Purification carboxy-terminal residues and resonance broadening was observed and Crystallization of a Complex of the y and e Subunits for several residues near a nitroxide label. An analysis of the possible of the FoFI-ATPase of Escherichia coll. J Mol Biol 1993, mechanism of proton transpo~ is presented. 229:1159-1162. 32. GRIVENME, FILLINGAME RH: Hairpin Folding of Subunit c of In the .same manner as in [27*], the T-subunit was overexpressed as a oo FIFo ATP synthase: 1H Distance Measurements to Nitroxide- fusion with GST; however, the fusion protein was not soluble. Con- Derivatized Aspartyl-61. Biochemistry 1994, 33:665-674. sistent with previous observations, if the e-subunit was expressed A continuation of the work described in [31°*]. A nitroxide analog concurrently a soluble complex was formed. The "i~--e dimer was of DCCD, N-(2,2,6,6-tetramethylpiperidine-l-oxy)-N'-cyclohexylcar- stable and was crystallized. Interestingly, the T-subunit lost the first bodiimide, was used to specifically label Asp61 of the E. coli subunit eleven amino acids from the amino terminus during processing. The c. The paramagnetic center caused increased 1H resonances of both crystals diffracted to at least 3 ,~ and belonged to space group P21212. putative transmembrane helices. This result was additional proof for The unit cell dimensions were a=161.9A, /0=44.1 A and c---63.4 A and the predicted hairpin structure. More importantly, the broadening of contained one ~-e dimer, which is still a reasonable size for solving 1H resonances provided a means to calculate distance constraints be- the structure at high resolution. tween the nitroxy group and assigned tH resonances. Using these 29. DUNNSD: The Isolated T Subunit of Escherichia coli F 1 AT- constraints along with inter- and intra-helical NOE determinations, Pase Binds the e Subunit. J Btol Chem 1982, 257:7354-7359. a hairpin structure with two slightly curved transmembrane helices was proposed. 30. VIK SB, DAO NN: Prediction of Transmembrane Topology. • of Fo Proteins from Eschertchta coil F1F o ATP Synthase Us- 33. CAPIEAUX E, RAPIN C, THINES D, DUPONT Y, GOFFEAU A: ing Variational and Hydrophobic Moment Analyses. Btochtm Overexpression in Eschertchia coil and Purification of an Biophys Acta 1992, 1140:199-207. ATP-Binding Peptide from the Yeast Plasma Membrane H +- The authors analyzed several sequences of subunit a and settled on ATPase. J Btol Chem 1993, 268:21895-21900. a topology containing six transmembrane crossings: this number has been a contentious issue. Using this topology of subunit a, and the more easily predicted topologies of subunits b and and C Fourier analysis of sequence variation was used to identify variable and con- DL Stokes and RK Nakamoto, Department of Molecular Physiology served faces of the F o transmembrane helices. This information was and Biological Physics, University of Virginia Health Sciences Center, used to construct a model for E. colt F o based on the same hypoth- Charlottesville, Virginia 22908, USA.