Structures of P-type and F-type ion pumps
David L Stokes and Robert K Nakamoto
U n i v e r s i t y of Virginia, C h a r l o t t e s v i l l e , 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
Pumping ions across biological membranes represents °
a crucial activity of cells. The resulting ion gradients are
used for a wide variety of purposes, including such essential functions as intracellular signaling, transport of
nutrients and synthesis of ATP. Given the huge dive>
sity in the function of these proteins, w e have chosen
to limit the review to the recent structural progress on
P-type and F-type ion pumps.
P-type ion pumps are named for the covalent phosphoenzyme formed as part of the reaction cycles of,
for example, Ca2+-ATPase, Na+/K+-ATPase, or H+/K +ATPase; F-type pumps are n a m e d from the original
FoF 1 nomenclature of the mitochondrial ATP synthase,
which also includes ATP synthases from chloroplasts
and bacteria. We have omitted the third major family,
V-type pumps from vacuoles and acidic organelles, as
little structural information has yet been derived from
this family.
P-type ion pumps
This family of ion pumps functions to establish and
maintain ion gradients across membranesl All members have an ~t-subunit of - 1 0 0 k D a that hydrolyzes
ATP and transports ions. Several members also have
a ~-subunit that influences their targeting to particular cell membranes and that may, or may not, contribute to ion transport. To date, no crystals suitable
for x-ray diffraction have b e e n reported , but two
new structures were recently determined by electron
microscopy: one for Ca2+-ATPase from sarcoplasmic
reticulum, and one for Na+/K+-ATPase from kidney.
Three-dimensional structures have previously been de-
termined for both of these molecules, but better specimen preparation of CaZ+-ATPase improved the resolution, and a new, more stable crystal form helped the
determination of the Na+/K+-ATPase structure. In addition, X-ray diffraction of non-crystalline CaZ+-ATPase
has investigated the structural effects of Ca 2+ binding.
Structure of Ca2÷-ATPase by electron microscopy and
X-ray diffraction
In the case of Ca2+-ATPase, vanadate has long been
known to induce crystallization, with three-dimensional reconstructions by electron microscopy having
previously been reported at 25,~ resolution [1-3]. For
the new reconstruction, Toyoshima et al. [4*'] used
frozen-hydrated specimens that b e t t e r preserved not
only the crystalline order, but also the cylindrical shape
of the long helical tubes obtained from the native membrane. Using helical reconstruction methods, a complete three-dimensional dataset was recovered, without 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
made for transmembrane densities based on a popular
folding model containing 10 transmembrane helices [5].
Furthermore, a cleft identified in the cytoplasmic head
was proposed as a potential ATP-binding site. In future work, a structure at slightly higher resolution (better than 10A) should reveal some secondary structure.
This could be obtained, either by more extensive processing of a larger number of tube images, as was done
for the acetylcholine receptor [6], or by reconstruction
from a different crystal form. In fact, a different crystal
form, which consists of an ordered stack of bilayers, is
currently being studied at higher resolution (-6-&) [7].
This second Ca2+-ATPase crystal form may also reveal
conformational differences between reaction interme-
Abbreviations
DCCD--dicyclohexylcarbodiimide; GST glutathione-S-transferaseNOE--nuclear Overhauser effect.
© Current Biology Ltd ISSN 0959-440X
197
198
Macromolecular assemblages
diates, as the crystallization conditions trap the enzyme
in a different reaction state.
Fig. 1. Balsa wood model of the Ca2+-ATPase at 14,~ resolution.
Dark grey regions represent intramembranous protein. The large
cytoplasmic domain is at the top of the model, while the small
luminal domain is at the bottom. The volume of the model is approximately 75% of theexpected volume of Ca2+-ATPase and the
scale bar represents 20 A. (Adapted from Toyoshima et al. [4"].)
X-ray diffraction of membrane pellets has been used by
Blasie and colleagues to determine a cylindrically averaged profile of Ca2+-ATPase across the bilayer. Their
most recent result [8"] comes after release of caged
Ca 2+ and shows small changes that are attributed to
Ca 2+ binding at three locations: within the bilayer at
putative Ca 2+ transport sites, on the cytoplasmic head,
and o n the luminal surface of the bilayer. These Ca 2+
sites correlate to sites of lanthanide binding (analogues
of Ca 2+) but the conformation of the protein itself also
changes u p o n Ca 2+ binding, which may account for at
least some of the differences observed.
Structure of Na+/K+-ATPase by electron microscopy
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 inducing 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 pearshaped 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 crystallization, perhaps because lattice stability requires the
additional set of intermolecular contacts. However, as
long as stacking is well ordered and molecular packing is reproducible from one crystal to the next, this
crystal form should be amenable to three-dimensional
reconstruction from multiple tilts.
Modeling the protein fold
A great deal of energy has b e e n spent trying to predict
the folding of the pump polypeptides based on their
amino acid sequences. A primary goal has been to
determine the number of transmembrane crossings
(commonly thought of as helices) and their location
in the structure. Currently, arguments are commonly
heard for models with either eight, or ten transmembrane helices (both amino and carboxyl termini are
known to be in the cytoplasm). This issue has been
addressed by a variety of methods (chemical labeling,
Structures of P-type and F-type ion pumps Stokesand Nakamoto 199
antibody binding, proteolytic cleavage) on Ca2+-ATPase, Na+/K+-ATPase, H+/K+-ATPase, and H+-ATPase.
Although virtually everyone agrees on the existence
and location of the first four transmembrane crossings,
the remaining four to six crossings remain highly controversial because there is little conserved sequence in .
this region and each protein nmst be considered individually. Clearly, any attempt to model protein folding
requires that the number of crossings be established.
Despite this critical uncertainty, the packing of helices
has been modeled using a new algorithm originally developed for the seven-helix receptors (e.g. rhodopsin
[16]). This method of analysis involves identification of
the conserved and variable faces of helices, its ultimate
goal being to arrange them so that variable faces contact the lipid and conserved faces interact with other
transmembrane helices. In fact, w h e n this analysis is
performed on a group containing both Ca2+-ATPase
and Na+/K+-ATPase [17"], many of the putative transmembrane helices (assumed to be 10) show such conserved and variable faces. This analysis could provide
important structural constraints for packing models o f .
transmembrane helices, such as that recently presented
by Inesi and colleagues [18"].
F-type ion pumps (ATP synthase)
The function of this family of ion pumps is to synthesize ATP from existing proton gradients. In contrast to
the single catalytic subunit of the P-type ATPases, the
FoF1 ATP synthase contains at least eight different kinds
of subunits - - five in the soluble F1, and three or more
in the membrane-bound Fo. The organization of these
subunits has been visualized by electron microscopy
of non-crystalline specimens (reviewed in [19",20]). In
addition, the F 1 sector, which is easily dissociated from
the membrane, has been crystallized and analyzed by
X-ray diffraction. The architecture of the Fo sector is
still mysterious; however, the fine structure of the proteolipid has been examined by NMR.
Structure of the F 1 complex by X-ray crystallography
Although the F 1 complex can be dissociated from the
membranous Fo and treated as a soluble protein, the
large size of F 1 is a considerable crystallographic challenge. The F1 complex consists of three copies each of
the major subunits ~ and [3, and single copies of the
minor subunits, y, 8 and e. The total molecular mass
is 371 kDa in the case of the bovine heart mitochondrial F 1 [21]. After years of crystallization studies, an
orthorhombic crystal form (P21212 t) of bovine heart
F 1 was presented last year by Walker's group [22"], and
the corresponding structure was published at 6.5 A resolution [23"]. Previously, Amzel's group [24] presented
a structure from trigonal crystals of rat liver F 1 at 3.6A
resolution. Even considering differences in resolution
and stages of refinement, there were major discrepan-
cies between the two structures, which may be traced
to the contribution of the minor subunits.
Amzel and Pedersen [25] observed that crystals of the
rat liver mitochondrial Fa belonged to space group R32,
and that the complex sat on a crystallographic threefold axis. Hence, each asymmetric unit contained only
one third of the F 1 complex, and the resulting structure necessarily revealed a highly symmetric particle
in which each ~-[3 pair was identical to the other
two. This crystal packing implies that the g-, 8- and
e-subunits, which appear only once in each Ft complex
and do not have internal three-fold symmetry, did not
participate in packing contacts and were thus free to
occupy random positions about the three-fold axis.
As a result, their contributions to the structure were
smeared around this three-fold axis. Despite this deficiency, the structure was refined and the authors
claim to have traced the chain for 900 of the nearly
1000 amino acids of the co- and [3-subunits, yielding an
R factor of 37%.
In contrast, the structure of bovine heart mitochondrial
F1 [23"] was very asymmetric with regard to the threefold axis, even though the bovine heart mitochondrial
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 asymmetric features in the resulting structure (Fig. 2a) were
the 40A 'stem' that extended from the bottom of the
complex, a 'pit' next to this stem, and a 15A depression with a trough at the top of the complex. In
addition, a long internal rod extended 90A along the
pseudo three-fold axis from the stern to the top of the
complex (Fig. 2b). This rod, which is likely to be an
~t-helix, may be part of the 8- or ~,-subunits, as both
are predicted to have high 0t-helical contents. Other
features appear to be related by three- or six-fold symmetry and thus are likely to belong to the homologous
ct- and [3-subunits. No attempts were made to delineate
individual subunits.
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
conditions. Whereas the rat liver enzyme was crystallized from a mother liquor containing ATP and potassium phosphate, the bovine heart crystals were made
in the presence of AMP-PNP, ADP and Mg 2+. ATP and
phosphate seem to prevent the minor subunits from
perturbing the three-fold symmetry of the ~t-[3 pairs,
whereas AMP-PNP, ADP, and Mg 2+ force asymmetry,
which can only be accommodated if the asymmetric
unit contains an entire F 1 molecule. This asymmetry
could result either from an indirect effect on the ring
of ~- and [3-subunits, or from direct participation of y-,
8-, or e-subunits in crystal contacts.
The influence of the different nucleotides on the conformation of the complex suggests that the two crystal
forms may have a functional significance. The differing patterns of crystallization are reminiscent of results
200
Macromolecular assemblages
(
(b)
et
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
show that the minor subunits change conformation in
response to the addition of nucleotides. These results
are consistent with mechanistic studies, which established a kinetic asymmetry amongst the three potential
catalytic sites on the [~-subunits [26]. Because the three
c~- and the three [3-subunits, respectively, have identical
sequences, asymmetry must be imposed by the minor
subunits. Furthermore, it is likely that the asymmetry
changes during the catalytic cycle as an integral part
of the catalytic and transport mechanism. In a similar manner to the P-type ATPases, structures derived
from the two different crystal forms may provide an
opportunity to understand the nature of the conformational changes. As yet, the hydrolytic states of the
nucleotides b o u n d within the crystals are not known,
so the published structures cannot be related to the catalytic cycle.
Crystallization of single F1 subunits
An alternative method for determining the structure of
t h e F 1 c o m p l e x is to crystallize single subunits or subcomplexes. Indeed, Cox and co-workers [27"] have
crystallized the e-subunit alone, and a y--e complex
[28"] from Escherichia coB. Instead of extracting subunits from FoF1, large amounts of polypeptides were
purified from fusion proteins. Fusions with glutathioneS-transferase (GST) were expressed, and after purifi-
cation, the desired portion was freed by cleavage at
an artificial thrombin site. Previously, Dunn [29] reported that isolated ~,-subunit remained soluble only
w h e n in complex with the e-subunit. In a manner
consistent with this observation, a soluble GST-~,-subunit fusion protein was obtained only w h e n expressed
concurrently with e. On the one hand, a potential problem with this approach may be in obtaining properly
folded subunits in the absence of other F 1 subunits. On
the other hand, a potential benefit may be the opportunity to reach true atomic resolution with individual
subunits, which could then be fitted into a lower resolution structure of the whole F 1 complex.
Structure of the Fo sector
As yet, very little experimental evidence is available regarding the organization of the F o complex. Recently,
Vik and Dao [30"] used the same hypothesis as Baldwin [16] and Green [17" ] (i.e. that variable faces contact lipid and conserved faces contact other helices) to
generate a model by sequence analysis. Vik and Dao
[30"].examined closely related sequences of Fo subunits
for hydrophobicity and amino acid variation. Using a
model of six transmembrane crossings for subunit a,
one for b and two for c, Fourier analysis of sequence
variation was used to identify variable and conserved
helical faces. The analysis indicated that subunit a has
two helices with variable faces, suggesting contact be-
Structures of P-type and F-type ion pumps Stokes and Nakamoto 201
t w e e n these two helices and the lipid; the remaining
four helices w e r e predicted entirely to contact other
helices. From the Fourier analysis, it was also predicted
that subunits b and c each had one variable face. O n
the basis of these predictions and previous results from
mutational studies, an arrangement of subunit a helices
was proposed. Most of the conserved faces of subunit
a helices w e r e positioned to interact with each other. In
addition, in this model, a single helix of subunit a contacts a cluster of 9-10 c subunits, and two other helices
of subunit a contact the two conserved helices from the
b subunits.
The only direct structural information on the Fo complex has b e e n obtained by Girven and Fillingame
[31",32"'] using multidimensional NMR to examine the
structure of the E. coli proteolipid subunit c. The purified polypeptide was studied in chloroform-methanolwater w h e r e it retained at least some characteristics of
the native protein [i.e. specific dicyclohexylcarbodiimide (DCCD) reactivity of the conserved Asp61 and
decreased DCCD reactivity in the Ile28Thr mutant] and
no apparent interactions between subunit molecules.
Using standard two-dimensional NMR, spin systems of
78 of the 79 amino acid side chains were assigned to
residue type, and 44 of these w e r e assigned to specific
residues in the sequence [31"]. Nuclear Overhauser effects (NOEs) were observed between the ends of the
predicted transmembrane helices suggesting that the
protein in solvent was folded as a hairpin, as it was
believed to fold in the membrane. Further proof of the
hairpin conformation was obtained by specific modification of Asp61 with a mixture of a nitroxide analog
of DCCD, N-(2,2,6,6-tetramethylpiperidine-l-oxy)-N'cyclohexylcarbodiimide [32",]. The paramagnetic nitroxide b r o a d e n e d many assigned 1H resonances in
both transmembrane helices and provided a m e a n s
to calculate distance ranges or limits. Using these constraints along with inter- and intra-helical NOE determinations, a hairpin structure with two slightly curved
transmembrane helices was proposed. The DCCD-reactive Asp61 on helix-2 was placed within the bilayer
very near the side chains of residues Ala24 and I1e28
on helix-1. The model was consistent with the effects
of chemical nmtagenic modifications made at these positions in the complex.
Conclusion
Determination of ion p u m p structures has b e e n an imm e n s e challenge; however, w e can look forward to
having reasonable structures for both P-type and Ftype p u m p s in the foreseeable future. For the P-type
pumps, electron microscopy provides g o o d prospects
for the determination of medium resolution structures
(6 ~_). Furthermore, different enzymatic states of both
Ca2+-ATPase and Na+/K+-ATPase have b e e n crystallized which will give insights into conformational
changes required for ion transport. For the F 1 complex,
X-ray crystallography may eventually achieve atomic
resolution, but the large size of the particle will make
this a slow process. It is not yet clear h o w the structure
of F o will be solved or h o w w e will c o m e to understand
the interactions between Fo and F 1.
What is apparent is that our ultimate picture of these
p u m p s will come from combining results from a
range of physical techniques. Crystallization of individual subunits from FoF1 [27°,28 "] or of specially constructed and expressed domains from P-type p u m p s
[33] m a y provide individual structural elements for
fitting into lower resolution structures. Modeling of
structure based on a variety of constraints may also
be necessary to fill gaps in the structural information. Once structures have b e e n established, we can
start to address h o w conformational changes carry out
the process of ion transport.
Acknowledgments
DL Stokes was supported in part by NIH grams AR40997 and
HL48807.
References and recommended reading
Papers of particular interest, published within the annual period of
review, have b e e n highlighted as:
•
of special interest
•of outstanding interest
1.
TAYLORKA, DUX L, MARTONOSI A: Three-Dimensional Reconstruction of Negatively Stained Crystals of the Ca++-ATPase from Muscle Sarcoplasmic Reticulum. J Mol Btol 1986,
187:417-427.
2.
TAYLORKA, HO MH, MARTONOSI A: Image Analysis of the
CaX+-ATPase from Sarcoplasmic Reticulum. A n n N Y A c a d
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3.
CASTELLANIL, HARDWICKEPM, VIBERT P: Dimer Ribbons in
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4.
•,
TOYOSHIMAC, SASABE H, STOKES DL: Three-Dimensional
Cryo-Electron Microscopy of the Calcium Ion P u m p
in the Sarcoplasmic Reticulum Membrane. Nature 1993,
362:469-471.
The authors studied an old crystal form of CaX÷-ATPase, which is
induced by vanadate in its native m e m b r a n e . 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
within, and o n 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 a n d of the ATP-binding pocket.
5.
MACLENNANDH, BRANDLCJ, KORCZAK B, GREEN NM: AminoAcid Sequence of a Ca 2+ + Mg2+-dependent ATPase from
Rabbit Muscle Sarcoplasmic Reticulum, Deduced from its
Complementary DNA Sequence. Nature 1985, 316:696-700.
6.
UNW1NN: Nicotinic Acetylcholine Receptor at 92{ Resolution. J Mol Biol 1993, 229:1101-1124.
7.
STOKF~SDL, GREEN NM: Structure of CoATPase: Electron Microscopy of Frozen-Hydrated Crystals at 6 A Resolution in
Projection. J Mol Biol 1990, 213:529-538.
8.
•
DELONGLJ, BLAISEJK: Effect of Ca 2+ Binding on the Profile Structure of the Sarcoplasmic Reticulum Membrane
202
Macromolecular assemblages
using Time-Resolved X-Ray Diffraction. Biophys J 1993,
64:1750-1759
Partially oriented m e m b r a n e pellets of sarcoplasmic reticulum were
e x a m i n e d by low-angle X-ray diffraction before, and after photolysis of caged Ca 2+. Before photolysis, -0.6 Ca 2+ ions per molecule
were bound, whereas -1.6 Ca 2+ ions per molecule were b o u n d after
photolysis. The resulting c h a n g e s to the X-ray pattern were small,
but after calculating the cylindrically symmetric profile of Ca2+-ATPase across the m e m b r a n e , these c h a n g e s were mostly attributed to
the binding of Ca 2+ at the three distinct sites o n the Ca2+-ATPase
molecule. Although t h e s e sites correlate with those f o u n d for lanthanides, which generate a substantially larger signal, changes in
Ca2+-ATPase conformation u p o n Ca 2+ binding probably account for
at least s o m e of the observed differences.
9.
••
SKRIVERE, KAVEUS U, HEBERT H, MAUNSBACHAB: Three-Dimensional Structure of Na,K-ATPase Determined from Membrane Crystals Induced by Cobalt-Tetrammine-ATP. J Str~tct
Btol 1992, 108:176-185.
A relatively n e w crystal form of Na+/K+-ATPase in the E1 conformation was used for reconstruction from multiple tilts at 25 A resolution.
Only three small negatively stained crystals were used for the reconstruction, but the four-fold symmetry helped fill in Fourier space,
yielding a structure with a n expected resemblance to Ca2+-ATPase.
The authors make plausible assignments for cytoplasmic, membranous, and extracellular parts of the molecule. Because unit cell parameters are stable, a study of this crystal form in the frozen-hydrated
state should be possible and should help confirm these assignments.
10.
11.
SKRIVER E, MAUNSBACH AB, HEBERT H, SCHEINER-BOBIS
G, SCHONER W: Two-Dimensional Crystalline Arrays of
Na,K-ATPase with N e w Subunit Interactions Induced by
Cobalt-Tetrammine-ATP. J Ultrastruct Mol Str~tct Res 1989,
102:189-195.
OVCHINNIKOV YA, DEM1N VV, BARNAKOV AN, KUZ1N AP,
LUNEV AM, MODYANOV NN, DZANDZHUGAYAN KN: ThreeDimensional Structure of (Na + + K+)-ATPase Revealed by
Electron Microscopy of Two-Dimensional Crystals. FEBS Lett
1985, 190:73-76.
12
MOHRAZM, SIMPSON MV, SMITH PR: The Tree-Dimensional
Structure of Renal Na,K-ATPase from Electron Microscopy.
J Cell Biol 1987, 105:1-8.
13.
HEBERT H, SKRIVER E, MAUNSBACH AB: Three-Dimensional
Structure of Renal Na,K-ATPase Determined by Electron Microscopy of Membrane Crystals. Febs Lett 1985, 187:182-186.
14.
HEBERT H, SKRIVER E, SODERHOLM M, MAUNSBACH AB:
Three-Dimensional Structure of Renal Na,K-ATPase Determ i n e d from Two-Dimensional Membrane Crystals of the p l
Form. J Ultrastn~ct Mol Struct Res 1988, 100:86-93.
15.
••
TAHARAY, OHNISHI S, FUJIYOSHI Y, KIMURAY, HAYASHI Y: A
pH Induced Two-Dimensional Crystal of Membrane-bound
Na+,K+-ATPase of Dog Kidney. F#2BS Lett 1993, 320:17-22.
A novel crystal form o f Na+/K+-ATPase is described, which the authors claim is present in 80% of m e m b r a n e fragments and which has
larger coherent crystalline areas than those of previous crystal forms.
Diffraction around 20A is s h o w n for both negatively stained and
frozen-hydrated crystals. The crystals consist of m e m b r a n e pairs,
which presents potential problems if the relationship between the
pairs is not strictly identical in all membranes. Assuming this pairing to be consistent, this n e w crystal form holds promise for future
three-dimensional reconstruction.
16.
BALDWINJM: The Probable Arrangement of the Helices in
G Protein-Coupled Receptors. EMBO J 1993, 12:1693-1703.
GREEN NM: Conservation Patterns Define Interactions between Transmembrane Helices of P-Type Ion Pumps. In The
Sodium Pump, Edited by Bamberg E, Schoner W. Darmstadt:
Steinkopff Verlag; 1993.
Sequence analysis of predicted t r a n s m e m b m n e helices from Ca 2+a n d Na+/K+-ATPase based on the hypothesis that variable residues
within the m e m b r a n e will generally face the lipid, w h e r e a s the conserved residues will interact with other transmembrane helices. Variability scores were assigned for small groups of very closely related
p u m p s , a n d the scores averaged across the diverse group of Ca 2÷ATPases a n d Na+/K+-ATPases. The analysis was based o n a m o d e l
with 10 t m n s m e m b r a n e helices a n d the faces thus determined will be
used in future attempts to pack transmembrane helices into a threedimensional structure. Many s u c h packing schemes can be generated
and ranked according to their compliance to the original hypothesis; other constraints based o n experimental evidence can t h e n be
e m p l o y e d to choose between the most probable schemes.
18.
•
INESI G, LEWIS D, NIKIC D, HUSSAIN A, KIRTI.EY ME: LongRange Intramolecular Linked Functions in the Calcium
Transport ATPase. Adv Enzym 1992, 65:185-215.
A review o f the enzymatic, spectroscopic, structural and mutagenic
studies o f Ca2+-ATPase with the aim of understanding the structural
basis for energy coupling b e t w e e n the ATP site and the distant ionbinding sites.
19.
•
CAPALDIRA, AGGELER R, GOGOL EP, WILKENS S: Structure of
t h e Escherlchla coil ATP Synthase and Role of the ~ and t~
Subunits in Coupling Catalytic Site and Proton Channeling
Functions. J Btoenerg Biomemb 1992, 24:435--439.
A review which summarizes m a n y electron microscopic and chemical labeling studies concerning the organization of the F 1 complex, as
well as the effect of various nucleotides on structure. The paper e m phasizes the conformational c h a n g e s of the F1 complex in r e s p o n s e
to b o u n d ATP, ADP and Pi. The authors suggest that the conformational 'switching' is a key to the coupling m e c h a n i s m b e t w e e n
catalysis a n d transport.
20.
PEDERSEN PL, AMZEI. LM: ATP Synthase: Structure, Reaction
Center, Mechanism, and Regulation of One of Nature's Most
Unique Machines. J Biol Chem 1993, 268:9937-9940.
21.
WAI.KERJE, FEARNLEY IM, GAY NJ, GIBSON BW, NOR'IYIROP
FD, POWELL SJ, RUNSWICK MJ, SARASTEM, TYBULEWICZVLJ:
Primary Structure and Subunit Stoichiometry of FI-ATPase
from Bovine Mitochondria. J Mol Btol 1985, 184:677-701.
22.
LUTI'ERR, ABRAHAMSJP, VAN RAAIJ i J , TODD RJ, LUNDQVIST
•
T, BUCHANANSK, LESLIE A, WALKERJE: Crystallization of F 1ATPase from Bovine Heart Mitochondria. J Mol Bgol 1993,
229:787-790.
This report d e ~ r i b e s crystallization of F 1 from conditions w h i c h
included AMP-PNP, ADP a n d Mg 2+. The crystals diffracted to 2.9A
and belonged to the space g r o u p P21212 t. The unit cell dimensions
were a=285A, b=108A and c=140A and the asymmetric unit was
large e n o u g h to accommodate the entire F 1 complex.
23.
••
ABRAHAMSJP, LUTTERR, TODD RJ, VAN RAAIJMJ, LESLIEAGW,
WALKERJE: Inherent Asymmetry of the Structure of F1-ATPase from Bovine Heart Mitochondria at 6.5,~ Resolution.
EMBO J 1993, 12:1775-1780.
This p a p e r presents the first m e d i u m resolution (6.5 A) structure for
which the entire F l complex is represented in the asymmetric unit.
Although n o attempt is m a d e to delineate the subunits, the structure
is remarkable for the significant asymmetry of the complex, w h i c h
at least in part m u s t be d u e to the minor subunits, T, 8 and e (see
Fig. 2). Some of the asymmetric features were a 40A stem at the
bottom o f the complex, which m a y be a part of the stalk observed
in m i t t • g r a p h s o f the whole FoF 1 complex, a pit next to the stem,
and a 15A depression at the top of the complex. Internally, several
features in three- and six-fold symmetry were observed along with
a 90 A rod extending along the p s e u d o three-fold axis.
24.
BlANCHEr M, YSER X, HULLIHENJ, PEDERSEN PL, AMZEL LM:
Mitochondrial ATP Synthase: Quaternary Structure of the F 1
Moiety at 3.6/~ Determined by X-Ray Diffraction Analysis. J
Biol Chem 1991, 266:21197-21201.
25.
AMZEL L i , PEDERSEN PL: Adenosine Triphosphatase from
Rat Liver Mitochondria: Crystallization and X-Ray Diffraction Studies of the Fl-Component of the Enzyme. J Biol
Chem 1978, 253:2067-2069.
26.
PENEFSKYHS, CROSS RL: Structure and Mechanism of FoF lType ATP Synthases and ATPases. Adv Enzymol 1991,
64:173-213.
27.
•
CODD R, COX GB, GUKS JM, SOLOMON RG, WEBB D: The
Expression, Purification and Crystallization of the e Subunit
17.
*
Structures of P-type and F-type ion pumps Stokes and Nakamoto
of the F 1 portion of the ATPase of Escherichia coll. J Mol
Btol 1992, 228:306-309.
A GST-e fusion protein was overexpressed and the e-subunit moiety
isolated and crystallized. The crystals diffracted to at least 2.9 A with
unit cell dimensions Of a=/?=94.9 A, a n d c=57.1 .~. The m u c h smaller
unit cell will be m u c h easier to solve, and at a higher resolution, than
the entire F 1 complex.
28.
Cox GB, CROMER BA, GUSS JM, HARVEY I, JEFFREY PD,
SOLOMONRG, WEBB, DC: Formation in vivo, Purification
and Crystallization of a C o m p l e x of the y and e Subunits
of the FoFI-ATPase of Escherichia coll. J Mol Biol 1993,
229:1159-1162.
In the .same m a n n e r as in [27*], the T-subunit was overexpressed as a
fusion with GST; however, the fusion protein was not soluble. Consistent with previous observations, if the e-subunit was expressed
concurrently a soluble complex w a s formed. The "i~--e dimer was
stable and was crystallized. Interestingly, the T-subunit lost the first
eleven amino acids from the amino terminus during processing. The
crystals diffracted to at least 3 ,~ a n d belonged to space group P21212.
The unit cell dimensions were a=161.9A, /0=44.1 A and c---63.4 A and
contained o n e ~-e dimer, which is still a reasonable size for solving
the structure at high resolution.
•
29.
30.
•
DUNNSD: The Isolated T Subunit of Escherichia coli F 1 ATPase Binds the e Subunit. J Btol Chem 1982, 257:7354-7359.
VIK SB, DAO NN: Prediction of Transmembrane Topology.
of Fo Proteins from Eschertchta coil F1Fo ATP Synthase Using Variational and Hydrophobic Moment Analyses. Btochtm
Biophys Acta 1992, 1140:199-207.
The authors analyzed several s e q u e n c e s of subunit a and settled on
a topology containing six t r a n s m e m b r a n e crossings: this n u m b e r has
been a contentious issue. Using this topology of subunit a, and the
more easily predicted topologies o f subunits b and and C Fourier
analysis of sequence variation was u s e d to identify variable and conserved faces of the Fo t r a n s m e m b r a n e helices. This information was
u s e d to construct a m o d e l for E. colt Fo based o n the same hypoth-
esis as Baldwin [10] and Green [11"] that variable faces contact lipid
and conserved faces contact other helices.
31.
o.
GRIVENME, FILLINGAME1Ll-I: Structure and Folding of Subunit
c of F1Fo ATP Synthase: 1H NMR Resonance Assignments
and NOE Analysis Biochemistry 1993, 32:12167-12177.
A review which presents a structural model of Fo subunit c based on
multidimensional NMR studies. Although a complete set of distance
constraints was not obtained, NOE's were measured for amino- and
carboxy-terminal residues and resonance broadening was observed
for several residues near a nitroxide label. An analysis of the possible
m e c h a n i s m of proton transpo~ is presented.
32.
GRIVENME, FILLINGAMERH: Hairpin Folding of Subunit c of
FIFo ATP synthase: 1H Distance Measurements to NitroxideDerivatized Aspartyl-61. Biochemistry 1994, 33:665-674.
A continuation of the work described in [31°*]. A nitroxide analog
of DCCD, N-(2,2,6,6-tetramethylpiperidine-l-oxy)-N'-cyclohexylcarbodiimide, was used to specifically label Asp61 of the E. coli subunit
c. The paramagnetic center caused increased 1H resonances of both
putative t r a n s m e m b r a n e helices. This result was additional proof for
the predicted hairpin structure. More importantly, the broadening of
1H resonances provided a m e a n s to calculate distance constraints between the nitroxy group and assigned tH resonances. Using these
constraints along with inter- and intra-helical NOE determinations,
a hairpin structure with two slightly curved transmembrane helices
was proposed.
oo
33.
CAPIEAUX E, RAPIN C, THINES D, DUPONT Y, GOFFEAU A:
Overexpression in Eschertchia coil and Purification of an
ATP-Binding Peptide from t h e Yeast Plasma Membrane H +ATPase. J Btol Chem 1993, 268:21895-21900.
DL Stokes a n d RK Nakamoto, Department of Molecular Physiology
and Biological Physics, University o f Virginia Health Sciences Center,
Charlottesville, Virginia 22908, USA.
203