(Received for publication, November 11, 1996, and in revised form, December 16, 1996)
From the Department of Pharmacology, Medical College of Ohio, Toledo, Ohio 43699-0008
An extensively trypsin-digested
Na+/K+-ATPase, which retains the ability
to bind Na+, K+, and ouabain, consists of four
fragments of the -subunit that contain all 10 transmembrane
domains, and the
-subunit, a fraction of which is cleaved at
Arg142-Gly143. In previous studies, we
solubilized this preparation with a detergent and mapped the relative
positions of several transmembrane helices of the subunits by chemical
cross-linking. To determine if these detected helix-helix proximities
were representative of those existing in the bilayer prior to
solubilization, we have now done similar studies on the membrane-bound
preparation of the same digested enzyme. After oxidative sulfhydryl
cross-linking catalyzed by Cu2+-phenanthroline, two
prominent products were identified by their mobilities and the analyses
of their N termini. One was a dimer of a 11-kDa
-fragment containing
the H1-H2 helices and a 22-kDa
-fragment
containing the H7-H10 helices. This dimer
seemed to be the same as that obtained in the solubilized preparation.
The other product was a trimer of the above two
-fragments and that fraction of
whose extracellular domain was cleaved at
Arg142-Gly143. This product was different from
a similar one of the solubilized preparation in that the latter
contained the predominant fraction of
without the extracellular
cleavage. The cross-linking reactions of the membrane preparation, but
not those of the solubilized one, were hindered specifically by
Na+, K+, and ouabain. These findings indicate
that (a) the H1-H2 transmembrane helices of
are adjacent to some of its
H7-H10 helices both in solubilized and
membrane-bound states, (b) the alignment of the residues of
the single transmembrane helix of
with the interacting H1-H2 and H7-H10
helices of
is altered by detergent solubilization and by structural
changes in the extracellular domain of
, and (c) the
three-dimensional packing of the interacting transmembrane helices of
and
are regulated by the specific ligands of the enzyme.
The coupled active transport of Na+ and K+
across the plasma membrane of most eucaryotic cells is carried out by
Na+/K+-ATPase. The enzyme consists of two
subunits, and
, both of which are essential to normal function
(1). That some transmembrane domains of the subunits must be involved
in cation transport is self-evident, and specific roles of several such
domains of the
-subunit in ion binding and transport have been
explored (1-6). There is one transmembrane domain in the
-subunit,
and the 10-span model of
is favored by most evidence (1).
To define the roles of the intramembrane domains in ion binding and
transport, it is clearly necessary to go beyond two-dimensional schemes
of folding and obtain information about the three-dimensional packing
of the transmembrane domains of Na+/K+-ATPase.
There is a paucity of such information, however, because it has been
difficult to apply high resolution structural methods to hydrophobic
helix-bundle proteins such as Na+/K+-ATPase.
Molecular modeling studies have been helpful (7-9). Limited
experimental data on the helix packing of
Na+/K+-ATPase have resulted from our recent
chemical cross-linking studies on enzyme preparations that were
subjected to controlled proteolysis and solubilized in a detergent
(10). These findings suggested contact between the intramembrane
H1,H2 helix pair and
H8-H10 intramembrane helices of , and
between these N-terminal and C-terminal helices of
and the single
intramembrane helix of
(10). The use of detergent-solubilized
membrane proteins in chemical cross-linking studies is both necessary
and problematic. On the one hand, solubilized preparations must be used
to rule out the possibility of cross-linking due to random collisions
of proteins and peptides that are highly concentrated within the
membrane phase (11-13). On the other hand, once the existence of a
stable noncovalent complex is established by the formation of a
cross-linked product in a solubilized preparation, it is necessary to
ensure that the detected protein-protein interaction is not entirely an
artifact of solubilization (11). The purpose of the present experiments
was to determine if the helix-helix proximities that we had detected in
solubilized preparations (10) were indeed representatives of the
interactions within the native membrane.
The purified membrane-bound
Na+/K+-ATPase of canine kidney medulla, with
specific activity of 1000-1600 µmol of ATP hydrolyzed/mg of
protein/h, was prepared and assayed as described previously (14). The
enzyme was exposed to trypsin (Sigma, Type III-S, bovine pancreas) in the presence of Rb+ and EDTA at pH 8.5 (10, 15) to remove large portions of the cytoplasmic domains of the
-subunit, and obtain a preparation containing an essentially intact
-subunit and several fragments of the
-subunit (10, 16, 17). This
preparation is often called the "19-kDa membranes" because its
cation occlusion capacity was originally thought to be due to a
C-terminal fragment of
with the apparent molecular mass of 19 kDa
(15). It is now evident, however, that several, if not all, peptides of
this preparation are involved in its functions (5, 16, 17). The
capacity of this digested preparation to occlude Rb+ was
verified before use by previously described procedures (15, 18).
For cross-linking, the digested enzyme was suspended (2 mg/ml) in a solution containing 10 mM Tris-HCl (pH 7.4), 0.25 mM CuSO4, and 1.25 mM o-phenanthroline, and incubated at 24 °C for 15 min. These conditions were chosen after preliminary experiments using the approach described previously (13). The reaction was terminated by the addition of EDTA to a final concentration of 30 mM, followed by SDS to a final concentration of 2%. Samples were subjected to electrophoresis on Tricine1/SDS-polyacrylamide gels (10). Unless indicated otherwise, sulfhydryl reagents were omitted from the buffers used for electrophoresis to preserve the cross-linked products. The stained peptide bands were quantified by a scanning densitometer (Bio-Rad). N-terminal analyses of the peptide bands were done as described previously (10).
Our previous conclusions regarding the intramembrane helix-helix interactions of Na+/K+-ATPase were based on the identification of cross-linked products obtained from enzyme preparations that were cleaved proteolytically, solubilized in digitonin, and oxidized in the presence of Cu2+ (10). The present experiments were initiated with the specific aims of determining (a) if the same or similar cross-linked products could be obtained in membrane-bound preparations that were not detergent-solubilized prior to cross-linking, and (b) if the functional specificities of the protein-protein interactions could be established by the sensitivities of the cross-linking reactions to ligands specific to Na+/K+-ATPase. Based on promising results of preliminary experiments, we focused attention on the cross-linking reactions of the membrane moiety of the trypsin-digested enzyme in the presence of Cu2+-phenanthroline complex.
The trypsin-digested preparation used here consists of four well
characterized fragments of the -subunit (10, 16, 19) that contain
the 10 transmembrane helices of the
-subunits, and an essentially
intact
-subunit, a fraction of which is cleaved at
Arg142-Gly143 (16, 19). The schematic structure
of this preparation is shown in Fig. 1, and the
resolution of its constituent peptides on Tricine/SDS gels is shown in
Fig. 2 (lane 1).
When the membrane-bound preparation was first exposed to
Cu2+-phenanthroline and then resolved on Tricine/SDS gels,
several of the original bands were reduced in intensity, and several
new bands appeared (Fig. 2, lane 2), indicating the
cross-linkings of some peptides. The most prominent, and the most
consistently observed, cross-linked products were the two designated as
bands a and b in Fig. 2. These bands were excised
from gels such as those of lane 2 of Fig. 2, electroeluted,
and subjected to N-terminal analysis. Also analyzed as controls were
the materials eluted from the positions corresponding to those of bands
a and b from the gels on which the uncross-linked
samples were resolved (Fig. 2, lane 1). These analyses
(Table I), in conjunction with the apparent molecular
mass of band a, showed that band a was the cross-linked dimer of the 22-kDa C-terminal fragment of containing the H7-H10 transmembrane helices (Fig. 1), and
the 11-kDa N-terminal fragment of
containing the
H1,H2 helix pair (Fig. 1).
|
Four sequences shown in Table I were identified upon N-terminal
sequencing of band b, indicating that this cross-linked
product contains the following four peptides in equimolar
stoichiometry: the 11-kDa N-terminal fragment of , the 22-kDa
C-terminal fragment of
, a fragment of
beginning at
Ala5, and a fragment of
beginning at
Gly143. That the trypsin-digested enzyme contains
beginning at Ala5, and that a fraction of this truncated
is also cleaved at Arg142-Gly143 has been
demonstrated (10, 16, 17, 19). Since the latter cleavage site (Fig. 1)
is between Cys125 and Cys148, which are
connected by a disulfide bridge (1), and since the bridge is expected
to remain intact under oxidative cross-linking reaction conditions, we
conclude that band b is the cross-linked trimer of 11-kDa
and 22-kDa fragments of
-subunit and a
-subunit that begins at
Ala5, is cleaved at Arg142-Gly143,
but is otherwise intact. Because we have not been able to detect
that is cross-linked either with the 11-kDa fragment alone or with the
22-kDa fragment alone, we suspect that formation of a disulfide bond
between the two
-fragments stabilizes a conformation of the
-subunit helix-bundle, which is the mandatory first step to the
subsequent direct contact of the Cys44 of
with an
intramembrane cysteine of the two
-fragments.
The above identifications of 11-kDa and 22-kDa fragments of in the
two most prominent cross-linked products are consistent with the
reduced intensities of 11-kDa and 22-kDa bands after exposure to
Cu2+-phenanthroline (Fig. 2, lanes 1 and
2). Closer examination of these gels shows, however, that
other peptides also may have participated in cross-linking reactions.
To date, we have been unable to characterize other discrete
cross-linked products.
The specificities of the cross-linking reactions were tested in
experiments, a representative of which are shown in Fig. 2. Formations
of the cross-linked products, and the disappearances of 11-kDa and
22-kDa fragments were prevented by Na+ and
K+ (Fig. 2, lanes 3 and 4), but not
by the same concentrations of choline and N-methylglucamine
(data not shown). Incubation in the presence of ouabain and
Mg2+ also prevented cross-linking (Fig. 1, lane
5), but Mg2+ alone was not effective (data not shown).
When the protective effects of different concentrations of
Na+ and K+ were quantitated,
K0.5 values were in the range of 10-20
mM for Na+ and less than 0.5 mM for
K+ (Figs. 3 and 4). The
relative values of these constants are in general agreement with the
relative K0.5 values for occlusions of
Na+ and Rb+ by this digested preparation (15),
and clearly indicate the specificities of the cation effects on the
cross-linking reactions. The concentration of ouabain for half-maximal
protection of cross-linking was reduced when Mg2+ was
present (Fig. 5), indicating that the well established
interactive effects of ouabain and Mg2+ on the native
enzyme (14) continue to exist within the peptide fragments of the
digested enzyme.
When the digitonin-solubilized preparation of the digested enzyme was cross-linked in the presence of Cu2+ as described previously (10) or in the presence of Cu2+-phenanthroline as in experiments of Fig. 2, no Na+, K+, or ouabain effects on the cross-linking reactions of the solubilized preparation were noted (data not shown).
The trypsin-digested preparation of the membrane-bound
Na+/K+-ATPase used here (Fig. 1) has no ATPase
activity, but retains the capacity to occlude Na+ and
Rb+ and to carry out a slow Rb+/Rb+
exchange after reconstitution (15). When this preparation is solubilized in digitonin and oxidatively cross-linked, a major product
is a dimer of the 11-kDa N-terminal fragment of and the 22-kDa
C-terminal fragment of
(10). We now show (Fig. 2 and Table I) that
such a dimer is also a major cross-linked product of the membrane-bound
version of this preparation. It has long been recognized that the
demonstration of similar cross-linked complexes of proteins in native
membranes and in solubilized preparations of the membranes is a good
indication of the existence of specific protein associations within the
native membranes (11, 13). The present findings and our previous data
(10), therefore, establish the proximities of the N-terminal and
C-terminal helices of
in the functional membrane. Although the
details of the contact domains remain to be mapped, as discussed
previously (10), the estimated locations of the sulfhydryl groups of
these helices (Fig. 1) suggest that the most likely interacting helices
are the H1,H10 pair and the
H2,H8 pair.
The other prominent cross-linked product of the membrane preparation
identified here is a trimer of an essentially intact and the above
two fragments of
(Fig. 2 and Table I). This is also similar to such
a trimer that we obtained previously from the detergent-solubilized
preparation (10). The two trimers, however, differ significantly in
respect to the nature of
that participates in cross-linking. In the
solubilized preparation the predominant reactant is the
that begins
at Ala5, but is otherwise intact (10), while the
predominant reactant in the membrane preparation is the same
N-terminally truncated
that is also cleaved at
Arg142-Gly143 between two half-cystines (Table
I). Since under the conditions we have used to prepare the digested
enzyme (i.e. at pH 8.5, the Arg142-Gly143 cleavage occurs in a small
fraction of total
content (16)), we conclude that without this
cleavage
does not cross-link with the
-fragments in the
membrane-bound preparation. Evidently, some spatial flexibility of the
-subunit chain, induced either by cleavage between the half-cystine
residues or by detergent solubilization, is required for close
juxtaposition of the single transmembrane cysteine of
with one of
the nearby transmembrane cysteines of
to allow the zero-length
cross-linking of the two sulfhydryl groups. The five intramembrane
sulfhydryl groups available for the formation of the two disulfides of
the cross-linked trimer are Cys44 of
and cysteines 104, 138, 930, and 983 of
(Fig. 1).
That the area of contact between the transmembrane helix of and one
or more transmembrane helices of
is altered significantly by a
cleavage in the extracellular domain of
is pertinent to a number of
studies that have provided considerable information on the effects of
various structural modifications of the extracellular domains and the
transmembrane domain of
on the assembly of the
,
-complex
(20-23), and on the functions of the enzyme (17, 21-24). We suggest
that, in the interpretation of these findings, the often neglected
possibility should be considered that a structural change of an
extramembrane domain of
may be transmitted to its transmembrane
helix, and then to the interacting helices of
.
A large number of studies on ligand-induced conformational transitions
of Na+/K+-ATPase in relation to its function
have been conducted over the years (25). Few of these, however, have
addressed such structural transitions of the transmembrane domains of
the enzyme. Experiments on hydrophobic labeling, followed by
proteolysis, have suggested the existence of different Na+-
and K+-dependent conformations of the
transmembrane domains (8, 26); a fluorescent label attached to a
specific transmembrane domain of the -subunit of the intact enzyme
has been shown to respond differently to various ligand-induced states
(27); and structural changes of some transmembrane helices, either by
chemical modification or by site-directed mutagenesis, have been shown
to alter specific ligand interactions with the enzyme (2-6). To our
knowledge, these and related previous studies, however, have not
provided experimental evidence for ligand-induced interactions among
the intramembrane domains. The sensitivities of the cross-linking reactions to Na+, K+, and ouabain demonstrated
in the present studies (Figs. 2, 3, 4, 5) clearly indicate that the highly
specific interactions of these ligands with the extensively digested
enzyme regulate the three-dimensional packing of the transmembrane
helices. Specifically, our data show that the conformational states
induced by the indicated ligands affect the alignments of the residues
of H1,H2 helix pair relative to those of the
H8-H10 helices, and the proximities of these
helices with the residues of one transmembrane helix of
. This does
not exclude the involvement of other transmembrane domains in
ligand-induced conformational changes of the helix-bundle. The use of
cysteine-scanning mutagenesis, other cross-linking reagents, and other
functionally competent fragments of the enzyme may reveal additional
ligand-sensitive transmembrane helix-helix interactions. Such studies
are in progress.
We thank Dr. J. Pohl, Emory University Microsequencing Facility, for amino acid analysis.