(Received for publication, August 18, 1994; and in revised form, October 14, 1994)
From the
The F moiety of rat liver ATP synthase has a
molecular mass of 370,000, exhibits the unique substructure
, and fully restores
ATP synthesis to F
-depleted membranes. Here we provide new
information about rat liver F
as it relates to the
relationship of its unique substructure to its nucleotide binding
properties, enzymatic states, and crystalline form.
Seven types of
experiments were performed in a comprehensive study. First, the
capacity of F to bind [
H]ADP, the
substrate for ATP synthesis and [
P]AMP-PNP
(5`-adenylyl-
,
-imidodiphosphate), a nonhydrolyzable ATP
analog, was quantified. Second, double-label experiments were performed
to establish whether ADP and AMP-PNP bind to the same or different
sites. Third, total nucleotide binding was assessed by the
luciferin-luciferase assay. Fourth, F
was subfractionated
into an
and a
fraction, both of which were
subjected to nucleotide binding assays. Fifth, the nucleotide binding
capacity of F
was quantified after undergoing ATP
hydrolysis. Sixth, the intensity of the fluorescence probe pyrene
maleimide bound at
subunits was monitored before and after
F
experienced ATP hydrolysis. Finally, the catalytic
activity and nucleotide content of F
obtained from crystals
being used in x-ray crystallographic studies was determined.
The
picture of rat liver F that emerges is one of an enzyme
molecule that 1) loads nucleotide readily at five sites; 2) requires
for catalysis both the
and the
fractions; 3)
directs the reversible binding of ATP and ADP to different regions of
the enzyme's substructure; 4) induces inhibition of ATP
hydrolysis only after ADP fills at least five sites; and 5) exists in
several distinct forms, one an active, symmetrical form, obtained in
the presence of ATP and high P
and on which an x-ray map at
3.6 Å has been reported (Bianchet, M., Ysern, X., Hullihen, J.,
Pedersen, P. L., and Amzel, L. M.(1991) J. Biol. Chem. 266,
21197-21201).
These results are discussed within the context
of a multistate model for rat liver F and also discussed
relative to those reported for bovine heart F
, which has
been crystallized with inhibitors in an asymmetrical form and has a
propensity for binding nucleotides more tightly.
Mitochondrial ATP synthases are comprised of two major units,
one called F and the other F
(for recent
reviews, see (1, 2, 3, 4, 5, 6) and 57).
The F
moiety spans the mitochondrial inner membrane and
directs protons to the F
moiety, which binds ADP and
P
and synthesizes ATP. The F
moiety of ATP
synthases from animal cells have molecular masses near 370 kDa and
contain five different subunit types in the unique stoichiometric ratio
. The presence of a single copy of the small
subunits (
,
, and
) for three
pairs may
impose asymmetry on the F
molecule(2, 57) . Alternatively, subunit
asymmetry may be induced within an otherwise highly symmetrical F
molecule primarily by nucleotide binding, particularly if the
small subunits are normally positioned in the center of the molecule
closely in line with the 3-fold axis of the
unit(2, 57) . There
is considerable evidence for asymmetry under noncatalytic conditions
from cross-linking(7) , subunit dissociation(8) , and
nucleotide binding studies(9, 10) . To account for the
participation of all three
pairs during ATP synthesis, the
position of either the small subunits (
,
, and
) or the
large subunits (
,
) are predicted to change relative to one
another (i.e. rotate or flicker). This assumed dynamic
behavior, for which there is now some cryoelectronmicroscopic
evidence(11) , has given rise to a model frequently referred to
as the ``rotating catalytic site'' model in which nucleotide
binding changes are inferred(6) .
Despite the availability
of a working model at the quaternary structural level, many questions
remain. First, there is the question of the total number of reversible
(exchangeable) nucleotide binding sites on F. Although
commonly stated to be
three(1, 2, 3, 4, 5, 6, 57) ,
most workers have not examined the simultaneous binding to F
of the substrate (ADP) and the product (ATP) of oxidative
phosphorylation. Second, it remains controversial as to whether
subunits contain two nucleotide binding sites (12, 13) or only a single nucleotide binding
site(14, 15) . Third, the role of
subunits in
contributing to both catalysis and reversible nucleotide binding to
F
remains controversial, with the preferred view being that
the
subunit optimizes the structure of
for catalysis (16, 17) while contributing partially (18, 19) or not at all (20) to catalytic
sites. Fourth, although a role for one or more of the small subunits
(
,
, and
) in altering nucleotide binding is inferred
from current
models(1, 2, 3, 4, 5, 6, 57) ,
experimental evidence is lacking, and it remains possible that
nucleotide binding may direct small subunit interactions. Finally, few
workers have given serious consideration to the possibility that at
least two distinct states of F
, catalytically active and
inhibited, with different structural features may be important,
respectively, in the function and regulation of the enzyme. The latter
possibility has taken on added interest in view of recent reports (21, 22) that rat liver and bovine heart F
exhibit differences in their crystalline forms.
The first
x-ray map of an F preparation, obtained on the rat liver
enzyme at 3.6 Å(21) , revealed several important features
of the molecule. First, subunits
and
were shown to
interdigitate in an alternating arrangement. Second, evidence for a
nucleotide binding region on
subunits near
/
interfaces
was obtained. Finally, the small subunits, although not detected, were
suggested to either reside in the center of the molecule or not to be
ordered. An x-ray map of the bovine heart enzyme at 6.5-Å
resolution (22) , although not distinguishing
and
subunits, depicts a molecule with features very similar to those of the
rat liver enzyme, but with some distinct asymmetrical features. These
studies implicate two distinct forms of F
ATPases.
To
help address some of the major questions described above, we have
carried out a comprehensive study involving a variety of different
approaches. The results obtained are discussed within the context of
current views about the relationship of the unique substructure of
F to its nucleotide binding properties, catalytic
functions, active and inhibited states, and crystalline form.
Table 1, part A,
shows that purified F precipitated twice with ammonium
sulfate retains about 1 mol of ADP and 1 mol of ATP/mol of
F
. In part B, it is seen, consistent with our earlier
findings(10, 36) , that addition of
[
H]ADP alone results in the net binding of
1
mol of ADP/mol of F
whereas the addition of
[
P]AMP-PNP alone results in the binding of
3 mol of AMP-PNP/mol of F
. Significantly, however, the
combined addition of the two differentially labeled nucleotides also
results in the net binding of
1 mol of ADP/mol of F
and
3 mol of AMP-PNP/mol of F
emphasizing that
the single detectable ADP site is separate and distinct from the three
sites for AMP-PNP. Previously, we had shown that ADP does not alter the
net binding of AMP-PNP, nor does AMP-PNP alter the net binding of
ADP(10) . Additionally, the addition of MgCl
or
CoCl
does not alters the stoichiometry of ADP or AMP-PNP
binding(10, 36) .
Results presented in Table 1, part C, address the question of whether the single ADP
site (part B) detected by radiolabeling is distinct from the endogenous
ADP site (part A) detected by the chemiluminescent assay. If the two
sites are identical, then prior incubation of F with ADP
followed by determination of total ADP by the chemiluminescent assay
should reveal only 1 mol of ADP/mol of F
. Conversely, if
the two sites are distinct, 2 mol of ADP/mol of F
should be
detected. As shown in part C the latter answer is obtained. As will be
revealed in studies described below, these two ADP sites reside on
different F
subunits. (In studies not presented here the
single endogenous ATP site was shown to be readily reversible
accounting for one of the three AMP-PNP sites.)
In summary (Table 1, part D), under nonhydrolytic conditions there are five
readily detectable nucleotide binding sites on rat liver F,
one reversible ADP site, three reversible AMP-PNP sites, and one site
for the nonexchangeable binding of ADP. These nucleotide binding
characteristics, together with those described earlier (10, 36) support the view that under the assay
conditions employed, rat liver F
behaves as an asymmetrical
molecule.
Figure 1:
A, loss of ATPase activity of F in the presence of MgCl
accompanied by sedimentable
protein. After precipitating F
(300 µg) twice with
ammonium sulfate at 25 °C, the enzyme was dissolved in 200 µl
of 50 mM Tris-Cl, pH 7.4. After dividing the sample into four
75-µl aliquots, 5 mM MgCl
was added and the
samples allowed to remain at 25 °C for the times indicated. Samples
were then subjected to centrifugation for 20 min at 12,000 rpm at 25
°C in a Sorvall RC 2B centrifuge, followed by protein
determinations on the supernatant and pellet fractions. B,
SDS-PAGE analysis of supernatant (S) and pellet (P)
fractions obtained in A. SDS-PAGE was carried out in slab gels
as described under ``Methods.'' The amount of
protein loaded on the gels was, respectively, 7.5 µg (leftpanel) and 15 µg (rightpanel).
Note: although the F
preparation used is over 95% pure,
overloading of the gel and the photography to enhance visualization of
the
and
subunits also enhances minor contaminants. In these
long slab gels the small subunits,
and
, tend to diffuse.
They are much more distinct when electrophoresis is carried out in a
Bio-Rad Mini-Protean dual slab cell (see Fig. 4, inset). No evidence for heterogeneity in the F
preparations used (e.g.
and
forms) was observed
when F
was subjected to electrophoresis in native gels at
acrylamide concentrations ranging from 3 to 15%. (In subsequent legends
to figures and tables the supernatant fraction is referred to as the
fraction and the pellet fraction as the
fraction.)
Figure 4:
Relative capacities of ADP and MgATP to
induce changes in the fluorescence of the probe pyrene maleimide.
F (150 µg) was incubated at 25 °C for 20 min in a
0.1-ml system containing 50 mM Tris-Cl, pH 7.4, and either 5
mM ADP or 5 mM ATP + 5 mM MgCl
. The incubation mixtures were then subjected to
column centrifugation (see ``Methods''). These two
different conditions result in the reversible binding of
1 mol of
ADP/mol of F
(Table 1) and 2.5 mol of ADP/mol of
F
(Table 3), respectively. The relative capacity of
pyrene maleimide to bind to these two different nucleotide-containing
preparations was then assessed fluorometrically exactly as described
under ``Methods.'' Inset, SDS-PAGE of
F
labeled with pyrene maleimide. Electrophoresis was
carried out in a Bio-Rad Mini-Protean dual slab cell in 15% acrylamide
according to the method of Laemmli(56) . The gel on the left was placed on a UV transmitter to visualize fluorescence
and subsequently stained with Coomassie Blue to visualize protein.
Pyrene maleimide labeling is not observed in the
,
, and
subunits and is observed only in the larger band corresponding to
the
and
subunits.
subunits contain no cysteine
residues and are not labeled by pyrene
maleimide.
Results presented in Fig. 2A show that
the fraction retains significant secondary structure as
revealed by circular dichoism spectroscopy, and similar to intact
F
, exhibits a high degree of
-helical character.
However, no ATPase activity could be detected in the
fraction even after electrophoresis under native conditions which
resulted in retention of ATPase activity in control F
. The
method used to detect ATPase activity within the gel following
electrophoresis (see ``Methods'') is based on the
reaction of lead nitrate with the P
produced in the ATPase
reaction to give a white precipitate of lead phosphate. The sensitivity
of the assay is limited only by time, and even after several days no
precipitate was observed in the gel resulting from electrophoresis of
the
fraction.
Figure 2:
A, circular dichoism spectra of
F and the
fraction.
Circular dichoism spectroscopy was carried out exactly as described
under ``Methods.'' The percent of secondary
structure was calculated from the program PROSEC(55) . B, native PAGE gels depicting F
and the
fraction when stained for protein
and activity. PAGE was carried out under native condition in
cylindrical gels and stained for protein and activity exactly as
described under ``Methods.'' Both F
(25
µg) and the
fraction (25 µg) were loaded in 50
mM Tris-Cl, pH 7.4, containing 50% glycerol. Gels containing
F
also included 10% glycerol, 5.0 mM ATP, and 3
mM MgCl
to minimize its propensity to
dissociate.
The finding that loss of ATPase
activity of intact F occurs simultaneously with loss of the
subunit (Fig. 1A), and the additional finding
that the fraction containing the
subunit exhibits no ATPase
activity (Fig. 2B) while retaining significant
structure (Fig. 2A), supports the view that the
subunit alone is not a catalytically active unit(38) .
Results tabulated in Table 2show that when 5 mM nucleotide is in the binding
assay, the four reversible nucleotide binding sites on F (i.e. the one ADP site, and the three AMP-PNP sites) are
accounted for within the
fraction. Additionally,
consistent with results obtained on intact F
(Table 1), ADP does not alter AMP-PNP binding nor does
AMP-PNP alter ADP binding. However, it will be noted that at 1 mM nucleotide, where the single ADP site is still recovered in the
fraction, only one of the three AMP-PNP sites can now
be accounted for (see values in parentheses in Table 2). The K
for the one ADP site and the one AMP-PNP site
remain near 1 µM (data not shown), values obtained for the
intact enzyme(10, 36) . However, K
values for the other two AMP-PNP sites have increased over
4-fold, indicating either that they also reside on
subunits (i.e. at
/
interfaces) or that they have been
damaged in the preparation of the
fraction.
Finally, Table 2summarizes results obtained on the fraction,
which is shown to retain the single endogenous, nonexchangeable ADP
site characteristic of intact rat liver F
(Table 2).
Previously, we have shown that the single tight Mg
site characteristic of liver (10) and heart (39) F
preparations is also recovered in the
fraction(10) . Because of the insolubility of this
fraction, it could not be tested for additional nucleotide binding by
the column centrifugation method.
Of special interest is the finding
that within the fraction the asymmetry in binding ADP
is retained, implicating the tight association of either the
or
subunits (or both) with a single
subunit. To investigate
this possibility, the
fraction was subjected to
molecular sieve HPLC chomatography. One major peak followed by a
trailing component well within the included volume was observed (Fig. 3A). Both the major peak and the trailing
component were collected, concentrated, and subjected to SDS-PAGE (Fig. 3B). Significantly, the major peak which
contained about two-thirds of the total starting protein migrated as a
single species corresponding to the
subunit. In contrast, the
trailing component corresponding to about one-third of the total
starting protein migrated as two species, one corresponding to the
subunit and the other to the
subunit. The
subunit was
not detected and may have bound irreversibly to the column.
Nevertheless, these results indicate that one of the three
subunits within the
fraction may be associated with a
single
subunit, the other two
subunits evidently migrating
as a dimeric species prior to the
unit (Fig. 3A).
Figure 3:
A, elution profile of the
fraction on a HPLC molecular sieve column. The
fraction, 61.5 µg in 50 µl of 50 mM Tris-Cl, pH 7.4,
containing 5.0 mM MgCl
was loaded on a Waters
Protein Pak 300 molecular sieve HPLC column and eluted with 100 mM Tris-Cl buffer, pH 7.4. The fractions designated I and II were
collected and concentrated in an Amicon-Centricon 10 device. B, SDS-PAGE of the total concentrates from fraction I and II
relative to that of control F
. SDS-PAGE was carried out as
described under ``Methods.''
The above findings indicate that the
fraction participates fully in the reversible binding
of 1 mol of ADP and 1 mol of AMP-PNP/mol of F
, and that the
fraction contains the one nonexchangeable ADP site. These
findings further indicate that the asymmetry of nucleotide binding
found in intact F
is preserved within the
and
fractions.
Results presented in Table 3show that during
ATP hydrolysis rat liver F binds up to 2.5 mol of ADP/mol
of F
or 1.5 mol more than the single mole that could be
added prior to catalysis. Therefore, ATP hydrolysis induces F
to provide nearly two additional sites for binding ADP that were
previously inaccessible to this nucleotide. Interestingly, the total
nucleotide content following ATP hydrolysis still approaches 5 mol/mol
of F
with only 1 mol being retained as ATP. The subunit
distribution of the bound nucleotides could not be identified under
these conditions as the MgATP-treated enzyme could not be
subfractionated into
and
fractions.
Results presented in Fig. 4show pyrene maleimide, which alone is essentially
nonfluorescent, induces a marked fluorescent enhancement upon binding
to F
-subunits. This enhancement is significantly
decreased in the F
preparation that contains a single,
reversible, mol of ADP/mol of F
but decreased much more in
the F
preparation that has experienced ATP hydrolysis and
bound ADP at two additional sites. Fig. 5presents the time
course of the fluorescence response to the addition of pyrene
maleimide. Here it is clear that over the time period monitored, the
two different ADP-F
forms exhibit less fluorescence upon
the addition of pyrene maleimide than control F
, and that
the form that has undergone ATP hydrolysis exhibits the least
fluorescence. Significantly, results also presented in Fig. 5show that an F
form that has been prepared as
described in Table 1in the presence of both ADP and AMP-PNP
reduces the fluorescence of pyrene maleimide to almost the same extent
as the form that has experienced ATP hydrolysis.
Figure 5:
Time course of the reactivity of pyrene
maleimide with F samples pretreated with ADP, MgATP, or
AMP-PNP + ADP. F
samples were treated exactly
as described in Fig. 4with 5 mM nucleotide or, in the
case of MgATP, with 5 mM ATP + 5 mM MgCl
and subjected to column centrifugation. The resultant F
samples containing bound nucleotide were then assessed
fluorometrically for their interaction with pyrene maleimide exactly as
described under ``Methods.''
These studies
implicate three distinct forms of rat liver F, the starting
preparation containing 2 mol of endogenously bound nucleotide/mol of
enzyme (Table 1, panel A), the form containing an
additional ADP bound at a reversible site within the
fraction (Table 1, panel B; and Table 2), and the
form that has either experienced ATP hydrolysis (Table 3) or been
treated to load both ADP and AMP-PNP (Table 1, panel B).
Rat liver F is routinely crystallized from a solution containing 5 mM ATP and 200 mM KP
, pH 7.5 (see ``Methods''). In this buffer the enzyme is maximally
catalytically active when MgCl
is added(40) .
Results summarized in Table 4, part A, show that, when crystals
obtained by adding ammonium sulfate to F
in 5 mM ATP and 200 mM KP
, pH 7.5, are redissolved
after several months, there is full retention of catalytic activity
both in Tris-Cl buffer and in the known activating buffer Tris
bicarbonate(31) . The critical importance of ATP in maintaining
rat liver F
in a stable form within the crystals was
demonstrated in experiments where crystals were washed in a medium
containing ammonium sulfate and P
but lacking ATP. Such
crystals, which now contain only ADP (<2 mol/mol of F
; Table 4, part B), yield upon redissolving an almost completely
inactive enzyme (Table 4, part A). Although it is not possible to
assess the exact stoichiometry (i.e. mol of ATP/mol of
F
) in the crystals while in the presence of 5 mM ATP, it seems likely that at least 1 mol of ATP/mol of F
is present in the crystalline enzyme. Rat liver F
as
isolated does contain
1 mol of ATP/mol of F
(Table 1), indicating that one tight site for binding ATP
is present.
These results emphasize that rat liver F crystals being used to obtain a high resolution structure
maintain the enzyme in an ATP-dependent stable form that can be readily
recovered with full retention of catalytic activity.
Results presented in Table 5show that when
initial ATPase rates are compared to that of the fully active
F form, the second most active F
form is F
, the enzyme treated with
crystallization buffer (5 mM ATP + 200 mM KP
, pH 7.5). F
and
F
also are highly active. In contrast, form
F
is 91% inhibited and F
is almost 60% inhibited. It is of interest to note that
F
, which has one site filled with ATP and
nearly four sites filled with ADP, is highly active, and only when
additional ADP is added directly to the assay to displace the bound ATP
(or fill the sixth site with ADP) to give F
is
there a dramatic inhibition. F
, which contains
both ADP and AMP-PNP, is also not fully inhibited. However, by adding
additional ADP to the assay F
, as
F
, is now inhibited by over 90%.
These
results emphasize that F derived by incubating
F
in crystallization buffer is almost fully
active, and that a form of F
containing a 4/1 ADP/ATP ratio
is almost fully active as well until additional ADP is added. In
contrast, F
, which has five sites filled,
3 with AMP-PNP and
2 with ADP, is
60% inhibited.
Results described here represent the first comprehensive
study of the nucleotide binding properties of intact rat liver
F, and one of the most comprehensive studies of this nature
on an F
preparation to date. Experiments were carried out
with F
in the presence of ADP alone, AMP-PNP alone, ADP and
AMP-PNP together, MgATP alone, and ATP and P
together. In
addition, F
preparations resulting from these treatments
have been assayed both for catalytic activity and for their capacity to
interact with the fluorescent probe pyrene maleimide. Additionally,
F
obtained from crystals currently being used to obtain a
high resolution structure have been analyzed for both catalytic
activity and nucleotide content. Finally, the stability of F
within the crystals has been determined. These studies are
fundamental to our understanding both of how ATP synthases from
mammalian tissues carry out ATP synthesis and of how these enzymes are
regulated by product inhibition. In addition, these studies are
fundamental in defining those enzymatic forms that an F
molecule can assume during its catalytic and regulatory modes and
will permit us to identify those forms on which three-dimensional
structures are being determined(21, 22) .
The
experimental results obtained on rat liver F are best
discussed and interpreted within the framework of the scheme presented
in Fig. 6. Here six different forms of the enzyme previously
designated in Table 5as F
,
F
, F
,
F
, F
, and
F
are presented. F
, an
active form (Table 5), is the isolated enzyme after twice
precipitating with ammonium sulfate. This form has
2 nucleotide
binding sites filled (Table 1, part A), one with a
nonexchangeable ADP recovered in the
fraction (Table 2, part B) and one with an ATP (Table 1, part A),
exchangeable with AMP-PNP and accounted for in the
fraction (Table 2, part A). The K
(
1
µM) of this site in binding AMP-PNP (10) is in the
same range as the K
(2.2 µM) of
AMP-PNP in inhibiting F
ATPase activity(41) . In
more conventional language, the very tight ADP site can be referred to
as ``noncatalytic''and the exchangeable ATP site as
``catalytic.'' As ADP is retained tightly bound to the
noncatalytic site even in the
fraction (Table 2, part
B), its binding domain is predicted to lie predominantly within an
subunit, although at an
/
interface consistent with our
earlier x-ray crystallographic studies(21) . Similarly, as the
AMP-PNP bound to the catalytic site is accounted for in the
fraction without a loss of affinity (Table 2,
part A), its domain is predicted to reside predominantly within a
subunit.
Figure 6:
Diagram depicting six different forms of
rat liver F predicted from results of
studies reported here. The data presented provide evidence for six
distinct forms of F
designated as
F
, F
,
F
, F
,
F
, and F
.
F
represents the enzyme as isolated after twice
precipitating with ammonium sulfate, F
the
enzyme after adding ADP to F
, and
F
after adding AMP-PNP to
F
. F
is the form
obtained after incubating F
with MgATP to
induce ATP hydrolysis, and F
results by adding
additional ADP to F
. F
results by adding ATP and high P
(200 mM) to
F
. Rat liver F
has been
crystallized under the latter conditions (see ``Methods''), which results in a catalytically
active F
molecule ( Table 4and Table 5) with
3-fold symmetry. In contrast bovine heart F
has been
crystallized in the presence of ADP and the inhibitors AMP-PNP and
sodium azide (46) to give an enzyme with distinct asymmetrical
features(22) . This form most closely resembles
F
in the diagram. The small subunits
,
, and
are not shown, although the
subunit is believed
to lie at the center of F
extending from the bottom to the
top of the molecule(22) . (See ``Discussion'' for a more detailed description of
the six F
forms depicted here. Additionally, please note
that to depict the purine moiety of ADP or AMP-PNP as acting at an
interface in some cases, it has been necessary to write these in
reverse as PDA or PNP-PMA, respectively.)
F is converted to
F
, also an active form (Table 5), by
adding ADP, which even at 5 mM fills only a single reversible
site (Table 1, part B). The K
of this site
is also near 1 µM(36) and is accounted for in the
fraction without a loss of affinity (Table 2,
part A). Moreover, ADP can be added to this site on F
without altering the binding of AMP-PNP or ATP bound at the
catalytic ATP site (Table 1, parts B and C). Therefore, this site
is predicted to reside on a second
subunit conformationally
distinct from the
subunit containing ATP. This reversible ADP
site is also predicted to be a catalytic site, now poised for ATP
synthesis. Other data presented here (Fig. 3) indicate that a
conformational change has occurred in the conversion of
F
to F
as pyrene
maleimide fluorescence is significantly reduced ( Fig. 4and Fig. 5).
F, an inhibited form (Table 5), is obtained either by adding ADP + AMP-PNP to
F
or only AMP-PNP to F
(Table 1, part B). The final enzyme that results has
five sites filled, two with ADP and three with AMP-PNP. One site
is filled with AMP-PNP by displacing the exchangeable ATP bound on a
subunit and can be accounted for in the
fraction (Table 2, A). The other two AMP-PNP sites cannot be accounted for
by binding predominantly to
subunits (Table 2) and are
assumed to be noncatalytic and to reside predominantly on
subunits at
/
interfaces. The conversion of
F
to F
by addition of
AMP-PNP induces a further decrease in the pyrene maleimide
fluorescence, i.e. a greater decrease than that produced by
adding ADP alone (Fig. 5). This indicates that
F
is conformationally distinct from both
F
and F
.
A second
inhibited form, F, is produced (Table 5)
by incubating F
with MgATP to fill almost five
sites (Table 3), one with ATP and the other four with ADP to
first produce the active form F
(Table 5), and then the inhibited form by adding more ADP
to the assay system. The subunit distribution of nucleotide on
F
is not known but is assumed to be very
similar to that characteristic of F
, the other
inhibited F
form where nucleotides are bound both on
subunits near
/
interfaces and on
subunits. Consistent
with this view, the pyrene maleimide fluorescence ( Fig. 4and Fig. 5) is reduced approximately the same amount in forms
F
and F
, the immediate
precursor of F
. Additionally, consistent with
this view are the earlier studies on rat liver F
by
Williams and Coleman(42) , who demonstrated that in the absence
of Mg
the photoaffinity probe benzophenone ATP labels
only
subunits, while in the presence of Mg
,
which induces ATP hydrolysis, both
and
subunits are
labeled.
Finally, a fourth active form, F,
is produced by adding ATP and high P
(200 mM) to
F
(Table 5). F
is
the rat liver form that has been crystallized and on which a
3.6-Å x-ray map has been obtained(21) . Although the
nucleotide composition of this F
form within the crystals
is not known, it is predicted that at least 3 mol of nucleotide/mol of
F
are present. This is because F
prior to crystallization loads 2.4 mol of nucleotide/mol of
F
with sufficient affinity to survive column centrifugation (Table 5) and because F
crystals require
ATP to maintain the enzyme in an active form (Table 4, part A).
F
obtained from extensively washed crystals retains neither
ATP nor activity and retains only ADP (Table 4, part B).
The
studies on rat liver F described above and summarized in Fig. 6provide an expanded framework on which to better
understand how F
ATPases participate in ATP synthesis and
how they are regulated. They also provide a better understanding of
which F
forms have been crystallized and the possible roles
that three-dimensional structures derived from one or more of these
forms may play in helping us understand the mechanism and regulation of
ATP synthesis. Finally, as it concerns conformational differences among
F
forms, although subtle, these exist. Therefore, these
studies may also provide insight into possible differences among
F
preparations from mammalian tissues.
Considering
briefly each of these points, it is first of interest to discuss the
mechanism of ATP synthases in relation to the studies described here.
In this case, F is of special interest as it
conforms in part to the predictions of the ``binding change''
mechanism (43, 44) where ATP bound at one
subunit is ready to be displaced upon coupling to the electrochemical
proton gradient, ADP bound to the second
subunit is ready to be
phosphorylated, and the vacant domain on the third
subunit awaits
the entry of ADP. Consistent with the presence of an
F
form of the enzyme being operative in energy
coupling are the recent studies of Turina and Capaldi(45) , who
show that communication between catalytic sites and the
subunit
is promoted by the additions of ATP or AMP-PNP, but not by the addition
of ADP.
As it concerns the relationship of these studies to the
regulation of ATP synthases, F may be
important. For example, upon removal or dissipation of the
electrochemical proton gradient, it can be seen that subsequent ATP
hydrolysis would first load F
at four sites with ADP and
one site with ATP keeping the enzyme active. At this point, F
is poised to go in either direction, i.e. ATP synthesis
if the electrochemical gradient is restored, or ADP inhibition if it is
not. In the latter case it seems likely that the ATPase inhibitor
protein (IF
) will play a critical role, perhaps by
promoting conversion of the ATP containing
subunit to one that
now prefers ADP. In support of this view, recent work has shown that
IF
does enhance nucleotide binding to
F
(34, 35) .
The relationship of these
studies to the F forms that have been crystallized becomes
immediately obvious by examination of Fig. 6. The bovine heart
enzyme has been crystallized in the presence of AMP-PNP and ADP (46) and would be predicted to be in an inhibited form similar
to F
with at least five nucleotide binding
sites occupied, and to exhibit subunit asymmetry as
reported(22) . In sharp contrast, the rat liver F
is crystallized under conditions (ATP and high P
) in
which the enzyme is in an active form, F
. This
form is fully active and remains fully active when recovered several
months later from crystals (Table 4). The x-ray diffraction data
of rat liver F
exhibits 3-fold symmetry(21) , and
no break in the symmetry has been detected to date even at higher
resolution with single crystals. This result would be predicted if
under these crystallization conditions, the major subunits become
structurally equivalent and the
subunit runs through the center
of the molecule close to the 3-fold axis. In this case, the only
possible asymmetry would be present in regions of
and
in
contact with
or with the two small subunits
and
,
which may also align closely with the
subunit in line with the
3-fold axis. The
and
subunits may not be ordered and
conform to the crystallographic symmetry.
Two fundamental issues
that remain unresolved concern the mechanism by which asymmetry is
induced, and whether an asymmetrical or symmetrical form of F is operative during ATP synthesis. Concerning the first issue we
must establish whether the small subunits (
,
, and
)
really induce asymmetry in the large subunits
and
or
whether nucleotides induce subunit asymmetry, which then promotes
binding of the small subunits. Concerning the second issue, is it
possible that, when subunit repositioning takes place to accommodate
the sequential participation of all three
pairs in the ATP
synthesis cycle, the F
molecule switches from an
asymmetrical to a symmetrical intermediate state, or vice
versa? It is of interest to note that F
would be predicted to be poised for ATP synthesis, as the final
crystallization medium due to significant ATP hydrolysis exhibits a
high ADP + P
/ATP ratio.
In any case, it seems
likely that two higher resolution structures of F will be
obtained: an inhibited asymmetrical form of the bovine heart
F
, F
, and a symmetrical, active
form of the rat liver enzyme, F
. Both
structures should be valuable in providing useful clues as to how
catalysis occurs and how product inhibition of the enzyme is induced.
They also should provide valuable information about the magnitude of
conformational changes that may occur in the transition from active to
inhibited form. Additionally, as the importance of cooperativity in
this complex protein remains a challenging, unresolved
issue(47, 48, 49, 50) , the need for
two different structural forms is evident. Certainly, F
ATPase is structurally more complex than hemoglobin. Therefore,
it is of interest to note from the recent very elegant studies of
Ackers and colleagues (51) that hemoglobin, once viewed simply
as involving two symmetrical states ``R'' and
``T''(52) , is now known to proceed through a number
of different states, some exhibiting symmetry and others asymmetry.
Finally, it should be noted that there may be some functionally
relevant differences among mammalian F preparations. In
contrast to bovine heart F
(53, 54) , where
either 6 mol of AMP-PNP/mol of F
or 3-5 mol of
ADP/mol of F
can be loaded under nonhydrolytic conditions,
rat liver F
loads no more than 3 mol of AMP-PNP/mol of
F
or 2 mol of ADP/mol of F
. Whether this
difference is due to amino acid sequence differences in
or
subunits, differences in the interaction of the large subunits with the
small subunits
,
, and
, or to posttranslational
modification is not clear. Currently, we are comparing closely the
properties of the bovine heart F
with rat liver
F
. Acknowledgments-We are grateful to Dr. Young Hee
Ko for photographing F
ATPase crystals and to Jackie Seidl
for processing the manuscript for publication.
Addendum-After the work reported here was processed
for publication, Dr. J. E. Walker kindly provided us with a preprint on
the structure of F-ATPase from bovine heart mitochondria
(Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E.
(1994) Nature370, 621-628). In confirmation of
our earlier x-ray model on rat liver F
at 3.6-Å
resolution (21) , the heart enzyme is of similar size, has
alternating
and
subunits, a nucleotide domain on
subunits at an
/
interface, and apparent disorder in the two
small subunits
and
. Consistent with experiments reported
here on rat liver F
where ADP + AMP-PNP produce an
inhibited enzyme with 5 nucleotides bound and predicted asymmetry, the
bovine F
structure crystallized with these nucleotides
present shows 5 bound nucleotides, exhibits subunit asymmetry, and is
believed to be inhibited. The rat liver F
x-ray map,
although exhibiting many common features with that of bovine heart
F
, has been crystallized as reported here under active
conditions, retains its activity after several months in the crystals,
and results in an x-ray map with 3-fold symmetry in which the three
pairs are predicted to be structurally equivalent.
Therefore, it seems likely that the two groups are working on two
different functionally relevant forms of the F
moiety of
ATP synthases, one which is active, and the other inhibited.