(Received for publication, December 13, 1995; and in revised form, February 21, 1996)
From the
Three intragenic second-site suppressors, P353L, T237I, and
L390F, were identified that suppressed two mutations in, and one
adjacent to, the P-loop in the -subunit of the yeast
F
-ATPase. The crystal structure of bovine
F
-ATPase (Abrahams, J. P., Leslie, A. G. W., Lutter, R.,
and Walker, J. E.(1994) Nature 370, 621-628) shows that
these suppressor residues are located in the nucleotide-binding domain.
Specific hypotheses have been formulated that suggest the
conformational coupling of the P-loop with the suppressor sites. P353L
is in a ``catch'' region, which forms unique interactions
with the
-subunit in the three different conformational states of
the catalytic site. The identification of this suppressor mutation
demonstrates genetically that the catch region is conformationally
coupled to the P-loop. T237I is shown to interact with Lys-209, which
occurs just after the P-loop. This suggests that this interaction
changes the conformation of the P-loop to suppress the initial
mutation. L390F interacts with Ala-181, which is adjacent to the
P-loop. The mechanism of this suppression is suggested to occur through
the interactions of L390F with Ala-181. These results identify critical
interactions that modulate the structure of the P-loop and thus the
biochemistry of the enzyme.
The mitochondrial ATP synthase is the major enzyme responsible
for the aerobic synthesis of ATP. The ATP synthase is composed of a
water-soluble portion, the F-ATPase (EC 3.6.1.34), and a
membrane portion, F
. The F
-ATPase has a subunit
composition of
with
an overall mass of
360,000 Da(1, 2) . The recent
determination of the high resolution (2.8 Å) structure of the
bovine F
-ATPase (3) and prior biochemical and
mutagenesis studies (for review, see (4) ) indicate that the
-subunits, together with small contributions from the
-subunits, compose the active sites of the enzyme.
The binding
change hypothesis for ATP synthesis proposed by Boyer et al.(5, 6, 7) suggests that the
energy-requiring step for ATP synthesis is the release of newly
synthesized ATP and not the phosphorylation step. The hypothesis
proposes that three catalytic sites in F participate in the
synthesis of ATP by a mechanism that involves conformational changes
mediating changes in affinity of the active sites for ATP. The binding
change hypothesis is supported by a number of biochemical studies (for
review, see (6) ) and recently by the high resolution crystal
structure of the protein (3) . The crystal structure indicates
that the three active sites are not identical: one site is occupied by
ADP (
), one site by AMP-PNP (
)(
), and one site is vacant
(
). The differences in
,
, and
are also demonstrated by
large conformational differences in the active-site domain of each
-subunit, including the structure and location of the P-loop
motifs.
The P-loop motif (8, 9) is present in many
nucleotide-binding proteins (9, 10) . The primary
sequence of the P-loop in the -subunit of F
is
Gly-Gly-Ala-Gly-Val-Gly-Lys-Thr. Crystallographic studies indicate that
the backbone structure of the P-loop is the same in many
nucleotide-binding proteins: p21
, elongation
factor Tu, myosin, RecA, adenylate kinase, and the
ATPase(3, 11, 12, 13, 14, 15) .
In p21
, the P-loop has extensive hydrogen
bonding with the
-,
-, and
-phosphates of
Mg
-GMP-PNP(12) . Biochemical studies of
mutants in Escherichia coli and yeast F
indicate
that the P-loop is critical for catalysis (16, 17, 18, 19) . Furthermore,
recent studies in this laboratory indicated that the primary structural
constraints of the P-loop in the
-subunit of the yeast ATPase
correspond very well with the known structure of
p21
(19) . As revealed by the crystal
structure of F
, the P-loop is in a dramatically different
conformation in
as compared with
and
, which suggests that the conformation of
the P-loop may explain the vacancy of
for
nucleotides. As such, changes in the position, geometry, and structure
of the P-loop of the mitochondrial ATPase may, in part, be responsible
for changes in the biochemistry of the active site during the catalytic
reaction cycle.
This study was initiated to identify residues in the
-subunit that interact with the P-loop. Intragenic suppressors
were isolated in strains with mutation codons coding for residues in,
and immediately adjacent to, the P-loop of the
-subunit of
F
. The primary structural constraints on these residues
were postulated to be determined by steric interactions with other
residues in the enzyme (19) . This study has identified
critical interacting residues in the
-subunit that appear to be
important conformational links to the P-loop in the active site of the
enzyme. These interactions may be important in the catalytic mechanism
for the transition from the high to the low affinity conformation, or
they may be important for modulating the activity of the enzyme to suit
the needs of the particular organism.
The following media were used for the growth of the yeast strains: YPD (1% yeast extract, 2% peptone, 2% glucose), YPG (1% yeast extract, 2% peptone, 3% glycerol), and SD (0.67% yeast nitrogen base without amino acids, 2% glucose). SD medium was supplemented with appropriate auxotrophic requirements at 20 mg/liter.
Figure 3:
Interaction pathway proposed for
suppressor P353L. The bovine numbering system was used in Fig. 3Fig. 4Fig. 5since the figures are derived
from the coordinates of the crystal structure of the bovine
F-ATPase(3) . A, P353L
(Pro-320
) is proposed to disrupt the hydrogen bond of
Asp-348 (Asp-315
) with Arg-370 (Asp-337
), which
disrupts the hydrogen bond of Arg-370 with the P-loop main chain
carbonyl. This structure is shown in the
conformation with bound Mg
-ADP. This structure
does not differ much from the
conformation. B, shown is the region corresponding to A in the
conformation. The regions were superimposed using
QUANTA Version 4.1.1 (29) as described under ``Materials
and Methods.'' The perspective shown is the same as that in A. The relative positions of the P-loop, Pro-353, Asp-348, and
Arg-370 are quite different in the
conformation as
compared with the
conformation, with shifts of 10
Å.
Figure 4:
Interaction pathway proposed for
suppressor T237I. Thr-237 (Ser-203) interacts with Lys-209
(Lys-175
) in the
conformation (A), but not the
conformation (B).
Mutation T237I is proposed to alter the conformation of the P-loop via
changes in the position or conformation of helix B. As described for Fig. 3, the regions were superimposed in the
and
conformers, and identical perspectives are
shown in A and B. Also shown is bound
Mg
-ADP in the
conformation (A).
Figure 5:
Interaction pathway proposed for
suppressor L390F. The relative positions of Leu-390
(Ile-357) and Ala-181 (Ala-147
) in the
(A) and
(B)
conformations are shown. Leu-390 is located in
-strand 9 of the
nucleotide-binding domain. The crystal structure indicates that Leu-390
interacts with Ala-181. The mechanism of suppression is suggested to
occur through the interactions of L390F with Ala-181, which distorts
strand 3 and alters the conformation of the P-loop. These interactions
are not largely different in the
(A),
, and
(B) conformations
despite large conformational changes in the region. As described for Fig. 3, the regions were superimposed in the
and
conformers, and identical perspectives are
shown in A and B. Also shown is bound
Mg
-ADP in the
conformation (A).
E. coli cells were transformed by electroporation as described (Bio-Rad). Yeast cells were transformed by electroporation (30) and by the LiAc/polyethylene glycol method(31) .
All DNA sequencing analysis was performed using
dideoxynucleotides (32) , Sequenase Version 2 (Amersham Corp.),
and S-dATP. Autoradiography was performed with Kodak XAR-5
film.
The primary structural constraints of residues in the
-subunit of the yeast mitochondrial ATPase P-loop suggested that
this P-loop has the same geometry as those of other nucleotide-binding
proteins, such as p21
(19) . Comparative analysis
of the structure of the p21
P-loop with GMP-PNP bound (12) with that of bovine
(3) indicated that these structures were nearly
superimposable, with a root mean square deviation of 0.41 Å. In
addition, the nucleotide and the Mg
ion are nearly
superimposed in the structures of p21
and
. These results suggest that the extensive hydrogen
bonding pattern of the p21
P-loop with the nucleotide (12) is also present in the bovine F
-ATPase. Thus,
small changes in the conformation of the P-loop may disturb the
hydrogen bonding and thereby alter the chemistry of the active site.
The initial goal of this study was the isolation of intragenic
suppressors of mutations at or adjacent to the P-loop, specifically at
residues 192 and 194 and residue 198. ()These residues, as
are all residues in the P-loop, are completely conserved between
-subunits of all F-type ATPases in the SWISS-PROT data base. (
)Residues 192, 194, and 198 were postulated to interact
sterically with other regions of the ATPase and thus may be important
for the conformational changes observed in the catalytic sites during
the reaction cycle(19) . The steric interactions need not be
limited to the most stable structure of the enzyme, but may occur
during the reaction cycle, as in the transition state of the enzyme.
Analysis of the crystal structure of bovine F
indicates
that residues 192 and 194 do not interact with any residues outside the
P-loop, while residue 198 may interact with the adenine ring of the
bound nucleotide(3) .
Table 1shows growth phenotypes
of yeast with various replacements at positions 192, 194, and 198 in
the -subunit of the ATPase. The results described in Table 1suggest that the size of the amino acid side chain is an
important determining factor in the function of the enzyme.
Particularly, position 198 appears to require a side chain that is
about the size of Val. If the side chain is too small, such as Ala or
Ser, or too large, such as Met or Lys, the enzyme is defective, and
with Gly at this position, the enzyme is inactive. The crystal
structure of bovine F
indicates that the side chain of
Val-198 is 3.8 Å from the adenine ring of the nucleotide. Thus,
Val-198 may serve to position the nucleotide or to stabilize the
binding of the nucleotide. In comparison, Tyr-378 is also 3.8 Å
from the adenine ring and serves as a major determinant in stabilizing
nucleotide binding(33) . As such, V198S would have a diminished
ability to act in this manner, and suppression of V198S would need to
compensate for this loss.
The requirements at positions 192 and 194 are not so apparent. However, a large number of replacements at these positions give dysfunctional phenotypes (temperature or cold sensitivity or slow growth), suggesting that the residues are not involved directly in the catalytic mechanism. Instead, these residues may serve structural roles or roles in the conformational coupling of the enzyme.
The dysfunctional mutants listed in Table 1were used to isolate intragenic suppressors. Because suppressors may be allele-specific, attempts were made to isolate suppressors from each of the mutants listed as dysfunctional in Table 1. Of these mutants, only A192V, V194Y, V194M, and V198S had strong enough phenotypes to isolate suppressors and provided a reversion or suppressor rate that allowed their isolation. Intragenic suppressors were isolated by a combination of methods as described under ``Materials and Methods'' and as summarized in Table 2. The number and type of intragenic suppressors isolated were very limited. For mutations V194Y and V194M, only suppressors T237I and P353L were isolated, respectively. For V198S, L390F was the only suppressor mutation identified, whereas for A192V, only revertants were isolated. This low number of intragenic suppressors may be due to limitations imposed by the genetic code or due to a severe limitation on the number of replacements that will suppress these mutations.
Fig. 1shows the growth phenotypes of the original mutants, the mutants with a suppressor mutation, and the suppressor mutations in a wild-type background. Despite the fact that the suppressors were isolated at one temperature, all of the suppressor mutations complemented the defective growth phenotype at 18 and 30 °C, while only T237I was effective at 37 °C. These data indicate that the suppressor mutations do not generally provide an enzyme with completely wild-type properties.
Figure 1: Growth phenotypes of the mutants and suppressors. A shows the key to the plates shown in B-D. Cells were diluted in water, placed on YPG medium, and incubated at 18 °C (B), 30 °C(C), and 37 °C (D). The first row shows the cells with the wild-type (wt) and null mutations in ATP2. For the second through fourth rows, the first columns show the phenotypes of the initial mutants, the second columns show the phenotypes of the initial mutant with the suppressor mutation, and the third rows show the phenotypes of the suppressor mutation in a wild-type background. The fifth rows show the growth phenotypes of L390A and L390G in an otherwise wild-type background. One-letter amino acid abbreviations are used to indicate the amino acid encoded by the codon.
If
the amino acid residue corresponding to the suppressor forms a critical
interaction with another residue, it would be expected that mutagenesis
of this residue in an otherwise wild-type background would give a
defective phenotype. This was tested by separating the suppressor
mutation from the original P-loop mutation (Fig. 1). Suppressor
L390F was defective at all temperatures, indicating that Leu-390 is an
important residue in the F-ATPase. In contrast, T237I and
P353L showed only moderate effects on the growth phenotypes, indicating
the replacements modify, but do not eliminate, the enzyme activity.
Another indicator of the critical importance of Leu-390 was demonstrated by site-directed mutagenesis of Leu-390 to Ala and Gly. Fig. 1shows that strains with L390A or L390G have a negative growth phenotype on glycerol medium. These results indicate that Leu-390 makes important contacts with at least one other residue. Identification of the P-loop suppressor mutant L390F indicates that this interaction can be transmitted to the P-loop in the active site of the enzyme.
The allelic specificity of suppressor L390F was tested since this specificity should provide some information on the mechanism of the suppressor mutation. Specifically, the suppressor should show allelic specificity if it acts by reversing the effect of the primary mutation. This is in contrast to a mechanism whereby a suppressor improves the overall state of the enzyme, thereby overcoming the dysfunctional defect. Fig. 2shows the growth phenotypes of the L390F mutation in the presence of 10 different residues at position 198. The results indicate that the suppressor mutation is allele-specific. At 30 °C, a nearly wild-type growth phenotype for L390F was observed with Cys, Lys, Met, or Thr at position 198. However, at all three temperatures, L390F suppressed only Cys or Thr at position 198. Gly-198 was nonfunctional with either Leu or Phe at position 390. These data support the previous conclusion that Val-198 makes critical steric interactions with another residue or with the bound nucleotide.
Figure 2: Allelic specificity of the Phe-390 suppressor. A shows the key to the plates shown in B-D. Cells were grown as described for Fig. 1. The first row 1 shows the cells with the wild-type (wt) and null mutations in ATP2. The second through fourth rows show the phenotype of the initial mutant, V198S, in an otherwise wild-type background, followed by cells with mutations at position 198 with the L390F suppressor mutation.
The hydrogen-bonding pattern of
Asp-348 with Arg-370 and of Arg-370 with Ala-192 is not present in the
conformation (Fig. 3B). In the
and
conformations, Arg-370 is 3.0
Å from the carbonyl oxygen of Ala-192, while this distance
increases to 4 Å in the
conformation. This
shift of 1 Å is sufficient to break the hydrogen bond between
Arg-370 and Ala-192. More dramatically, Asp-315 is 12.6 Å from
Arg-370 in the
conformation, while they hydrogen-bond
in the
conformation. Thus, these interactions are
specific for the conformations with nucleotide bound to the active site
and may be important for stabilizing the
and
conformations. This hypothesis is supported by the
fact that Pro-353 is in a catch region that interacts with the
-subunit in the
conformation (3) .
Furthermore, Pro-353, Arg-370, and Asp-348 are conserved in all 56
sequences of F-type ATPases in the SWISS-PROT data base,
suggesting that these residues are critical.
Mutagenesis of the residue that corresponds to Asp-348 in E. coli to Val (D301V) resulted in an enzyme that was defective in assembly of the ATPase complex(34) . Therefore, this residue is important, minimally, for forming a stable structure of the ATPase. There have been no reports on mutagenesis of residues corresponding to Pro-353 or Arg-370, but it is of interest to determine their roles in the structure and function of the ATPase. The identification of a suppressor mutation in the catch region that suppresses a mutation in the P-loop demonstrates genetically that the two regions are conformationally coupled. Further studies are required to determine conclusively if Pro-353, Arg-370, and Asp-348 are critical in the conformational coupling cascade during the catalytic cycle.
Lys-209 is not a well conserved
residue, but the size of the residue appears to be important (cf.Fig. 6). In E. coli, for example, the
corresponding residue is Ile. This indicates that the charged group is
not critical, but the steric interactions may be important. Replacement
of Lys-209 with Val in yeast results in an enzyme that has a 3-fold
increase in the K for ATP and GTP and a 3-fold
decrease in the k
for ATP binding. However, there
was no change in the V
for ATP hydrolysis as
compared with the wild-type enzyme. (
)This single change to
a residue that is similar to that found in the E. coli enzyme
significantly modified the kinetics of the yeast enzyme. Possibly, Ile
at this position in the E. coli enzyme can account for some of
the biochemical differences observed between the E. coli and
mitochondrial enzymes(35, 36, 37) .
Figure 6:
Corresponding residues from a number of
species and enzymes (see Footnote 3) that are proposed to interact. The
residues that correspond to Lys-209 and Thr-237 are shown in A, and residues that correspond to Ala-181, Leu-390, and
Ser-161 are shown in B. Sequences were aligned using MaxHom (28) and the SWISS-PROT data base. The primary sequences of the
-subunit of the ATPase were ordered into mitochondrial, bacterial,
blue-green algae (BG), and chloroplast sequences, as shown.
There were only partial sequence data available for sequence 27, and
the space indicates that the sequence is not known. The dot in sequence 34 (A) indicates that the MaxHom
program did not align this residue with the data base
sequences.
The interaction of Thr-237 with Lys-209 is proposed to be important for modulating the activity of the enzyme. The importance is supported by the following data. First, T237I suppresses a P-loop mutation and thus must be able to alter the conformation of the active site. Since the P-loop forms multiple hydrogen bonds with the nucleotide, changes in the conformation of the P-loop have the potential of altering the hydrogen-bonding network. Second, T237I in an otherwise wild-type background is defective at 18 and 37 °C, but not at 30 °C as compared with the wild-type strain (Fig. 2). This indicates that the T237I interaction in the wild-type background modifies, but does not eliminate, the activity of the enzyme. Finally, mutagenesis of Lys-209 to Val has significant effects on the biochemistry of the ATPase. The variations of the residues at these two positions are proposed to modify the activity of the enzyme for the needs of the organism.
Fig. 6indicates that residues corresponding to Ala-181 and Leu-390 in other species or enzymes may, in part, define biochemical differences between these enzymes. The putative interacting residues can be placed into two groups: the mitochondrial or bacterial and the blue-green algae or chloroplast ATPases. The exact identity of the corresponding residues is variable, but they are generally limited to hydrophobic interactions, such as Ile with Ala, Ala with Ile, or Gln with Met. For blue-green algae or chloroplast ATPases, the pair is limited to Arg (or Lys in one case) and Met. It is not clear how an Arg/Met pair could fit in the same space as the Ala/Ile pair in the structure of the bovine enzyme. However, Asp or Glu at position 161 is always coincident with Arg at position 181, and the carboxylate may form a salt bridge with the guanidinium group of Arg. These differences in primary sequence may provide unique properties to the chloroplast and blue-green algae enzymes(38, 39) . Mutagenic and biochemical studies on these residues in the yeast or bacterial enzyme should provide insight into the function of this putative salt bridge.
Although suppressor
mutations of mutants in the P-loop of the E. coli ATPase have
been reported(40, 41) , there are a number of
important differences compared with this study. In the E. coli study, the initial mutations were in Gly-149 (Gly-190 in yeast).
In the bovine enzyme, this residue has very unusual dihedral angles
that only allow Gly at this position(3) . Assuming that the
geometry of the P-loop is the same for the E. coli enzyme,
then the geometry of the P-loop must be altered in the mutations at
this residue. Suppressors of these mutations may be limited to those
that can have a broad effect on the conformation of the P-loop.
Furthermore, the primary structural constraints of the E. coli P-loop are different from those of yeast. Although there has not
been an extensive examination of the P-loop constraints in E.
coli, Ser can replace Gly-149, and mutant F has nearly
normal activity(35, 36) . This is in contrast to the
yeast enzyme, where the corresponding mutation, G190S, severely impairs
F
activity(19) . A third important difference is
that the primary sequence of the
-subunit from E. coli is
more divergent from bovine than is yeast. The biochemical differences
between the E. coli and mitochondrial enzymes must certainly
be defined by the differences in their primary structures. However,
primary structural differences may also change possible replacements
that could suppress any given mutation, such as mutations in the P-loop
mutations. For these reasons, the suppressors identified in the E.
coli enzyme need not correspond to those identified in yeast.
The suppressors of mutations in the P-loop in the E. coli studies were G172D, S174F, D192V, and V198A. These residues are
located at the beginning of -sheet 4, in
-sheet 4, in helix
C, and at the end of helix C, respectively. Interestingly, these
suppressors are clustered and are in the region of the yeast P-loop
suppressor T237I. The clustering of these suppressor mutations provides
additional evidence that helix C and
-sheet 4 affect the
conformation of the P-loop and thus the biochemistry of the enzyme.
Although some residues may diverge because their importance is
relatively minor in defining the biochemistry of an enzyme, there must
be other residues that diverge due to the different requirements of the
organism or organelle. Suppressor studies may be an important tool for
the identification of residues that are important in determining the
biochemistry of the enzyme. The suppressors identified in this study
all originated from mutations that were in or near the P-loop of the
mitochondrial ATPase. As such, they all are able to correct a defect
located in this critical motif. Since two of the putative interacting
pair of residues are not strictly conserved between species and
enzymes, Thr-237/Lys-209 and Ala-181/Leu-390, it is suggested that this
divergence can be responsible, in part, for their biochemical
differences. Of course, there are five different subunits that compose
the F ATPase, each with divergent residues. Any, or many,
of these differences may contribute to the biochemical differences
observed between enzymes. However, certainly not all of the residues
that are divergent are important for modulating the kinetics of the
enzyme, as is postulated for the interacting pairs Thr-237/Lys-209 and
Ala-181/Leu-390.
The P-loop undergoes dramatic conformational
changes in the or
to the
conformation ( (3) and Fig. 3Fig. 4Fig. 5) and possibly in the transition
from the high to the low affinity site in the catalytic mechanism. The
understanding of the conformational coupling pathway from the proton,
to F
, to F
, to the catalytic site requires
molecular details of essential residues that interact and trigger this
transition. Suppressor studies may be an important tool to identify
interacting residues, such as Asp-348 and Arg-370, that are key
components in the conformational coupling pathway.