From the Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The multicopy subunit c of the
H+-transporting F1F0 ATP
synthase of Escherichia coli is thought to fold across the
membrane as a hairpin of two hydrophobic -helices. The conserved
Asp61, centered in the second transmembrane helix, is
essential for H+ transport. In this study, we have made
sequential Cys substitutions across both transmembrane helices and used
disulfide cross-link formation to determine the oligomeric arrangement
of the c subunits. Cross-link formation between single Cys
substitutions in helix 1 provided initial limitations on how the
subunits could be arranged. Double Cys substitutions at positions
14/16, 16/18, and 21/23 in helix 1 and 70/72 in helix 2 led to the
formation of cross-linked multimers upon oxidation. Double Cys
substitutions in helix 1 and helix 2, at residues 14/72, 21/65, and
20/66, respectively, also formed cross-linked multimers. These results
indicate that at least 10 and probably 12 subunits c interact in a
front-to-back fashion to form a ring-like arrangement in
F0. Helix 1 packs at the interior and helix 2 at the
periphery of the ring. The model indicates that the Asp61
carboxylate is centered between the helical faces of adjacent subunit c
at the center of a four-helix bundle.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
F1F0 ATP synthases catalyze the formation
of ATP utilizing the energy of a transmembrane H+
electrochemical gradient, generated by electron transport complexes and
other ion pumping systems. Closely related ATP synthases are found in
the plasma membrane of eubacteria, the inner membrane of mitochondria,
and the thylakoid membrane of chloroplasts. The enzyme is a
multisubunit complex with distinct extramembranous and transmembrane
domains, termed F1 and F0, respectively. Ion movement through F0 is coupled to ATP synthesis/hydrolysis
at sites in F1 (1, 2). The simplest F1 sectors,
as found in Escherichia coli, consist of five subunits in an
3
3
stoichiometry. Homologous
subunits are found in mitochondria and chloroplasts. A high resolution
structure of a substantial part of bovine F1 shows the
three
- and three
-subunits to alternate around a central core
through which subunit
extends and protrudes (3). The structure fits
well with the binding change mechanism proposed by Boyer and co-workers
(4), where each of the three
-subunits alternates between the loose
binding of ADP plus Pi, tight ADP plus Pi
binding and ATP synthesis, and ATP release during catalytic turnover.
Several recent studies now show that subunit
rotates within the
core of the hexagonally arranged
3
3
complex to presumably drive the binding changes in each
-subunit
(5-7). Both subunit
and
appear to rotate as a unit (8). The
mechanism of coupling H+ translocation through
F0 to
subunit rotation is unknown.
The E. coli F0 is the simplest type found in
nature. It consists of three subunits with a stoichiometry of
a1b2c10 + 1 (9). Electron
microscopic studies suggest that the a and b subunits pack at the
periphery of a complex of subunit c (10). Subunit b, with a single
transmembrane helix and larger cytoplasmic domain, is proposed to
associate with subunit to make up a stator that binds and fixes the
F1
3
3 catalytic
head group to F0 (11, 12). Subunit a is thought to fold
through the membrane with five or six transmembrane helices and play a
key role in the H+ transport (2, 13). Structural and
genetic studies indicate that subunit c folds in the membrane as a
hairpin of two hydrophobic
-helices connected by a polar loop on the
F1 binding side of the membrane (2, 14). A conserved Asp or
Glu (Asp61 in E. coli) centered in the second
transmembrane helix is essential for H+ transport (2, 15).
The most compelling evidence for a direct role of the side chain
carboxyl in cation binding has come from work on the related
Na+-translocating enzyme of Propiogenium
modestum (16, 17) and on a modified E. coli enzyme that
binds Li+ (18). The coupling of proton movements to binding
changes in F1 appears to occur by an interaction between
the loop region of subunit c with subunits
and
(15, 19, 20).
Several models have been proposed whereby ATP synthesis in the
F1 domain is coupled to proton movements through
F0 via movements of subunit c relative to the multicopy
subunit a (21-23).
Information on the structural organization of F0 subunits is essential if we are to understand how H+ transport is coupled to ATP synthesis. In this study, Cys was substituted into subunit c in order to determine the arrangement of subunits by disulfide cross-linking. The cross-linking data support the structural model of monomeric subunit c as determined by NMR (24).1 A ringlike arrangement of 12 subunits c with helix 1 in the center and helix 2 at the periphery is indicated. The subunits interact with the front face of one subunit packed against the back face of the next subunit with the Asp61 carboxylate centered within a four-helix bundle between adjacent subunits c. The model provides insights and limitations into how H+ translocation can be coupled to the rotation and synthesis of ATP within F1.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Oligonucleotide-directed Mutagenesis and Plasmid Construction-- The plasmids used in this study are derivatives of plasmid pDF163, which contains the wild type uncBEFH genes (bases 870-3216)2 (25). Plasmid pNOC, a derivative of plasmid pDF163 with a C21S substitution in subunit b, was used as a template for the introduction of all the Cys substitutions described. All three of the F0 subunits coded by plasmid pNOC lack Cys. Plasmid pNOC was constructed by a rapid site-directed mutagenesis procedure (26). An antisense oligonucleotide, 5'-GGCGGCCATACGTACTTCATGGAGAACAG-3', corresponding to positions Leu19-Pro27 of subunit b was synthesized to incorporate a single base change (underlined) to create a Ser codon at position 21 and to overlap the nearby SnaB1 restriction enzyme site (italics). The polymerase chain reaction (PCR)3 was then performed using this primer and a sense oligonucleotide primer designed to the coding strand (bases 1540-1560), upstream of the PstI restriction enzyme site (1561-1566), using plasmid pDF163 DNA as the template. The PCR product was then digested with PstI and SnaB1 restriction enzymes and ligated to the equivalent sites of plasmid pDF163 to generate plasmid pNOC. The substitution was verified by sequencing the entire fragment.
PCR mutagenesis procedures were used to generate other Cys substitutions. Where possible a one-step PCR strategy was employed by taking advantage of restriction enzyme sites in the vicinity of the desired substitution. The majority of helix 1 substitutions were constructed in this fashion using BsrG1 (bases 1911-1916, overlapping amino acid positions 9-11) and AvaI (bases 1976-1981, overlapping amino acid positions 31 and 32) sites. Briefly, if the substitution was within 10 amino acid residues of the restriction site, a primer was designed so as to incorporate the substitution and the restriction site. Residues 20-30 were substituted using antisense primers incorporating the Cys codon and AvaI site. Amplification was carried out using a sense primer 5' to the PstI site (bases 1561-1566). The PCR product was then digested with PstI and AvaI and ligated into the respective sites of plasmic pNOC. Primers designed for substitution of residues 12-17 incorporated the BsrG1 site. Amplification was achieved with an antisense primer to bases 2172-2189, 3' to the nearby HpaI site (bases 2162-2167). This enabled cloning of the product into the BsrG1 and HpaI sites of plasmid pNOC. For the remaining substitutions and those in helix 2, a two-stage PCR mutagenesis procedure was used (26). This procedure requires a specific mutagenic primer and two wild type primers, 5' and 3' to the region of interest. In this case, the sense primer 5' to the region was designed to bases 1540-1560 so that the PstI site was incorporated into the PCR product. The antisense primer 3' to the region was designed to bases 2303-2319, so that the HpaI and SnaB1 (bases 2256-2262) sites were incorporated into the PCR product. The first PCR step involves use of the mutagenic primer with one of the wild type primers. This first product then serves as a megaprimer for the second round of PCR with the second wild type primer. The product was then digested with PstI and HpaI or SnaB1 and ligated into the respective sites of plasmid pNOC. Double Cys substitutions were introduced in combination in helix 1 and helix 2 by subcloning. The PstI/AvaI fragment from a plasmid carrying a single Cys substitution in helix 1 was ligated into the respective sites of a pNOC plasmid derivative carrying a helix 2 Cys substitution. Correct subcloning was confirmed by DNA sequencing. To generate double Cys substitutions in helix 1, or double Cys substitutions in helix 2, the PCR mutagenesis procedures described above were employed using one of the single Cys mutant DNA as the template with a primer generated to create the second Cys substitution. All substitutions were verified by sequencing the entire subcloned fragment.Expression and Cross-link Analysis--
A chromosomal
uncBEFH deleted strain, JWP109 (pyrE41, entA403,
ArgH1, rspsL109, supE44,
uncBEFH),4 was
transformed with plasmid pNOC and its Cys-substituted derivatives. Complementation was tested by transferring transformant colonies to
minimal medium 63 plates (27) containing 22 mM succinate, 2 mg/liter thiamine, 0.2 mM uracil, 0.2 mM
L-arginine, 0.02 mM dihydroxybenzoic acid, and
100 mg/liter ampicillin. Control plates contained 0.2% glucose instead
of succinate.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of Single Cys Substitutions on Function--
Single Cys
substitutions were introduced at positions 8-31 of helix 1 and
positions 53-75 and 78 of helix 2 (Table
I). The Cys substitutions coded in
plasmid pNOC, which carries the uncBEFH genes encoding
subunits a, c, b, and of F1F0 respectively,
were transformed into the
uncBEFH strain JWP109. Growth
of transformants was tested on succinate minimal medium, where growth
depends on a functional oxidative phosphorylation system. Most of the
substitutions promoted growth, although the extent varied considerably
(Table I). Notably, substitution of conserved Gly and Ala residues in helix 1 and of Asp61 and Pro64 in helix 2 resulted in lack of growth on succinate.
|
Disulfide Cross-link Formation by Single Cys Substitutions-- Membrane vesicles were treated with the oxidant CuP and analyzed by gel electrophoresis and immunoblotting. Residues within the N-terminal region (positions 8-11) and C-terminal region (positions 73-75 and 78) formed high yield, disulfide cross-linked homodimers in the presence of oxidant (Fig. 1 and Fig. 2). The F53C protein also formed a high yield cross-linked homodimer. Significant spontaneous homodimer formation occurred at positions L8C, L9C, and V78C. These positions are predicted to be close to where the helices emerge from the hydrophobic core of the membrane, where they may be more susceptible to spontaneous oxidation. The consecutive stretch of cross-links from residues 8-12 and 73-75 are consistent with these regions being very flexible, with a greater susceptibility to thermal collision, perhaps because they lie at the N and C termini of the protein.
|
|
|
Cross-link Formation between Helix 1 Double Cys Substitutions-- Double Cys substitutions were introduced into helix 1 to see if cross-linked multimers of subunit c could form (Table II). As discussed above, single Cys substitutions at positions 15, 26, and 30 result in formation of cross-linked homodimers in high yield, and these residues fall on the same face of helix 1 in the NMR structure. We reasoned that Cys substitution at two of these positions should provide information on (i) whether homodimer formation in the single Cys substitutions was due to two helices 1 coming together face to face along this surface (Fig. 3B) or (ii) whether these faces of the two helices neighbor each other as would be the case in a ring type arrangement (Fig. 3C). If the helices are arranged face-to-face (Fig. 3B), then only homodimer formation would occur, whereas in a ring type arrangement cross-linked multimers, up to the number of subunits c in the F0, should be detected. All combinations of double Cys substitutions at positions 15, 26, and 30 led to an extensive ladder of subunit c cross-linked multimers (Fig. 4; Table II).
|
|
|
Cross-link Formation between Helix 2 Double Cys
Substitutions--
The Cys substitutions in helix 1 are consistent
with subunit c being arranged in a ring, implying that a similar
arrangement must exist for helix 2. A variety of double Cys
substitutions were generated in helix 2, falling at different offsets
around the helix (Table II; Fig. 3D). Double Cys
substitutions between positions 67-72 were of particular significance,
since this region was not subject to high yield dimer homodimer
formation in the respective single Cys substitutions. Further, the
region starting at Met65 appears to be of a continuous and
regular -helix in the NMR structure (Fig. 3D). Of the
eight mutants constructed, L70C/L72C formed multimers upon oxidation
(Fig. 6). The propensity of L70C/L72C to
form a cross-linked multimer can satisfy a number of arrangements of
helix 2 relative to helix 1. However, an oligomeric arrangement of
subunit c in a ring with the interacting faces of helix 1 and helix 2 packed as in the NMR model and with helix 1 on the inside and helix 2 on the outside fits well with the cross-linking data (Fig.
3E). This places the critical Asp61 toward the
interior, positioned within a four-helix bundle.
|
Cross-link Formation between Helix 1 and Helix 2 Double Cys Substitutions-- Cysteines were introduced into both helix 1 and helix 2 to determine their orientation with respect to each other (Table III). We reasoned that a Cys on helix 1 of one subunit c should be able to form a "diagonal" cross-link with a Cys on helix 2 of a neighboring subunit c in the oligomeric ring structure and thus elicit the formation of cross-linked multimers.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Singly and doubly Cys-substituted mutants of subunit c were generated here to determine by disulfide cross-link formation the arrangement of subunits in F0. The results confirm that subunit c is folded in a hairpin-like structure with two transmembrane helices. Double Cys substitutions that formed cross-linked multimers on oxidation but showed little or no homodimer formation as single Cys substitutions provide the most compelling evidence for a ringlike arrangement of subunits, as shown in Fig. 3E. The defining double Cys substitutions include the A14C/M16C, M16C/G18C, and G18C/L19C pairs in helix 1 and the L70C/L72C pair in helix 2. Multimers formed by "diagonal" cross-linking between helix 1 and helix 2 define the orientation of helices with respect to each other and provide further evidence for a ringlike arrangement. The key double Cys substitutions that fall into this category are A14C/L72C and A21C/M65C. The cross-linking data fit remarkably well with the proposed NMR model of monomeric subunit c (24).1 In fact, the NMR model was used as a basis for predicting residues that were likely to cross-link in the experiments described above.
The formation of cross-linked homodimers from single Cys substitutions in helix 2 conflict with the model shown in Fig. 3E. Notably, the stretch of cross-linking is centered around Asp61. Cross-link formation may reflect structural flexibility in this region that is necessary to function, possibly a swiveling of helix 2 relative to helix 1 in events related to proton transport. As mentioned under "Results," it also seems possible that Cys replacement of Gly or Ala residues, which are normally packed at the interface between helices in the monomer, may result in packing of the larger side chain at either side of the interface. If there is some breathing in the packing of helices, the position of the sulfhydryl may change from side-to-side by a dynamic equilibrium. Cys side chains packed on opposite sides of the interface would obviously be susceptible to disulfide bond formation. This could account for the cross-linked homodimers seen in the G58C and A62C mutants. We have also carried out cross-linking studies in the presence of aminoxid WS35, a detergent that was previously used to solubilize and reconstitute intact F1F0 (33). The addition of detergent resulted in high yield homodimer formation with all mutant membranes exhibiting even minor cross-linking with oxidant alone and also cross-linking with membranes in some mutants where cross-linking was not observed in the absence of detergent. The structure of solubilized F0 must be somewhat different, i.e. more flexible than the structure in the lipid bilayer.
The high yield, cross-linked homodimers that form in the single Cys
substitutions of helix 1, V15C, I26C, and I30C, fall on one face of an
-helix in the NMR structure, i.e. on the face that lines
the inner core of the ring shown in Fig. 3E. The
predominance of Gly and Ala in residues to either side of this face
reduces the cross-sectional diameter throughout helix 1. For this
reason, we depict helix 1 as being smaller in diameter than helix 2 in the schematic model (Fig. 3E). In a ring type arrangement,
this is expected from from geometric packing constraints. The side chains of the face protruding into the central core are in close proximity with those in the neighboring helix 1, and preliminary modeling indicates C
-C
distances of
9-10 Å, i.e. short enough to allow disulfide cross-link formation with only minor movements of helices. The opposing face of
helix 2, i.e. the one positioned to the outside of the ring, is also hydrophobic with multiple branched chains. Residues in these
two opposing faces are hydrophobic but not highly conserved (32), which
is consistent with both surfaces being exposed to the lipid milieu of
the membrane. In other membrane proteins, the surfaces involved in
protein-protein contact show the greatest sequence conservation (34).
Met11, Val15, and Leu19 on the
interior helical face are selectively labeled by
3-(trifluoromethyl)-3-(iodophenyl)diazirine, in addition to other
selectively labeled residues elsewhere in the protein, which led to the
suggestion that these residues were on one face of an
-helix that
was exposed to the fatty acyl phase of the lipid bilayer (35). In the
ring of subunit c suggested here, the diameter of the space in the
central core is
25 Å, and it seems likely that lipid molecules will
be present and account for the
3-(trifluoromethyl)-3-(iodophenyl)diazirine accessibility. Similar
situations with centrally located lipid are seen with the
bacteriorhodopsin trimer of the purple membrane (36) and presumed for
the homooligomeric ring of the light-harvesting complex LH2 (37). The
arrangement proposed here correlates well with recent scanning force
microscopy studies, where F0 appears as a ringlike
structure surrounding a central dimple when viewed from the cytoplasmic
side (38, 39).
A variety of experiments support the idea that Asp61 in
E. coli and equivalent residues in other species bind and
release protons in the transport step coupled to ATP synthesis (15,
40). The essential Asp61 in helix 2 of the E. coli protein can be moved to position 24 in helix 1 with retention
of function (41). The NMR model of monomeric subunit c shows
Ala24 of helix 1 in close proximity to Asp61 of
helix 2, but the side chains point to opposite surfaces of the packed
helices.1 How then is function retained in the Asp
interchange mutant? In the model depicted here, the Ala24
and Asp61 side chains are positioned within the center of a
four-helix bundle formed by the front and back face of two adjacent
monomers (Fig. 3E). The interchange of the essential
carboxyl from one position to another can be rationalized in such an
arrangement. The model can also be used to rationalize the position of
essential liganding residues in subunit c of the
Na+-translocating F1F0 ATPases and
a Li+ binding variant of the E. coli
F1F0 ATPase. Kaim et al. (17) have
shown that residues Gln32, Glu65, and
Ser66 are essential for Na+ binding in the
P. modestum enzyme, i.e. residues at positions equivalent to residues 28, 61, and 62 in E. coli. The
conserved residues in P. modestum (Gln, Glu, and Thr) have
been identified at equivalent positions within the transmembrane
helices of subunit c in the
Na+-F1F0 ATPase of
Acetobacterium woodii (42). In both enzymes, Pro is found at
a position equivalent to Ala24 in E. coli. In
the model depicted here, each of these side chains points toward the
center of the four-helix bundle formed by neighboring subunits. The
model also explains why VD61AI AE61S(G/A)
substitutions in E. coli subunit c enables Li+
binding (18); i.e. the Ser62 hydroxyl can serve
as a liganded group opposite the Glu61 carboxyl.
The F1F0 ATP synthases share many structural
similarities with a family of so called vacuolar or
V1V0 ATPases. The V1V0
ATPases function as primary proton pumps to acidify intracellular
organelles and extracellular spaces (43, 44). The subunit c of
V1V0 H+-ATPases is approximately
twice the size of its counterpart in the F1F0
ATPases and appears to have evolved by duplication of a progenitor gene
(45). Four transmembrane helices are predicted. Helices 1 and 3 and
helices 2 and 4 of this larger subunit c show homology with helices 1 and 2 of the subunit c of the F1F0 ATP synthases, respectively. Helices 1 and 3 of V-type subunit c show a
striking enrichment and sequence conservation of Gly and Ala residues,
similar to that seen in F-type subunit c. A model has been proposed for
this multicopy subunit based on electron microscopy image analysis and
chemical modification (46-48). In the model, six subunits c, each
folding as a bundle of four -helices, come together to form a
hexameric complex with a central pore. The model does differ in
critical respects from the one presented here. However, the prediction
that helix 1 lines the pore of the oligomer (48), based upon Cys
substitution analysis and chemical modification, is borne out in
E. coli subunit c model. It seems likely that the
arrangement of the V-type subunit c will be like that proposed here,
with helices 1 and 3 lining the inner ring and helices 2 and 4 lining
the outer. The loss of the conserved carboxylate in the second helix of
the V-type subunit c would effectively halve the number of proton
binding sites. This loss would lower the H+/ATP ratio,
allowing ATP-driven H+ pumping to generate greater
electrochemical gradients while preserving the overall structural
features of the complex (49). Based on the precedent with the E. coli subunit c (41), attempts have been made to generate a
functional enzyme after exchange of the essential carboxyl from helix 4 to helix 3, but they have
failed.5 The model shown here
suggests that an interchange between helix 4 and helix 1 is more likely
to work, with the proton binding site at the center of a four-helix
bundle and helix 4 packing close to helix 1. Finally, the model would
predict that in the Na+-translocating
V1V0 ATPase of Enterococcus hirae
(50), the Na+-liganding groups should fall between helix 4 and helix 1. The Na+-liganding residue Gln32 in
P. modestum appears to be replaced by Ser30 in a
conserved Gly- and Ala-rich sequence (32).
The arrangement of subunit c presented here provides insights and
limitations on how proton movement can be coupled to ATP synthesis/hydrolysis. The coupling takes place by rotation of the
-subunit within the core of the
3
3
subunit (5-7). This has led to proposals that the oligomeric ring of
subunit c rotates past a static, peripherally located subunit a in the
membrane sector (5, 22, 23, 51). In the models, the
proton-translocating carboxyl is located on the outside of the ring,
and upon protonation it is envisioned as moving into the hydrocarbon
region of the lipid bilayer. In the ring arrangement shown here, the
proton or ion would be coordinated in the center of the four-helix
bundle rather than being exposed directly to the lipid milieu. Subunit a would in some way be expected to cause an opening and closing of the
four-helix bundle with resultant ion uptake and release. We should note
that it is still unproven that the subunit c oligomer rotates in the
membrane. Alternatively, others have suggested that the membrane sector
may remain fixed relative to the
and
subunits in F1
(15, 48). In such a model, only subunit
and the associated subunit
are predicted to move at the polar loop surface of subunit c.
Conformational changes would be relayed from the site of ion
binding/release to the polar loop of subunit c, and conformational
changes in the polar loop then drive movement of the
complex
from loop to loop in a circular fashion. The key question obviously
remaining is whether rotations take place within the membrane
sector.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM23105 and a grant from the Human Frontiers Science Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This paper is dedicated to Dr. E. Brown.
To whom correspondence should be addressed: Dept. of Biomolecular
Chemistry, 587 Medical Sciences Bldg., University of Wisconsin, Madison, WI 53706. Tel.: 608-262-1439; Fax: 608-262-5253.
1 Girvin, M. E., Rastogi, V. K., Abildgaard, F., Markley, J. L., and Fillingame, R. H. (1998) Biochemistry, in press.
2 The unc DNA numbering system corresponds to that used by Walker et al. (52).
3 The abbreviations used are: PCR, polymerase chain reaction; CuP, Cu(II)-(1,10-phenanthroline)2; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
4 Jiang, W. P., and Fillingame, R. H. (1998) Proc. Natl. Acad. Sci. U. S. A., in press.
5 N. Nelson, personal communication.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|