Originally published In Press as doi:10.1074/jbc.C200168200 on April 9, 2002
J. Biol. Chem., Vol. 277, Issue 23, 20117-20119, June 7, 2002
ACCELERATED PUBLICATION
Substitution of a Single Amino Acid Switches the
Tentoxin-resistant Thermophilic F1-ATPase into a
Tentoxin-sensitive Enzyme*
Georg
Groth
,
Toru
Hisabori§¶,
Holger
Lill
, and
Dirk
Bald**
From the Department of Plant Biochemistry,
Heinrich-Heine Universitat, D-40225 Dusseldorf, Germany, the
§ Chemical Resources Laboratory, Tokyo Institute of
Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan, the
¶ Yoshida ATP System Project, Exploratory Research for Advanced
Technology (ERATO), Japan Science and Technology Corporation (JST),
5800-3 Nagatsuta-cho, Midori-ku, Yokohama 226-0026, Japan, and the
Department of Structural Biology, Faculty of Earth and Life
Science, Vrije Universiteit Amsterdam, De Boelelaan 1087, 1081 HV
Amsterdam, The Netherlands
Received for publication, March 20, 2002, and in revised form, April 2, 2002
 |
ABSTRACT |
In contrast to the homologous bacterial and
mitochondrial enzymes the chloroplast F1-ATPase
(CF1) is strongly affected by the phytopathogenic inhibitor
tentoxin. Based on structural information obtained from crystals of a
CF1-tentoxin co-complex (Groth, G. (2002) Proc. Natl.
Acad. Sci. U. S. A. 99, 3464-3468) we have replaced residues
Ser66 and 
Arg132 in the
33
subcomplex of the thermophilic
F1-ATPase from Bacillus PS3 by the
corresponding residues of the chloroplast ATPase to confer tentoxin
sensitivity to the thermophilic enzyme. The mutation Arg132 
Pro, proposed to relieve steric
constraints on tentoxin binding, did not have any significant effect.
However, mutation Ser66 Ala, predicted to provide a
crucial hydrogen bond with the inhibitor, resulted in tentoxin
inhibition of ATP hydrolysis comparable with the situation found with
the chloroplast enzyme.
| |
INTRODUCTION |
Tentoxin is a cyclic tetrapeptide derived from phytopathogenic
fungi of the Alternaria species, causing chlorosis in
sensitive plant species. It acts as an inhibitor of the chloroplast
F0F1-ATP synthase from these species, but not
of the homologous enzymes from other bacteria and animals (1-4). In
membrane-bound F0F1-ATP synthase, both ATP
synthesis and ATP hydrolysis are inhibited by tentoxin (1), with the
soluble F1 subcomplex, which is not capable of ATP
synthesis; ATP hydrolysis is inhibited (2). Although binding studies
suggested an uncompetetive manner of inhibition by interference with
cooperative release of nucleotides from the enzyme (2, 5), the precise
mechanism of tentoxin is not known. Based on labeling studies, one high
affinity inhibitory binding site and additionally one to two low
affinity binding sites have been proposed (6, 7). Binding of tentoxin
to low affinity sites relieves inhibition caused by binding to the high
affinity site (6, 7).
The F1 subcomplex of F0F1-ATP
synthase is also referred to as F1-ATPase and consists of
the subunits 33
. Its
33 subunits make up the smallest
entity capable of continuous ATP hydrolysis (8, 9). High resolution
structures of the 33 complex from
bovine heart mitochondria as well as of the
33 region from the thermophilic
Bacillus PS3 and from spinach chloroplast revealed an
alternating, hexagonal arrangement of the three and three subunits (10-12). These subunits consist of three domains: N-terminal -barrels, a central nucleotide-binding domain, and a C-terminal bundle of -helices (10).
Recent results obtained by co-crystallization of spinach chloroplast
F1-ATPase
(CF1)1 and
tentoxin shed more light on the binding of the inhibitor (13) and
showed that tentoxin is bound at the -interface in a cleft near
the N-terminal -barrel domains. The structure of the
CF1-tentoxin complex suggests a critical role of residue
Asp83 for tentoxin binding and/or inhibition, which has
been concluded from mutagenesis experiments in the past (14, 15), but
it displayed at the same time structural differences in the vicinity of
Asp83 between CF1 and tentoxin-resistant
F1-ATPases, e.g. from Escherichia coli (EF1) or from the thermophilic
Bacillus PS3 (TF1).
Another critical region for tentoxin inhibition seems to be located in
the chloroplast ATPase subunit. Studies using chimeric 33 complexes that have been assembled
from subunits originating from the tentoxin-sensitive CF1
and from the insensitive Rhodospirillum rubrum
F1-ATPase (16, 17) indicated that the poorly conserved residues 120-133 might be crucial.
In this report we superimposed the structures of the
CF1-tentoxin complex (13) and the corresponding parts of
TF1 (11) to pinpoint crucial amino acid residues involved
in tentoxin binding. We predicted that Ser66 and
Arg132 of TF1 (corresponding to
Ala81 and Pro133 of CF1) play
a central role in conferring tentoxin resistance to TF1. To
test these predictions based on the static picture provided by the
crystal structure, we prepared two mutants of the
33 complex of the tentoxin-insensitive
TF1, where the two critical residues Ser66
and Arg132 were replaced by alanine and by proline,
respectively, as found in the corresponding position of
CF1. The results of this mutagenesis study show how
inhibition of ATP hydrolysis responds to subtle changes of the protein structure.
| |
EXPERIMENTAL PROCEDURES |
Chemicals--
Tentoxin was purchased from Sigma, a pyruvate
kinase/lactate dehydrogenase mixture was obtained from Roche Molecular
Biochemicals, and restriction enzymes were from New England
Biolabs. All other chemicals were of analytical grade.
Bacterial Strains--
Plasmid construction and amplification
was done using the strain E. coli JM109; for overexpression
of TF1, 33 E. coli JM103
uncB-D was used.
Plasmid Construction--
The pkkHC5 expression plasmid coding
for the , , and subunits of the thermophilic
Bacillus PS3 F1-ATPase, carrying a decahistidine tag at the N terminus of the subunit and a single cysteine in the
subunit (18) (the protein encoded is referred to as "wild-type" TF1 33 here) was digested
with the restriction enzymes PstI and EcoRV (for
the mutation Ser66 Ala) and EcoRI and
EcoRV (for the mutation Arg132 Pro). The
resulting fragments were then ligated into pBluescript SK vector
(Stratagene), previously cut with the same restriction enzymes.
Directed mutagenesis was done by the full-circle polymerase chain
reaction method with a PCR machine (Tpersonal, Biometra) according to
the suggestions from the QuikChange site-directed mutagenesis
kit (Stratagene). The primer used were
5'-ACAGTACGGACGATCGCCATGGCGGCCACAG ACGGCCTCATC-3' (forward) together with
5'-GATGAGGCGTCTGTGGCCGCCATGGCGATCGTCCGTACTGT-3' (backward) for the mutation Ser66 Ala and
5'-CGCGCCCGATTGAAAGCCCTGCCCCGGGCGTTATGGACC-3'
(forward) with
5'-CCGGTCCATAACGCCCGGGGCAGGGCTTTCAATCGGGCGCG-3'
(backward) for the mutation Arg132 Pro. The codons
carrying the mutation and one base each introducing an additional
NcoI (in case of Ser66 Ala) or
SmaI (in case of Arg132 Pro) recognition
site are underlined. Positive clones were identified by digestion with
SmaI or NcoI, respectively, cloned into the
equivalent position of the expression vector pkkHC5 and verified by DNA
sequencing (ABI Prism 310, PerkinElmer Life Sciences).
Overexpression and Protein Purification--
The expression
vector was transformed into E. coli JM103uncB-D and
cultivated as described in Refs. 9, 18, and 19. Cells were harvested,
disrupted by sonification, centrifuged, and the supernatant was
subjected to a heat shock for 20 min at 60 oC (9).
Proteins were purified with a nickel-nitrilotriacetic acid (Ni-NTA,
Quiagen) affinity column (18, 19) and stored as an ammonium sulfate
precipitate (70% saturation) at 4 °C.
ATP Hydrolysis Activity Measurement--
ATP hydrolysis activity
was determined using an ATP regenerating system (20) with an UV-VIS
spectrophotometer (Lambda 40, PerkinElmer Life Sciences). The
absorption at 340 nm of a reaction mixture containing 50 mM
MOPS/KOH, pH 7.0, 50 mM KCl, 4 mM
MgCl2, 2 mM phosphoenolpyruvate, 0.2 mM NADH, 2 mM ATP, 40 µg/ml pyruvate kinase,
40 µg/ml lactate dehydrogenase was measured for 1 min, then the ATP
hydrolysis reaction was started by addition of 2 µg of
33, and the absorption change was
observed for 8 min. Activities were calculated from the slope 2 min
after starting the ATP hydrolysis reaction using the extinction
coefficient of NADH at 340 nm of 6230 M
1
cm1.
| |
RESULTS AND DISCUSSION |
Superimposition of the CF1 and TF1
Structures at the Tentoxin Binding Site--
A superimposition of the
structure of the CF1-tentoxin complex (13) with the
structure of the 33 subcomplex of
TF1 (11), which is shown in Fig.
1, B and C,
revealed that the positions of residues Leu65,
Val75, and Leu238, which probably form
important hydrophobic contacts with the inhibitor, are essentially
conserved as well as the position of the crucial residue
Asp83 in the subunit (Fig. 1B). Calculation
of potential hydrogen bonds showed that the carboxyl side chain of
Asp83 is hydrogen-bonded to the amide hydrogens of
leucine 2 and glycine 4 in the tentoxin molecule, which aligns the
inhibitor in the binding cleft formed at the -interface (13). In
TF1 this critical interaction is probably impaired by a
potential hydrogen bond formed between Asp68
(Asp83 of CF1) and the hydroxyl group of
Ser66 (corresponding to Ala81 in
CF1), which prevents correct tentoxin binding (13). In
addition the superimposition of the two F1 structures
clearly visualizes the potential critical role of residue
Pro133 in tentoxin binding in CF1. In
TF1 this residue is replaced by arginine
(Arg132), whose bulky side chain seems to block access
to the tentoxin binding site (Fig. 1C).
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Fig. 1.
Structure of the tentoxin binding site.
A, location of the tentoxin binding site
(circle) near the N-terminal -barrel domain of one
- pair from spinach chloroplast F1-ATPase
(CF1) (12, 13). Subunit is colored in
yellow, and subunit is shown in green.
B and C, stereo images of the tentoxin binding
pocket of CF1 (13), complexed with one molecule tentoxin
(backbone and residue numbers in blue), superimposed with
the corresponding part of F1-ATPase from the thermophilic
Bacillus PS3 (TF1, backbone and residue numbers
in red) (11). The positions of the residues mutated in this
study, Ser66 (B) and Arg132
(C), are indicated.
|
|
Tentoxin Sensitivity of Mutant
33--
ATP hydrolysis activities of
the thermophilic 33 complexes carrying
the mutations Ser66 Ala
(33(S66A)) or Arg132
Pro (33(R132P)) were measured
with an ATP regenerating system and compared with wild-type
TF1 33 (Fig.
2). The specific activity in the absence
of tentoxin was 9-10 units/mg for all three enzymes, comparable with
values previously reported for wild-type TF1
33 (9, 18). These results indicate
that the mutations per se do not significantly influence ATP
hydrolysis activity. After preincubation with tentoxin, ATP hydrolysis
by TF1 33 (S66A)
declined remarkably (Fig. 2). Significant inhibition was observed using
>1 µM tentoxin; the concentration dependence of
inhibition was exceptionally steep when 5-20 µM tentoxin
were used. Half-maximal inhibition was obtained with 5-10
µM tentoxin, and maximal inhibition of 65-70% was
achieved with about 20 µM of the inhibitor. Increasing
the tentoxin concentration up to 100 µM did not enhance
the inhibition, and concentrations >100 µM led to a
slight re-activation of the enzyme.
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Fig. 2.
ATP hydrolysis activity of mutant
TF1 in the presence of tentoxin. ATP hydrolysis
activity was measured with an ATP regenerating system coupled to
oxidation of NADH. The samples were preincubated with the indicated
concentrations of tentoxin at room temperature for 1 h.
Closed circles, TF1
33 (S66A); open circles,
TF1 33 (R132P);
closed triangles, wild type. For details, see
"Experimental Procedures."
|
|
In contrast, only a slight decrease of activity, amounting to about
10% inhibition, was observed when 20-100 µM tentoxin
were added to wild-type TF1
33 or the mutant TF1
33 (R132P) (Fig. 2).
The degree of inhibition, about 70% determined for TF1
33 (S66A), is comparable with
values reported earlier for the Mg-ATPase activity of chloroplast
F1-ATPase (16, 17) and for chimeric mutants constructed by
reconstitution of mutated subunits derived from R. rubrum and and subunits derived from CF1 (16).
The KI value measured here was significantly lower
than the KI value of about 108
M published for CF1 (6, 7), but comparable with
values reported for the above-mentioned chimeric enzymes (16). A
re-activation of the enzyme as observed here in the presence of
tentoxin concentrations >100 µM was previously also
reported for CF1 and explained by the binding of a second
and possibly a third tentoxin molecule to the F1 complex,
which by an unknown mechanism may relieve inhibition.
The Role of Asp83 for the Binding of
Tentoxin--
Functional binding of tentoxin seems to depend
essentially on correct hydrogen bonding between the amide hydrogens
from the tentoxin backbone and residue Asp83 (13). In
the thermophilic F1 this important hydrogen bonding is
obviously affected by a potential hydrogen bond formed between Asp68 and the side chain of the adjacent residue
Ser66 (3.3 Å). A similar competition for intermolecular
(Asp-TTX) and intramolecular hydrogen bonding (66-68) is
avoided in the tentoxin-sensitive CF1 complex as the
chloroplast subunit contains alanine in the equivalent position of
the binding site (Ala81). For the same reason tentoxin
sensitivity can probably be achieved in the F1 complex from
Chlamydomonas reinhardii simply by the replacement of
Glu83 by aspartate (15), as a proline residue, which has
no capability to form hydrogen bonds, is located in the adjacent (n-2) position.
A steric effect on the binding of the inhibitor caused by the side
chain located in position 81 seems unlikely as alanine and serine show
about the same surface volume of 89 Å3. In addition the
even more bulky threonine (surface volume 116 Å3) or
proline side chain (surface volume 113 Å3) is found in the
equivalent position of the binding side in the tentoxin-sensitive
F1 complex from Syneccococcus PC6301 or in the
Chlamydomonas Glu83 Asp F1
mutant (15). Thus the structural requirement in the subunit for
effective tentoxin binding is apparently to avoid any intramolecular
hydrogen bonding with the crucial aspartate in position 83.
Steric Blockage of the Tentoxin Binding Site Caused by
Arg132--
Although the available structural
information (Fig. 1C; see also Ref. 13) strongly suggested
that in wild-type TF1 33 the bulky side chain Arg132 blocks access to the
tentoxin binding niche; its replacement by proline did not have a
significant effect on tentoxin sensitivity. Furthermore, the double
mutant TF1 33
(R132P/S66A) displayed the same tentoxin sensitivity as the
single mutant TF1 33
(S66A) (data not shown), indicating that steric hindrance by this
bulky side chain is not a predominant factor for tentoxin binding. The reason why the mutation failed to show the result expected from the
CF1 and TF1 structures might be related to a
substitution of Leu125 by proline in the native
TF1 complex (surface volume: Leu, 167 Å3; Pro,
112 Å3), which might compensate for the effect caused by
the more bulky arginine side chain in position 132. In addition, the
wild-type-like activity of the mutant might be explained by dynamic
movements of this part of the subunit during tentoxin binding,
which are not visible in a static protein structure, but might be
resolved by a set of intermediate structures (alternative
conformations) or by dynamic studies.
Requirements for Tentoxin Binding in F1--
The point
mutation Ser66 Ala is sufficient to achieve maximal
inhibition, underscoring the importance of the ability of the crucial
residue Asp68 (Asp83 in CF1)
to form hydrogen bonds with the tentoxin peptide backbone. The results
from our mutagenesis studies indicate that prerequisites for inhibition
by tentoxin are a tentoxin binding cleft, an aspartate side chain for
correct hydrogen binding, and the absence of other residues that might
interfere with this crucial hydrogen bond. The comparably high
KI value determined here for tentoxin inhibition
indicates that other amino acid residues, which probably are located in
the subunit, also influence the affinity for tentoxin. Experiments
to elucidate the role of these residues in fine-tuning the affinity for
tentoxin are presently under way in our laboratory.
The results presented in this paper demonstrate in a remarkable way the
feasibility of functional predictions based on structural information,
e.g. for the design of special characteristics in a target
protein. On the other hand, they also stress that, as it comes to
functional considerations, structural dynamics should be taken into account.
| |
ACKNOWLEDGEMENT |
We are indebted to Petra Voeller (Free
University Amsterdam) for excellent technical assistance.
| |
FOOTNOTES |
*
This work was supported by the Deutsche
Forschungsgemeinschaft (GR1616/4-1).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.
**
To whom correspondence should be addressed.
Published, JBC Papers in Press, April 9, 2002, DOI 10.1074/jbc.C200168200
| |
ABBREVIATIONS |
The abbreviations used are:
CF1, chloroplast F1-ATPase;
TF1, F1-ATPase obtained from thermophilic Bacillus
PS3;
33 (S66A) and
33(R132P), thermophilic
33 complexes carrying the mutations
Ser66 Ala or Arg132 Pro, respectively;
MOPS, 4-morpholinepropanesulfonic acid.
| |
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Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
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ABSTRACT
INTRODUCTION
