From the Department of Biochemistry and Biophysics, Göteborg University and Chalmers University of Technology, S-405 30, Göteborg, Sweden
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ABSTRACT |
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Conformational changes in proton pumping
transhydrogenases have been suggested to be dependent on binding of
NADP(H) and the redox state of this substrate. Based on a detailed
amino acid sequence analysis, it is argued that a classical
dinucleotide binding fold is responsible for binding
NADP(H). A model defining
A,
B,
B,
D, and
E of this
domain is presented. To test this model, four single cysteine mutants
(cf
A348C, cf
A390C, cf
K424C, and cf
R425C) were introduced
into a functional cysteine-free transhydrogenase. Also, five cysteine
mutants were constructed in the isolated domain III of
Escherichia coli transhydrogenase (ecIIIH345C, ecIIIA348C,
ecIIIR350C, ecIIID392C, and ecIIIK424C). In addition to kinetic
characterizations, effects of sulfhydryl-specific labeling with
N-ethylmaleimide,
2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid, and diazotized
3-aminopyridine adenine dinucleotide (phosphate) were examined.
The results are consistent with the view that, in agreement with the
model, Proton-translocating nicotinamide nucleotide transhydrogenase
couples the reversible stereospecific transfer of hydride equivalents from the 4A position of NADH to the 4B position of NADP+
with translocation of one proton (Ref. 1; see also Ref. 2) from the
periplasmic space to the cytosol, according to the following reaction.
-Ala348,
-Arg350,
-Ala390,
-Asp392, and
-Lys424 are located in or close to the NADP(H) site.
More specifically,
-Ala348 succeeds
B. The remarkable
reactivity of
R350C toward NNADP suggests that this residue is close
to the nicotinamide moiety of NADP(H).
-Ala390 and
-Asp392 terminate or succeed
D, and are thus,
together with the region following
A, creating the switch point
crevice where NADP(H) binds.
-Asp392 is
particularly important for the substrate affinity, but it could also
have a more complex role in the coupling mechanism for transhydrogenase.
INTRODUCTION
Top
Abstract
Introduction
References
For reviews on transhydrogenase, see Olausson et al.
(2), and Jackson (3).
Membrane-bound transhydrogenase is composed of three domains. In the
Escherichia coli enzyme, domain I
(ecI,1 1 to ~
404) and
domain III (ecIII, ~
260 to
462) are exposed to the cytosol and
contain the binding sites for NAD(H) and NADP(H), respectively. Domain
II (~
405 to
510 and
1 to ~
260) spans the membrane.
Domain I (dI) from E. coli (4, 5), Rhodospirillum rubrum (rrI) (6), and bovine (7), and domain III (dIII) from
E. coli (5), R. rubrum (8, 9), and bovine (7, 9)
have been overexpressed, purified, and partially characterized. So far,
domain II has not been expressed as a separate entity. Interestingly,
dII is not required for transhydrogenation to occur (decoupled from
proton translocation), as initially shown by Yamaguchi and Hatefi (7).
Mixtures of recombinant dI and dIII from the same species or from
different species catalyze decoupled "forward" and "reverse"
reactions (cf. Reaction 1) and the so-called "cyclic reaction" (which involves the reduction of bound NADP+ by
NADH, followed by the oxidation of bound NADPH by AcPyAD+)
(5, 8, 9). From a recent study, it was observed that mixtures of rrI
plus rrIII and rrI plus ecIII behaved similarly (10). They catalyzed
high cyclic reaction rates (about the same as those observed in the
complete E. coli and R. rubrum enzymes) that were limited by
the transfer of hydride equivalents (10) and slow reverse reaction
rates that, with excess rrI under usual assay conditions, were limited
by the release of NADP+ (5, 8). With this knowledge at
hand, it is now possible to use the rrI plus ecIII system to complement
mutagenesis experiments performed on the complete enzyme. In addition
to substrate binding affinities and hydride equivalent transfer rates,
release rates of NADP+ and relative affinities between
domains are properties that can be studied in mixtures of rrI and ecIII.
A three-dimensional model of the NAD(H)-binding site in E. coli transhydrogenase has been predicted (11). It adopts the structure of a classical dinucleotide binding domain
and constitutes approximately half of the residues in ecI. The
structure of alanine dehydrogenase (L-AlaDH), a
homologue of transhydrogenase domain I, has recently been solved (12).
The monomeric unit of L-AlaDH consists of two structurally
similar domains, one of which adopts a classical dinucleotide binding
fold responsible for NAD(H) binding, thus supporting the general
features of the model (11).
Since proton translocation is very likely associated with
conformational changes that affect binding and release of NADP(H) (5,
13, 14), information about the structural architecture of the
NADP(H)-binding site is essential for understanding the proton pumping
mechanism. It has been proposed that ecIII adopts a structure
resembling the classical fold found in e.g. glutathione and thioredoxin reductases (2).
In the present report, the development of a model comprising parts of
the NADP(H)-binding domain is described. The prediction was tested by
site-directed mutagenesis. In this context, the availability of a
functional cysteine-free transhydrogenase (cfTH) (15) has been
important. Using this transhydrogenase, single cysteine mutants were
introduced into strategic positions. By probing the various mutants
with the sulfhydryl-specific reagents NEM and MIANS,
information about the location of the mutations relative to the
substrate was gained. Furthermore, the reactivities of certain cysteine
mutants of cfTH and ecIII enzymes toward diazotized AADP and AAD
(analogues of NADP and NAD, respectively, where the amide group has
been exchanged for a diazonium moiety) were examined; the aim was to
find residues that would be spatially close to the nicotinamide ring.
The results of all labeling experiments are discussed in view of the
predicted model.
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EXPERIMENTAL PROCEDURES |
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Site-directed Mutagenesis--
The plasmid, here denoted pCLNH,
carries the gene coding for the cfTH to which an N-terminal histidine
tag has been added.2 This
plasmid served as the DNA template for the construction of three
cysteine mutants, cfA348C, cf
A390C, and cf
R425C. The plasmid
(here denoted pEcIII) coding for the N-terminal histidine-tagged ecIII,
was used as template for the introduction of a set of three single
cysteine mutants, i.e. ecIIIH345C, ecIIIR350C, and
ecIIID392C. The pEcIII was constructed from the pNHis plasmid (5). The primers (MedProbe, Norway), GCGTCGAACCATGGAAGCCAGGAAGTGGGTGAGCACCGC and
GTAAAACGACGGCCAGT (M13 Fp), with pNHIS as template, were used for
polymerase chain reaction amplification of the fragment coding for
residues
286-
462. The NcoI/BamHI fragment
was excised from pNHIS and replaced by the NcoI- and
BamHI-treated polymerase chain reaction-amplified fragment
to yield pEcIII. The Quikchange mutagenesis kit (Stratagene) was used
for the preparation of these six mutants, and the protocol from the
manufacturer was strictly followed. The cf
K424C mutant was made by
polymerase chain reaction mutagenesis (16-19), using a
PauI/AatII cassette. The ecIIIA348C mutant was achieved by excision of the BssHII/SmaI
cf
A348C mutant fragment and subsequent transfer to pEcIII. The
ecIIIK424C mutant was made essentially as the original ecIII (5), but
using the cf
K424C mutant plasmid as template. The full-length genes
were sequenced to verify the correctness of all mutant products.
Expression and Purification of Enzymes--
Domain I from
R. rubrum was expressed and purified essentially as
described (6, 20). Expression and solubilization of the E. coli transhydrogenase enzymes and domains were carried out
essentially as described (21). However, cells were resuspended in 30 mM sodium phosphate buffer containing protease inhibitor (CompleteTM EDTA-free, Boehringer Mannheim), pH 7.5; cells
were disrupted using an X-press (AB Biox, Göteborg, Sweden). In
the case of the ecIII mutants, the proteins were purified as described
(5). Following the solubilization and subsequent centrifugation of the
cfTH enzyme and the cfTH mutants, the supernatant was added to a
nickel-nitrilotriacetic acid resin (2-3 ml of resin/g of membrane
pellet), preequilibrated with buffer A (30 mM sodium phosphate, 1 mg/ml Brij 35, 1 mg/ml Thesit, pH 7.5), containing 0.5 M NaCl and 7 mM imidazole. This batch was
incubated with gentle mixing until more than 80% of the protein had
bound to the nickel-nitrilotriacetic acid resin (after about 1 h).
Regular column chromatography followed, where the gel was first washed
with 10-15 column volumes of buffer A containing 0.7 M
NaCl and 30 mM imidazole. In a second wash (5-10 column
volumes), the salt and imidazole concentrations were decreased to 0.1 M and 10 mM, respectively. The protein was
eluted by increasing the imidazole concentration to 150 mM
in a single step. The transhydrogenase-containing fractions were
pooled, diluted with one volume of buffer A, and applied to a 6-ml
Resource Q column, pretreated with 20 ml of buffer A, 30 ml of buffer B
(buffer A containing 1 M NaCl), and finally 40 ml of buffer
A. The column was washed with 15 column volumes of 200 mM
NaCl in buffer A. The protein was eluted by increasing the NaCl
concentration from 200 to 500 mM in a narrow 5-ml gradient
to yield a high concentration of the protein; the
transhydrogenase-containing fractions were pooled, frozen in liquid
nitrogen, and stored at 80 °C.
All mutants displayed a purity greater than 90% as judged by SDS-polyacrylamide gel electrophoresis using 10-20% gradient gels (Novex, Germany) (not shown). All experiments were performed at 25 °C.
Labeling of Sulfhydryl Groups by NEM and MIANS--
NEM and
MIANS (Molecular Probes, Inc., Eugene, OR) were dissolved in 50%
methanol prior to use. The concentration of MIANS was determined
optically using an absorption coefficient of 17,000 M1 cm
1 at 322 nm. To determine
the time-dependent effect of these probes on reverse and
cyclic transhydrogenation rates, normally 200 µM NEM or 4 µM MIANS were added to the enzyme. When labeling
experiments were carried out in the presence of substrates, these were
incubated together with the protein for 5 min before the labeling
reagent was added.
Activity Assays-- Transhydrogenation reactions of cfTH and cfTH mutants were assayed in cfTH buffer (50 mM sodium phosphate, 1 mM EDTA, 50 mM NaCl, 0.1 mg/ml Brij 35, pH 7.0). The dIdIII assay buffer (20 mM each of Mes, Mops, Ches, and Tris, 50 mM NaCl, pH 7.0) was used for the reconstituted rrI plus ecIII system. Reverse and cyclic transhydrogenase activities were measured optically as described (5, 22).
MIANS Binding Measured by Fluorescence--
The negatively
charged MIANS molecule becomes fluorescent when it reacts with
sulfhydryl groups. Therefore, the kinetics of MIANS labeling of single
cysteine mutants was monitored by a SPEX model FL1T1 2
spectrofluorometer. Excitation and emission slits were both 4.3 nm. The
excitation and emission wavelengths were set to 330 and 418 nm,
respectively. Reactions were initiated by the addition of MIANS to a
final concentration of 4 µM. The absorbances of the
substrates or substrate analogues at 330 and 418 nm were measured, and
inner filter effects were corrected for according to the method
developed by Kubista et al. (23).
Labeling of Single Cysteine Mutants by Diazotized
AAD(P)--
Diazotization of the substrate analogues AADP and AAD was
performed according to the protocol developed by Fisher et
al. (24), and the concentrations of the products were determined
optically using an absorption coefficient of 19,600 M1 cm
1 at 263 nm (24) for both
substrate analogues.
The experiments designed to analyze the effects of NNAD(P) on cyclic
reaction rates catalyzed by ecIII mutants are described in the legend
to Fig. 5. In the case of the ecIIIK424C mutant, 1.8 µM
ecIIIK424C, 860 µM NNAD(P), and 0.40 µM rrI
were used. Similar procedures (see figure legend of Fig. 5) were
followed when the effects of NNAD(P) on reverse activities catalyzed by cfA348C and cf
A390C were investigated. 530 µM
NNAD(P) was added to 16.7 µM cf
A348C, and 530 µM NNAD(P) was added to 4.3 µM cf
A390C; 5-µl samples were withdrawn at selected times and added to 1 ml of
cfTH buffer; reverse reaction rates were measured using 500 µM NADPH and 500 µM
AcPyAD+.
Trypsin Digestion-- Purified, detergent-dispersed, cfTH, and cfTH mutants were diluted with cfTH buffer to a concentration of about 1.5 mg/ml. NEM (1 mM) or MIANS (1 mM) was incubated for 1 h with the protein before the addition of trypsin. NADPH (1 mM) or NADP+ (5 mM) was added to the protein sample 5 min prior to the initiation of proteolysis. The proteins were digested at ambient temperature with trypsin at a mass ratio of 1:30 of trypsin:transhydrogenase, with and without NEM or MIANS and in the absence and presence of NADPH or NADP+. The reactions were terminated after 45 min by the addition of soybean trypsin inhibitor, at the mass ratio 2:1 of trypsin inhibitor/trypsin. Samples were analyzed by SDS-polyacrylamide gel electrophoresis using 10-20% gradient gels.
Determination of Protein Concentration-- The concentration of proteins were determined by BCA using bovine serum albumin as standard (25).
Determination of the NADP(H) Content in ecIII and ecIII
Mutants--
The concentration of NADP+ was determined by
a modified Klingenberg procedure previously described (5). The
concentration of NADPH was deduced from optical spectra of the ecIII
enzyme samples using an extinction coefficient of 6200 M1 cm
1 at 339 nm. The NADP(H)
concentrations were compared with the corresponding BCA-determined
protein concentrations in order to determine the amount of NADP(H)
bound per ecIII protein.
Measurements of Dissociation Constants--
Equilibrium
dialysis, performed essentially as described (26), was used to measure
dissociation constants of cfTH and efTH mutant proteins for NADPH.
Proteins, with and without 1 mM NEM, were incubated for 30 min (cfA390C) or 2 h (cfTH and cf
K424C) at ambient
temperature, before they were dialyzed against 1 liter of dialysis
buffer (20 mM Mops, 50 mM NaCl, 0.1% Brij, pH
7.0) at 0 °C for 12-16 h. The protein was then concentrated to a
final volume of at least 0.9 mg ml
1. 60 µl of protein
and 60 µl of buffer or buffer plus NADPH were applied to opposite
sides of the dialysis membrane. The dialysis chamber was rotated at
about 60 rpm at 4 °C. After 6 h, 40-µl aliquots from each
chamber were transferred to 1 ml of a buffer containing 10 mM Mops, pH 7.0. The fluorescence of the sample was
measured at the emission wavelength of 460 nm using an excitation wavelength of 340 nm. Oxidized glutathione and glutathione reductase were added to determine the fluorescence contribution from NADPH.
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RESULTS |
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Structural Model of the NADP(H)-binding Domain of E. coli
Transhydrogenase--
Sequence similarity searches such as FASTA and
BLAST (27) failed to unambiguously detect any proteins homologous to
domain III of transhydrogenases. Therefore, modeling by homology was not applicable, and information about structural arrangements must rely
on alternative procedures. Secondary structure and accessibility predictions in combination with multiple sequence alignments represent important tools in this context. The shaded
residues in the E. coli transhydrogenase sequence
shown in Fig. 1 signify positions that
are conserved (36% of all residues in dIII) among transhydrogenases from 11 different sources. The result of the secondary structure and
accessibility predictions by the PHD program (28, 29) is presented in
the same figure. The patterns that emerge can be compared with
conserved features observed in other NADP(H)-binding proteins with
known structures. Among these proteins, the classical
dinucleotide binding domain is the most common structure, but
alternative NAD(P)(H)-binding folds exist (30, 31). There are several
characteristics of the transhydrogenase sequences (Fig. 1), suggesting
that indeed domain III comprises a classical dinucleotide binding fold,
and henceforth, strands and helices are labeled according to standard
nomenclature for such a fold (32, 33). First, many essential properties
of the Wierenga fingerprint are maintained. Second, the predicted
secondary structure also emphasizes that this region should adopt a
Rossman
fold. Third, at the C-terminal end of
B, one or
more positively charged residues are found to interact with the
2'-phosphate group on the NADP(H) molecule in e.g.
glutathione reductase, trypanothione reductase, 17-
-hydroxysteroid
dehydrogenase, 6-phosphogluconate dehydrogenase, and NADPH-cytochrome
P-450 reductase. The
-His345 and
-Arg350
residues in E. coli transhydrogenase are, according to the
prediction, in appropriate positions to stabilize the 2'-phosphate in a
related manner.
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The information in Fig. 1 can be used for further tracing of the
secondary structure elements that make up the dinucleotide binding
fold. For example, residues -His354 to
-Leu359 are likely to be part of
C.
C is less
evident and has therefore not been assigned. The minimal
-sheet in a
classical dinucleotide binding domain contains four strands,
A,
B,
C, and
D. Since the switch point, creating the cleft into
which the substrate is fitted, is situated between the C-terminal ends
of
A and
D, these strands should exhibit conserved features. The
hydrophobic character of
A and the glycine-rich consensus at the
switch point are well recognized, but the features of
D have been
analyzed in less detail. From analysis of the six NADP(H)-binding
proteins, glutathione reductase, trypanothione reductase,
17
-hydroxysteroid dehydrogenase, 6-phosphogluconate dehydrogenase,
ferredoxin reductase, and NADPH-cytochrome P-450 reductase (see Fig.
1), it is observed that they all carry a strictly conserved glycine
residue located at the C-terminal end of
D or succeeding
D,
approximately where the switch point is formed; furthermore, the strand
is hydrophobic, and in glutathione reductase and trypanothione
reductase a well conserved aspartic acid residue is located at the N
terminus of
D. The lengths of the
D strands vary. Residues
-Asp383 to
-Gly389 in E. coli
transhydrogenase are suggested to constitute
D. The three
-strands (
A,
B, and
D) making up the dinucleotide binding core have now been assigned. Some NAD(P)(H)-binding proteins do not
contain additional
-strands in this fold (30). In transhydrogenase, however, the stretch
418-
424 is strongly predicted as a
-strand; it is hydrophobic; its N-terminal residues are conserved,
which is reasonable since they should be close to the substrate; the conserved
-Arg416 could correspond to well conserved
charged residues preceding
E in several other proteins; and finally,
in the "PREDB" program, designed to predict
-strand contacts,
-Asp383 to
-Gly389 and
-Gln418 to
-Lys424 were strongly
predicted to make contacts as parallel
-strands.3
The result of the prediction is summarized as a visual guide in Fig. 2. Below, on the one hand, experiments intended to test this model will be described, and, on the other hand, the model will be used with the aim to help rationalize the properties of several mutants.
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Single Cysteine Mutants Inserted into Cysteine-free E. coli
Transhydrogenase--
Four single cysteine mutants, cfA348C,
cf
A390C, cf
K424C, and cf
R425C, were constructed, expressed,
and purified as described under "Experimental Procedures." These
residues should, according to the prediction, be in or near the
NADP(H)-binding site. Some kinetic and thermodynamic properties of the
mutants are given in Table I. None of the
mutated residues was essential, but cf
A348C, cf
K424C, and
cf
R425C displayed markedly reduced reverse transhydrogenation activities. In addition to yielding the lowest activities, cf
R425C also gave the most elevated KmNADPH value,
approximately 8-fold higher than that displayed by cfTH. The
substitution cf
A348C was intended to change the properties of
transhydrogenase as little as possible; still, however, both reverse and cyclic transhydrogenation rates were significantly affected, confirming the importance of this highly conserved region in
transhydrogenase.
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Sulfhydryl-specific Labeling by NEM and MIANS--
Both NEM and
MIANS contain the maleimide moiety, which reacts specifically with
sulfhydryl groups (34). In the first set of experiments (not shown),
the role of the size of the reagent was investigated by comparing the
relative inhibitory effects on reverse transhydrogenation when the
small NEM probe was bound to the mutant cysteine with the effect when
the larger MIANS molecule was bound. Both MIANS and NEM inhibited the
reverse reaction rate catalyzed by the cfA348C mutant; 25 and 43%
activity remained after a 2-h incubation with 4 µM MIANS
and 0.2 mM NEM, respectively. In the case of the cf
A390C
mutant, both NEM and MIANS caused more than 95% inhibition after
1 h. The cf
K424C mutant was more affected by MIANS (about 10%
remaining activity after 1 h) than by NEM (about 40% remaining
activity after 1 h). Interestingly, NEM had no inhibitory effect
on the cf
R425C mutant, whereas binding of MIANS caused about 55%
inhibition (after 1 h). At the times chosen for comparisons (see
above), the rate of further inhibition was minimal (cf. Fig.
3).
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If NEM or MIANS labeling occurs in or close to the NADP(H) site, the
rate of inhibition would be expected to be slower in the presence than
in the absence of NADPH. The results of such comparisons are shown in
Fig. 3. The presence of 500 µM NADPH had the following
effects on the inhibition rates. It slightly decreased the inhibition
rate of cfA348C by MIANS (Fig. 3A); it displayed a strong
protective effect on cf
A390C from both MIANS labeling (Fig.
3B) and NEM labeling (not shown); and it also considerably
lowered the MIANS inhibition rate of cf
K424C (Fig.
3C); however, no protection, or even a slight stimulation, was observed for the cf
R425C mutant (Fig. 3D).
As MIANS reacts with sulfhydryl groups, it becomes fluorescent (34).
Therefore, binding of MIANS to the mutant can be followed fluorometrically. This provides for an alternative approach to that
described above for studying the effect of substrates on MIANS binding
rates. As can be seen in Fig. 3, A-D, the trends were
similar to those found by the activity measurements. Thus, NADPH
protected cfA390C (Fig. 3B) most effectively, but it also caused decreased binding rates for the cf
A348C (Fig. 3A)
and cf
K424C (Fig. 3C) mutants, whereas no protection was
observed for the cf
R425C (Fig. 3D) mutant. Qualitatively,
the NADP+ molecule displayed similar effects on MIANS
binding as the reduced substrate (Fig. 3, A-D).
Trypsin Cleavage--
Transhydrogenase displays a characteristic
cleavage pattern by trypsin. The -subunit is normally only
susceptible to proteolysis in the presence of NADP(H) to yield a
distinguishing 30-kDa fragment (35), allowing a qualitative evaluation
of NADP(H)-induced conformational changes in the
-subunit (results
not shown). In the absence of MIANS or NEM, this tryptic fragment was
produced only upon NADP(H) binding for the four cysteine mutants
described above. After preincubation of the cf
A348C mutant
with MIANS, the
-subunit became very sensitive to trypsin
digestion even in the absence of NADP(H), indicative of a structural
perturbation. Treatment with NEM had no effect on the cleavage pattern
of the cf
A348C mutant but rendered the
-subunit of the cf
A390C
mutant resistant to trypsin digestion even in the presence of NADP(H);
NEM also drastically reduced the ability of NADP(H) to induce the
tryptic 30-kDa fragment for the cf
K424C mutant. In the case of the
cf
R425C mutant, neither NEM nor MIANS caused an altered
NADP(H)-dependent trypsin cleavage pattern.
Dissociation Constants for NADPH--
The results of the catalytic
activity assays and the proteolysis experiments suggest that MIANS and
NEM binding to the cfA390C and cf
K424C mutants may influence
NADP(H) binding. Therefore, measurements of the dissociation constants
of NADPH (KdNADPH) in the absence and presence of
bound NEM were performed by equilibrium dialysis (see "Experimental
Procedures" and Ref. 26). The data are presented in Fig.
4, and the resulting
Kd values are included in Table I. The values of
KdNADPH in the absence of NEM for the cf
A390C
and the cf
K424C mutants were approximately 5 and 6 µM
(11.5 µM in a second preparation of the cf
K424C
mutant), respectively; no bound NADPH was detected for either of these
mutants in the presence of NEM in the concentration range of NADPH
tested (see Fig. 4), suggesting that NEM caused a decrease of their
binding affinities for NADPH by at least 2 orders of magnitude. The
cfTH enzyme displayed an affinity for NADPH (1-2 µM)
that was unaffected by NEM (Table I and Fig. 4); this value is similar
to that found for wild-type
transhydrogenase.4
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Probing the NADP(H)-binding Site by Single Cysteine Site-directed Mutagenesis in Domain III of E. coli Transhydrogenase-- Five single cysteine mutants of ecIII, ecIIIH345C, ecIIIA348C, ecIIIR350C, ecIIID392C, and ecIIIK424C, were expressed and purified according to the scheme described under "Experimental Procedures."
A characteristic feature of the isolated dIII from various species is that they have high affinities for the NADP(H) substrates, which was manifested by the fact that normally almost 100% of the protein molecules contain bound substrate, the majority of which was in their oxidized state (5, 8, 9). In order to determine if the mutations introduced in ecIII affected the affinities for NADP(H), the substrate contents were measured in all mutants (see "Experimental Procedures"). From the results shown in Table II, it was noticed that the mutants behaved differently from the nonmutant ecIII. First, a smaller fraction of the ecIII mutants carried substrates. Second, the fraction of reduced substrate was greater. The most striking observation, however, was that the purified ecIIID392C mutant did not contain any measurable amounts of bound substrates. Certainly, this particular mutation must have led to strongly decreased affinities for NADP(H).
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Protein-protein titrations, keeping the ecIII mutant concentration fixed and varying the rrI concentration, were performed for each mutant, both for the cyclic reaction and for the reverse reaction (not shown). From the titration curves, maximal rates were estimated, as well as the concentration of rrI required to reach half of the maximal rates. In the case of the cyclic reaction, this rrI concentration is dependent on the affinity between the two domains, whereas in the reverse reaction it is determined by the relative kinetics of the component mechanistic steps, including dI and dIII association and dissociation (8, 10). The results from the protein-protein titrations are summarized in Table III.
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Reverse Reaction Catalyzed by rrI plus ecIII and rrI plus ecIII Mutant Mixtures-- Every mutant exhibited elevated reverse transhydrogenation rates compared with the ecIII reference, the most pronounced effect demonstrated by the ecIIID392C mutant (approximately 14 times the rate catalyzed by rrI plus ecIII) and the least effect displayed by the ecIIIH345C and ecIIIR350C mutants (approximately 2.5 times the rate of the wild-type control). The rates of release of NADP+ must, at a minimum, be as fast as the turnover of the reverse reaction rate. Therefore, as an approximation, the mutants may be ranked according to effects on NADP+ release rates as follows: ecIIID392C > ecIIIA348C > ecIIIK424C > ecIIIH345C = ecIIIR350C. The ratios of the rrI concentrations required to reach half-maximal rates to the fixed mutant concentrations, did not vary extensively between the various mutants but were all high compared with the ratio displayed by the native ecIII (Table III). Possibly, a high ratio may correlate with a high rate of reverse transhydrogenation.
Cyclic Reaction Catalyzed by rrI plus ecIII and rrI plus ecIII Mutant Mixtures-- All of the single mutants catalyzed relatively high rates of cyclic transhydrogenation, ranging from about 20% for the ecIIID392C mutant to about 70% for the ecIIIA348C mutant as compared with wild-type ecIII. This indicated that the abilities of these mutants to transfer hydride equivalents, which have been shown to be rate-limiting for the cyclic reaction under the present conditions (10), have not been dramatically affected. Three protein-protein titrations using various fixed concentrations of ecIII and variable rrI were performed in order to provide for an adequate analysis of the affinities between rrI and the intact ecIII and between rrI and the ecIII mutants. By comparing the half-saturating rrI concentration for a particular mutant with the appropriate (similar fixed concentration of ecIII as mutant) corresponding rrI concentration for nonmutant ecIII (Table III), it was possible to determine if the interdomain affinity had changed. Significant decreased affinities were detected only for the ecIIIH345C and the ecIIID392C mutants (Table III).
The Effect of Diazotized AAD(P) on Single Cysteine
Mutants--
Several NAD(P)-binding proteins have been shown to be
irreversibly inhibited by diazotized AAD(P) at neutral pH, and the
conclusions were that cysteines close to the nicotinamide moiety had
been modified (24, 36-40). As expected, AADP was a competitive
inhibitor with respect to the NADP(H)-binding site in E. coli transhydrogenase with a Ki of about 100 µM, and AAD at a concentration of 1 mM did
not compete with NADPH binding (not shown). Since the investigated
mutants were targeted toward the NADP(H)-binding site, they were
subjected to diazotized AADP (NNADP) treatment. To test if the effects
observed were specific, each mutant was also treated with the same
concentration of diazotized AAD (NNAD). Cyclic transhydrogenation
activities catalyzed by the rrI plus ecIII system were measured for
ecIII mutants, whereas reverse transhydrogenation activities were
analyzed for cfTH mutants. The results of these experiments are
presented in Fig. 5. Neither the
ecIIIH345C (Fig. 5A), nor the ecIIIK424C (not shown) mutant was specifically inhibited by NNADP; for the latter mutant, both NNAD
and NNADP caused an initial 25% drop in activity, whereafter the
activity remained essentially constant. The ecIIIA348C mutant was
significantly inhibited by both NNADP (about 93% inhibition after
2 h) and NNAD (about 65% inhibition after 2 h) (Fig.
5B), but with some preference for the NNADP molecule. The
corresponding mutant in the intact enzyme, i.e. cfA348C,
was much less affected by similar concentrations of the reactive
substrates (about 20 and 30% inhibition by NNAD and NNADP,
respectively) (not shown). Possibly, this could be a consequence of the
much lower affinity of the intact enzyme for NADP(H) relative to that
of the isolated ecIII. The ecIIIR350C mutant reacted rapidly with even
very low concentrations of NNADP, and at these low concentrations
hardly any reactivity with NNAD was observed (Fig. 5C).
Also, the ecIIID392C mutant reacted preferentially with NNADP, and the
cyclic reaction rate was decreased by about 85% after 1 h,
compared with about 48% in the presence of NNAD (Fig. 5D).
The cf
A390C mutant in the intact enzyme was not affected much by
either NNADP or NNAD (approximately 20% inhibition after 3 h for
both reagents) (not shown).
|
The results suggest that -Ala348,
-Arg350, and
-Asp392 are located in, or
very near, the NADP(H)-binding site, since the corresponding cysteine
mutants displayed a greater reactivity toward the NNADP than toward the
NNAD molecule.
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DISCUSSION |
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As described under "Results," theoretical considerations
strongly suggest that domain III in H+-TH adopts a
classical dinucleotide binding structure responsible for the binding of the NADP(H) substrate. The
A,
B,
B,
C, and
D secondary structures were assigned to specific residues in the
E. coli transhydrogenase (Fig. 2). Based on this model, cysteine residues were introduced into an otherwise cysteine-free transhydrogenase (cfTH), targeting positions that would be
spatially close to the substrate.
The cfA348C and cf
A390C mutants were chosen solely with the aim
of providing structural information, and the desire was to disturb the
function of the enzyme as little as possible. However, the replacement
of an alanine by a cysteine could induce strain in the backbone
structure, since the two residues have different preferences for
and
angles (41), which could explain the relatively large
inhibitory effect of the cf
A348C mutant on reverse transhydrogenation (about 20% activity compared with cfTH; see Table
I). The
-Ala348 residue is located in a highly conserved
region predicted to be in a loop conformation close to the 2'-phosphate
group of NADP(H) (Figs. 1 and 2). The structural importance of this
region previously recognized (42, 43) is further substantiated by the
fact that binding of MIANS to the cf
A348C mutant caused an increased
sensitivity of the
-subunit to trypsin digestion, in the wild-type
enzyme only caused by NADP(H). It was reported by Bragg et
al. (42, 43) that replacements of the positively charged residues
-His345 and
-Arg350 caused a dramatic
loss of transhydrogenase activities, of which the former was suggested
to reflect structural alterations. The decreased binding rates of MIANS
in the presence of NADP(H) suggest that
-Ala348 is
physically either in or near the substrate binding site or that the
conformational changes known to be induced by substrate binding (14,
35) result in a shielding effect.
The pronounced ability of NADP(H) to protect the cfA390C mutant from
reacting with MIANS (Fig. 3B) and NEM (not shown) and the
fact that reaction with NEM yielded an enzyme derivative virtually unable to bind the NADPH substrate support the predicted location of
-Ala390 at the end of
D in the NADP(H)-binding site.
Necessarily,
-Asp392, which has been shown to be crucial
for reverse transhydrogenation (44), must also be in or very near the
NADP(H) site.
The -Lys424 and
-Arg425 residues are
conserved among the known transhydrogenase sequences (Fig. 1). Both
amino acids are charged, and the former is a possible candidate for
direct, and the latter for indirect, participation in vectorial
protonation events. Furthermore, it was earlier proposed that the two
residues could be located close to the nicotinamide moiety of the
NADP(H) molecule and by their positively charged nature possibly
stabilize the binding of reduced substrate relative to the oxidized
species (5). Therefore, these sites were found to be interesting
targets for mutagenesis. For similar reasons as described for the
cf
A390C mutant, the present results (see Fig. 3C) are
clearly consistent with the view that
-Lys424 is located
within a few Å of the NADP(H) substrate. In a recent study, it was
found that replacing
-Lys424 with an arginine strongly
repressed NADP(H) binding (45). It is suggested that this effect and
the NEM effect on the cf
K424C mutant both are of steric nature. In
contrast, except for the high KmNADPH value for
R425C, there was no evidence that
-Arg425 interacted
directly with NADP(H) (Fig. 3D), although it should be close
to the binding site according to the model. A likely explanation for
this is that the side chain of
-Arg425 does not point
toward the substrate. Thus, even a large molecule can react with the
thiol group of the cf
R425C mutant without dramatically disturbing
the ability of NADP(H) to bind and induce the conformational
rearrangements that upon trypsin digestion yield the characteristic
30-kDa fragment. However, all mutants of
-Arg425 that
have been characterized (Ref. 45; see Table I), exhibited a substantial
loss of transhydrogenase activities. Although not essential, this
residue may still be important for the catalytic mechanism of the enzyme.
A second set of cysteine mutations was introduced into ecIII, which in
the nonmutant state is devoid of cysteines. As described in the
Introduction, the rrI plus ecIII hybrid system offers a possibility to
study properties such as substrate affinities, hydride equivalent
transfer rates, substrate release rates, and domain I-domain III
interactions in a semiquantitative manner. Mutants in domain III of the
intact enzyme that were either difficult to study for various reasons
(see below) or found to be particularly interesting were expressed as
corresponding cysteine mutants in ecIII. The H345C mutant was
previously shown to poorly assemble into membranes (42, 43) and was
here found to be difficult to purify (not shown). None of the various
-Asp392 mutants analyzed so far catalyzed reverse
transhydrogenation (44). Although not essential,
R350C seems to be
an important residue for catalysis, but its role is unclear (43).
Together with these three residues,
-Ala348 and
-Lys424 were also mutated for further characterization
of the NADP(H) site.
All ecIII mutants displayed decreased affinities for NADP(H), which were reflected by the enhanced rates of reverse transhydrogenation (see below) and by the lower amounts of bound substrates (see "Results"). Possibly, the increased levels of reduced substrate bound to the purified mutant were also a consequence of increased release rates of NADP+. Note also that greater levels of rrI were required to reach half-maximal rates for the ecIII mutants than for the nonmutant ecIII and that the increased reverse reaction rates seemed to be correlated with this fact. It has been reported previously that for both rrI plus rrIII (8) and rrI plus ecIII mixtures (10), rrI can perform several rounds of the following cycle before NADP+ is released from ecIII: binding of NAD+, association with ecIII, transfer of the hydride equivalent from NADPH on ecIII to NAD+ on rrI, dissociation from ecIII, and release of NADH. Thus, the less time NADP+ spends on the ecIII enzyme, the fewer ecIII each rrI can productively visit during the time of one turnover of ecIII. In summary, all mutants affected the binding site to various degrees, as anticipated from the model and in accordance with the results obtained for the cfTH mutants.
The two mutant hybrid mixtures rrI plus ecIIIH345C and rrI plus ecIIID392C also catalyzed significantly lower cyclic reaction rates than the reference rrI plus ecIII mixture, about 25 and 18%, respectively (Table III). Since the cyclic reaction is at least partly limited by hydride equivalent transfer (10), which is also direct (46), it is possible that these mutations caused an unsuitable orientation of the substrates to give an efficient catalysis. All other mutants displayed only minor effects.
Of all the ecIII mutants, ecIIID392C displayed the most dramatic
difference from wild-type ecIII behavior. Most, if not all, of its
properties can be explained by its decreased affinity for substrates,
e.g. the fact that it was purified in the absence of bound
substrates (Table II), the high rates of reverse transhydrogenation (Table III), and the 10-min incubation with NADP(H) required to achieve
maximal rates. However, once the NADP(H) substrate was bound, it was
able to transfer hydride equivalents to NAD(H) bound to rrI. Also, it
was interesting to note that the pH dependence of the reverse reaction
catalyzed by rrI plus ecIIID392C was distinctly different from the pH
profiles displayed by the wild-type rrI plus ecIII and the other two
mutant mixtures analyzed, i.e. rrI plus ecIIIH345C and
ecIIIR350C (not shown). It has been hypothesized that D392 could be
directly involved in vectorial proton transport (44). The present
results do not exclude this possibility.
The coupling between proton movement from the periplasmic side to the
cytosolic side, on the one hand, and the production of NADPH and
NAD+ from NADP+ and NADH, on the other hand,
are probably governed by conformational changes. Binding of NADP(H) to
dIII certainly gives rise to structural rearrangements (35).
Furthermore, the conformation of ecIII has been reported to be
dependent on whether the bound NADP(H) is oxidized or reduced (5). One
or several residues that mediate this redox-dependent
conformational change should therefore be located in close proximity to
the nicotinamide ring of NADP(H). In an attempt to localize such
residues, the cysteine-specific reactive NAD(P) analogues, diazotized
AAD(P) (36), were used to probe several cysteine mutants. Diazotized
AADP caused a more rapid inhibition of the ecIIIA348C, ecIIIR350C,
ecIIID392C, and possibly cfA348C mutants than did diazotized AAD
(see Fig. 5), supporting the possibility that these residues are
located in or near the NADP(H)-binding site. Interestingly, ecIIIR350C
displayed a pronounced reactivity toward diazotized AADP. Judging from
the model presented in Fig. 2, this result was surprising. One
possibility is that
-Arg350 is not involved in the
stabilization of the 2'-phosphate group as postulated (Ref. 2; Fig. 2;
and see "Results") and thus is misplaced in the model. If
-Arg350 is very close to the nicotinamide ring of
NADP(H), it could have an important function in mediating a putative
conformational change triggered by the redox state of the NADP(H)
substrate (5). A second possibility is that the diazotized AADP
molecule binds to the cysteine mutant as it approaches the site. A
third, but less likely, possibility is that NADP(H) does not bind to
ecIII as depicted in Fig. 2, which is the common mode of NAD(P)(H)
binding to classical
dinucleotide binding domains.
These possibilities are presently being investigated.
In conclusion, theoretical predictions and a cysteine scanning approach
proved to be a fruitful combination in order to yield a low resolution
structure of the NADP(H)-binding site in energy-transducing transhydrogenase. The results suggest that dIII adopts a classical NAD(P)(H) binding fold, where the residues -Ala348,
-Arg350,
-Ala390,
-Ala392,
and
-Lys424 are in or near the NADP(H) site.
-Asp392 is a critical residue for appropriate substrate
binding, and
-Arg350 may be close to the nicotinamide
ring of NADP(H).
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ACKNOWLEDGEMENTS |
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We thank Prof. J. B. Jackson (University of Birmingham, UK) for the pCD1 plasmid containing the rrI gene and for helpful advice on the technique of equilibrium dialysis to measure substrate dissociation constants.
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FOOTNOTES |
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* This work was supported by the Swedish Natural Science Research Council.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: Dept. of Biochemistry
and Biophysics, Göteborg University and Chalmers University of
Technology, Box 462, S-405 30, Sweden. Tel.: 46-31-7733921; Fax:
46-31-7733910; E-mail: jan.rydstrom{at}bcbp.gu.se.
2 J. Meuller and J. Rydström, unpublished observations.
3 T. Hubbard, personal communication.
4 J. Rydström, X., Hu, and J. B. Jackson, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
ecI and ecIII, domain I and domain III constructs, respectively, of E. coli
transhydrogenase;
cfTH, cysteine-free transhydrogenase;
cfTH mutants
(e.g. cfK424C), mutants that have been introduced into
cfTH;
dI, dII, and dIII, domain I, II, and III, respectively, of
membrane-bound transhydrogenases in general;
rrI and rrIII, domain I
and III, respectively, of R. rubrum transhydrogenase;
pEcIII, a plasmid carrying the ecIII gene (see "Experimental
Procedures");
ecIII mutants, mutants that have been introduced into
ecIII;
AcPyAD+, 3-acetyl pyridine adenine dinucleotide
(oxidized form);
AAD, 3-aminopyridine adenine dinucleotide;
AADP, 3-aminopyridine adenine dinucleotide phosphate;
NNAD, diazotized AAD;
NNADP, diazotized AADP;
NEM, N-ethylmaleimide;
MIANS, 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid;
Mes, 4-morpholineethanesulfonic acid;
Mops, 4-morpholinepropanesulfonic
acid;
Ches, 2-(cyclohexylamino)ethanesulfonic acid.
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REFERENCES |
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