From the
The periplasmic histidine-binding protein, HisJ, is a receptor
for the histidine permease of Salmonella typhimurium.
Receptors of this type are composed of two lobes that are far apart in
the unliganded structure (open conformation) and drawn close together
in the liganded structure (closed conformation). The binding of the
ligand, in a cleft between the lobes, stabilizes the closed
conformation. Such receptors have several functions in transport:
interaction with the membrane-bound complex, transmission of a
transmembrane signal to hydrolyze ATP, and receiving a signal to open
the lobes and release the ligand.
In this study the mechanism of
action of HisJ was further investigated using mutant proteins defective
in ligand binding activity and closed form-specific monoclonal
antibodies (Wolf, A., Shaw, E. W., Nikaido, K., and Ames G. F.-L.(1994) J. Biol. Chem. 269, 23051-23058). Y14H is defective in
stabilization of the closed form, does not assume the closed empty
form, and assumes an altered closed liganded form. T121A and G119R are
similar to Y14H, but assume a normal closed liganded form. S72P binds
the ligand to the open form, but does not assume a recognizable closed
form. S92F is defective in the ability to undergo conformational change
and to stabilize the closed form. All other mutant proteins appear to
fall within one of these four categories. The biochemical
characterization of these mutant proteins agrees with the structural
analysis of the protein. We suggest that mutant proteins that do not
assume the normal closed form, in addition to their defect in ligand
binding, fail to interact with the membrane-bound complex and/or to
transmit transmembrane signals.
Bacterial periplasmic permeases are complex transport systems
that translocate a wide variety of compounds in Gram-negative bacteria
such as Escherichia coli and Salmonella typhimurium.
Present knowledge about the mechanism of action of these systems has
been reviewed (Doige and Ames, 1993; Shuman and Panagiotidis, 1993;
Higgins, 1992). The histidine periplasmic permease is well
characterized and both its composition and mechanism of action are
typical of periplasmic permeases in general. This permease is composed
of a receptor, the periplasmic histidine-binding protein (HisJ), and a
membrane-bound complex (QMP complex) (Kerppola et al., 1991).
HisJ has several functions: it binds histidine with high affinity (K
The three-dimensional x-ray
structure of HisJ from both S. typhimurium and E.
coli, and of the homologous lysine-, arginine-, ornithine-binding
protein (LAO)
The scheme shown in Fig. 1describes the working hypothesis we currently use for this
purpose. The receptor alternates between four forms: closed empty
(J
The four forms that HisJ normally assumes in solution are
shown in Fig. 1. We previously identified monoclonal antibodies
(mAbs) that specifically recognize the closed form of HisJ
(closed-form-specific mAbs, (Wolf et al., 1994)). Such mAbs
are used in this study to determine whether mutant proteins are in the
closed or open form. Binding of radiolabeled histidine is used to
distinguish between the liganded and unliganded forms. By using a
combination of these reagents, we examined the ability of each of the
mutant proteins to assume the four forms.
Forty mutant HisJ proteins
with a variety of properties were tested for their affinity for
histidine and 13 were found to be defective in binding ability. shows that the K
The interaction
between all 13 mutant proteins and two types of mAbs was examined. Two
closed form-specific mAbs, 7A2 and 9D2, and two control mAbs that are
not conformation sensitive, 1A3 and 2A5 (Wolf et al., 1994),
were used to perform an immunoblot analysis in the presence of
histidine (). 1A3 and 2A5 interact with wild type and with
all the mutant proteins equally well, indicating that none of the
substitutions results in a disastrous structural damage. In contrast,
7A2 and 9D2 interact poorly with most of the mutant proteins. The
possibility that 7A2 and 9D2 are unable to interact with altered HisJ
mutant proteins in general is eliminated because they have been shown
to interact well with 12 other HisJ mutant proteins (S108A, K112E,
K133E, E136K, D144N, D149A, D149N, T151A, D156A, K171E, and K176E),
which have normal K
Verification that Y14H undergoes a
conformational change was obtained by monitoring tryptophan
fluorescence (Miller et al., 1983; Zukin et al.,
1986). HisJ has a single tryptophan residue, Trp-130, that is located
far from the ligand-binding site. In wild type, unliganded HisJ
fluorescence has a maximum at 330 nm and increases with increasing
histidine concentrations, reaching a maximum that corresponds to an
enhancement of 58% over unliganded protein (Fig. 3a);
neither the wavelength where maximum emission occurs nor the shape of
the spectrum changes. Such an enhancement of fluorescence indicates
that HisJ undergoes a conformational change resulting in exposure of
Trp-130 to a different environment (Zukin et al., 1986). In
Y14H, the fluorescence also increases with increasing histidine
concentrations, although the maximal level of enhancement is much
smaller (15%) and higher histidine concentrations are required for
saturation (Fig. 3b). It should be noted that since
Tyr-14 is located far from Trp-130, the substitution should not affect
the environment of Trp-130 significantly; thus, the smaller
fluorescence enhancement supports the notion that the closed form of
Y14H is abnormal.
T120M also interacts poorly with 9D2 and this interaction is
not stimulated by histidine; 9D2 does not allow histidine trapping by
T120M, indicating that T120M does not assume the closed liganded form.
Thus, T120M behaves like S72P and therefore presumably does not undergo
the conformational change either.
In the case of G119E, the
immunoblot analysis shows that it interacts well with 7A2 and 9D2. The
ELISA experiments show that it has a poor interaction with 9D2 in the
absence of histidine (data not shown) and that it improves in its
presence (); 9D2 allows histidine trapping. Thus G119E
behaves like G119R and T121A and therefore, presumably, it assumes the
proper closed liganded form, but is defective in its stabilization.
The working hypothesis for the mechanism of action of
periplasmic permeases proposes that the receptor has several functions:
ligand binding, interaction with the membrane-bound complex,
transmitting a signal through the latter to hydrolyze ATP, and
receiving a signal to release the ligand. Past genetic and biochemical
evidence has led to the notion that these receptors have separate
ligand-binding and interaction domains (Kustu and Ames, 1974;
Prossnitz, 1989; Hor and Shuman, 1993; Dean et al., 1992).
Receptors defective in any of these functions would be inactive in
transport. An important conclusion of the present study is that in many
transport-negative mutants, the respective receptors with defective
binding ability also have an altered closed form. Since the closed form
is the most likely candidate to initiate the subsequent steps in
transport, as shown by its ability to stimulate ATP hydrolysis by the
membrane-bound complex (Davidson et al., 1992; Bishop et
al., 1989; Hoshino et al., 1992; Richarme et
al., 1992),
From the
biochemical results and the summary of structural properties described
in I and Fig. 8it is apparent that all the other
mutant proteins analyzed belong to one of the several categories
discussed above.
It appears that the
liganded form interacts with the membrane-bound complex better than the
unliganded form (as demonstrated by cross-linking experiments).
Another implication of the interaction between the
receptor and the membrane-bound complex is the manner by which the
ligand is released from the receptor in order to be translocated. This
should be an active process because the energy of binding is about 10
kcal/mol (as calculated from the K
Periplasmic permeases belong to
the large superfamily of traffic ATPases (also known as ABC
transporters) that includes several medically important eukaryotic
representatives, such as the cystic fibrosis transmembrane regulator
(CFTR) and P-glycoprotein (Doige and Ames, 1993; Higgins, 1992). It is
reasonable to suggest that advances resulting from an understanding of
the mechanism of action of periplasmic permeases would be useful for
the understanding of the eukaryotic systems. Even though eukaryotic
traffic ATPases have not been found to use a soluble receptor component
(see Doige and Ames (1993) for discussion of this aspect), the
possibility remains that in some cases, through evolution, a soluble
receptor has been fused into the membrane portion of some eukaryotic
transporters. An important precedent for this kind of event has emerged
recently with the discovery that large extracellular portions of some
eukaryotic receptors, namely a group of metabotropic glutamate
receptors (O'Hara et al., 1993) and the Ca
Plasmid pFA54, in strain GA432, carries wild type hisJ (cloned from TA271) under the control of the tac promoter. All mutations are chromosomal, except for hisJ9103 and hisJ9106 which were created by in vitro mutagenesis (plasmids pFA187 and pFA190, respectively, in strains
GA249 and GA241) under the dhuA
The E. coli and S.
typhimurium HisJs are 98% identical and the S. typhimurium HisJ and LAO are 70% identical (Kang et al., 1991). All
the substitutions described in Table I are in residues that are
identical in all of these proteins, except for residue 121 which is
threonine in both HisJs and serine in LAO. The above analysis is based
on the structures of S. typhimurium LAO (Oh et al.,
1993, 1994a) and of E. coli HisJ (Yao et al., 1994),
which have been solved to a higher resolution than that of S.
typhimurium HisJ. The second column describes the properties of
the wild type residue. Only interactions that involve the ligand, or
occur between lobes or between lobes and the hinge region are
described. A poor K
We thank Jack F. Kirsch for the use of the fluorimeter
and Carolyn Chi for performing the transport assays.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
= 30 nM), interacts
with the membrane-bound complex, triggers a signaling pathway that
results in the hydrolysis of ATP (and/or GTP) and in the release of the
ligand from the receptor. This series of events results in
translocation of the ligand to the cell interior (Prossnitz et
al., 1988; Oh et al., 1994b; Bishop et al.,
1989; Prossnitz et al., 1989; Joshi et al., 1989;
Petronilli and Ames, 1991). Although there is considerable information
on the mechanism of ligand binding by these receptors, little is known
about their other functions. The goal of this investigation is to gain
further understanding of these various functions through a
structure/function analysis of HisJ using mutant proteins defective in
transport. We discuss the significance of our findings for the
mechanism of transport in general.
(
)from S. typhimurium have
been solved at high resolution (HisJ, 1.9 Å; LAO, 1.8 Å)
(Oh et al., 1994b; Yao et al., 1994;
Kang et al., 1991; Oh et al., 1993). The structures
resemble those of other periplasmic receptors (Quiocho, 1990; Zou et al., 1993; Mowbray and Cole, 1992; Tame et al.,
1994), which are composed of two lobes, connected via two or three
peptide stretches, and separated by a deep cleft in the unliganded
protein; the binding site is located in the cleft. When the ligand is
bound, the lobes are placed close to each other and the ligand is
completely buried. The stabilization of the closed liganded form
involves interactions of the ligand with several side chain residues
and with the peptide backbone from both lobes, and interactions between
the two lobes via water molecules (Oh et al., 1993). The
conformational change from the open to the closed form involves a large
scale, rigid body movement of one lobe relative to the other. The
existence of these two forms is supported by the solution of the
structures of both LAO and the maltose-binding protein, each in their
liganded closed and empty open forms (Oh et al., 1993, 1994a;
Sharff et al., 1992).
), open empty (J
), open
liganded (J
-H), and closed liganded
(J
-H), with H being the model ligand (histidine)
and J the model receptor, HisJ; notice that the protein alternates
between the closed and open forms regardless of the presence of ligand.
Evidence for various steps in this scheme has been supplied by
thermodynamic, kinetic, biochemical, and x-ray crystallography studies
(for reviews, see Furlong(1987), Quiocho(1990), Adams and Oxender(1989)
and Ames(1986)). Besides the resolution of several structures both in
the open unliganded and closed liganded forms, other relevant findings
are the following. (i) The closed empty form of the
C4-dicarboxylate-binding protein from Rhodobacter capsulatus was shown to exist by thermodynamic and kinetic studies (Walmsley et al., 1992a, 1992b). (ii) The closed empty form of HisJ has
been shown to exist in solution by using monoclonal antibodies (Wolf et al., 1994). (iii) The unliganded glucose/galactose-binding
protein from S. typhimurium has been crystallized in the
closed empty form (although this form is not identical to the closed
liganded form) (Flocco and Mowbray, 1994). (iv) The maltose-binding
protein liganded with a large ligand,
-cyclodextrine, has been
crystallized in the open liganded form (Sharff et al., 1993).
(v) A crystal of unliganded leucine/isoleucine/valine-binding protein
that was soaked in mother liquor containing leucine assumed an open
liganded form (Sack et al., 1989). Thus, strong evidence
points to the existence of the closed empty and open liganded forms.
The present study provides additional evidence in favor of such a
hypothesis and new insights into its mechanism of action.
Figure 1:
Scheme describing the mechanism of
action of binding proteins.
Numerous
other proteins with diverse biological functions have been shown by
x-ray crystallography also to undergo a large hinged domain motion: e.g. calmodulin, glutamate dehydrogenase, adenylate kinase,
the cAMP-dependent protein kinase, and others (Gerstein et
al., 1994; Bennett and Huber, 1984). Understanding the mechanism
of action of periplasmic binding proteins is not only important to
understand how they mediate transport, but can also provide a model for
understanding in general the mechanism of action of proteins that
undergo large conformational changes upon ligand binding.
Bacterial Strains
All bacteria are derivatives
of S. typhimurium LT2. All HisJ mutants are part of the
laboratory collection and were obtained by one of the following
methods: inability to grow on D-histidine as a histidine
source (Ames et al., 1977), resistance to the inhibitory
analog azaserine (Ames et al., 1977), and in vitro mutagenesis.
Binding Proteins Preparation
Wild type HisJ and
Y14H proteins were obtained by osmotic shock (Lever, 1972a) from
strains GA432 and GA408 carrying plasmids pFA54 and pFA247, which
contain the respective hisJ genes under tac promoter
control. They were purified by a two-step ammonium sulfate
precipitation (Noel et al., 1979), followed by
diethylaminoethyl-cellulose high performance liquid chromatography
(Nikaido and Ames, 1992). HisJ and Y14H were over 90% pure as judged by
SDS-polyacrylamide gel electrophoresis. They were foundto be free of
ligand by thin layer chromatography (Nikaido and Ames, 1992) using an
imidazole reagent to detect histidine (Ames and Mitchell, 1952). Mutant
proteins (listed in ) were prepared by osmotic shock and
dialyzed extensively to remove histidine. To estimate the K for histidine binding the mutant
proteins were further concentrated and partially purified (through the
carboxymethylcellulose step) as described (Nikaido and Ames, 1992). The
HisJ concentration in the osmotic shock fluids was estimated by ELISA
using polyclonal antibodies and pure wild type HisJ as a standard.
Immunoblot Assay
SDS-polyacrylamide gel
electrophoresis (12.5% acrylamide) was performed using a Bio-Rad
mini-gel system, followed by immunoblotting using a semidry transfer
apparatus (Hoefer, San Francisco, CA) onto an ``Immobilon P''
membrane (Millipore Corp.). The membranes were treated with polyclonal
antibody (diluted 1 to 3000) or monoclonal antibody (mAb) at
concentrations calculated from calibration curves to be 60, 160, 80,
and 30 ng/ml for mAbs 1A3, 2A5, 7A2, and 9D2, respectively. The second
antibody was either goat anti-mouse IgG or goat anti-rabbit IgG, both
coupled to horseradish peroxidase (Bio-Rad), and the color was
developed with an ECL kit (Amersham). The protein bands were quantified
by a KRATOS DM3000 spectrodensitometer (Schoeffel Instruments) and were
found to be within a linear range.
Enzyme-linked Immunosorbent Assay (ELISA)
The
assay was performed as described (Friguet et al., 1990) using
purified unliganded wild type HisJ and Y14H or osmotic shock fluids
obtained from other mutant strains (10 ng/well) and performing all
steps at 37 °C for 1 h. The antibody concentration was varied as
indicated. When histidine was present, it was mixed with the mAb before
addition to the microtiter wells. The second antibody was a 500-fold
dilution of either goat anti-mouse IgG or goat anti-rabbit IgG, both
coupled to alkaline phosphatase (Bio-Rad); the incubation time was 1 h
at 37 °C. The alkaline phosphatase reaction was followed at 405 nm,
using an assay kit (Bio-Rad).
Fluorescence Measurements
Unliganded pure wild
type HisJ or Y14H (2.8 µM in MOPS buffer (10 mM,
pH 7.0)) were incubated at 25 °C. Fluorescence measurements were
performed with a Perkin-Elmer LS50B Luminescence spectrometer. The
emission spectra were recorded using an excitation wavelength of 296
nm. The widths of the excitation and emission slits were 5 mm.
Histidine-binding
Assay
L-[H]Histidine binding was
measured in MOPS buffer (10 mM, pH 7.0) by either equilibrium
dialysis or filtration through nitrocellulose membrane filters, as
described previously (Lever, 1972b). Unless specified differently,
dialysis was performed for 17 h at 4 °C. The filtration assay was
performed at room temperature and the filters were washed twice with
600 µl of buffer each, which essentially eliminates retention of
histidine in the absence of mAb. The K
for ligand binding was calculated from Scatchard and
Lineweaver/Burk plots. It should be noted that, like in the case of
wild type HisJ (Wolf et al., 1994), only mAbs 9D2 and 7A2
allow histidine trapping by the mutant proteins, while 1A3 and 2A5 do
not.
Miscellaneous Procedures
Protein determination and
the production of monoclonal antibodies have been described (Lowry et al., 1951; Wolf et al., 1994).
values for
histidine of these 13 proteins are grossly defective, with values
between 0.2 and 7 µM, i.e. 7- to 230-fold worse
than the K
of the wild type (0.03
µM). The respective bacterial strains were also analyzed
for their transport activity and all are defective, with apparent K
values for transport between 20 and 900
nM, i.e. 3- to 150-fold worse than that of the wild
type (6 nM). These mutant proteins may be defective in the
ability to undergo the conformational change (Steps 1 or 3), to
stabilize the closed liganded form (J
-H), or to
bind histidine to the open form (J
+ H
J
-H; Step 2); a possible alternative is that the
substitution stabilizes the closed empty form.
values for histidine
binding (Wolf et al., 1994). The possibility that the
substitutions that result in poor interaction with 7A2 and 9D2 have
direct effects on the mAb epitopes is also unlikely because all of
those substitutions are in residues that are buried (with intrinsic
solvent accessibility, <12%; ). Thus it is likely that
most of the mutant proteins do not assume or stabilize the correct
closed form. Each of the mutant proteins was further analyzed and the
properties are described below. Mutant Y14H is described in more
detail, as an introduction to the various experimental approaches.
Y14H
Does Y14H Undergo a Conformational Change and Assume
the Correct Closed Liganded Form?
Y14H binds histidine with a K of 2.0 µM, which is about
70-fold worse than that of the wild type ( (Oh et
al., 1994b)). The fact that Y14H assumes a liganded form gives no
information as to whether it assumes the closed liganded or the open
liganded form, i.e. as to whether the protein has undergone a
conformational change. The poor reaction with the closed form-specific
mAbs described in already indicates that Y14H probably
assumes a closed form that is abnormal and/or unstable. This finding
can be made quantitative by ELISA experiments by determining the extent
of interaction between Y14H and 9D2. Fig. 2, panel a, shows the reactivity of wild type HisJ with respect to mAb
concentration; the interaction is much better in the presence than in
the absence of histidine (solid versus dashed lines),
confirming previous results (Wolf et al., 1994). An
intermediate concentration of mAb was then chosen to test the effect of
varying the concentration of histidine (panel d). The
histidine concentration corresponding to half-maximal interaction
between the wild type and 9D2 is 30 nM. Since this value is
similar to the K
of HisJ for histidine as
measured by equilibrium dialysis, these results must reflect a complete
reaction between 9D2 and the closed liganded form. Thus, in agreement
with previous results, 9D2 recognizes the closed form.
Figure 2:
Effect of histidine on the interaction of
9D2 with wild type and mutant proteins, as measured by ELISA. ELISA
conditions were as described under ``Materials and Methods.''
Wells were coated with pure unliganded wild type (WT; solid
circles) or Y14H (open squares), or crude osmotic shock
containing S72P (open triangles), S92F (solid
squares), G119R (solid triangles), and T121A (open
circles) (10 ng/well). In all subsequent figures these symbols
remain the same, unless otherwise specified. a-c, titration of
mAb 9D2 in the presence and absence of histidine: the coated proteins
were treated with increasing concentrations of 9D2, as indicated on the abscissa, in the absence (dashed line) or presence (solid line) of histidine (1 mM). d, titration of histidine at fixed mAb concentration: 9D2
concentrations were 30 ng/ml for wild type, S92F, G119R, and T121A, and
300 and 600 ng/ml for Y14H and S72P, respectively. High mAb
concentrations were used because the latter mutant proteins did not
interact well with 9D2. The ordinate indicates the absorbance
relative to the maximum reaction at saturating histidine
concentrations. Histidine concentrations higher than 50
µM, up to 1 mM, were tested, but are not shown;
they can be calculated from panels a-c. The concentration of
histidine which led to half-maximal interaction was calculated from a
Scatchard plot.
Y14H also
interacts progressively better with increasing concentrations of 9D2
and, similarly to the wild type, the reaction is improved by the
presence of histidine (Fig. 2, panel a). This indicates
that Y14H can indeed assume the closed conformation. However, the
reaction is weaker than the one observed with the wild type, which may
indicate that the closed form is altered or unstable. When the
histidine concentration is varied in the presence of intermediate
concentrations of 9D2, the half-maximal effect occurs at 4.0 µM (Fig. 2, panel d); this value is comparable to the K value measured for Y14H by equilibrium
dialysis. At very high histidine concentrations (1 mM), when
all the protein molecules are liganded, the interaction is weaker than
with wild type (panel a), indicating that the closed
conformation is abnormal.
Figure 3:
Effect of histidine on the fluorescence
emission spectra for wild type and Y14H. The change in fluorescence
intensity of pure wild type or Y14H (2.810
M in MOPS buffer (10 mM; pH 7.0)), all
unliganded, is shown on the ordinate. The effect of L-histidine on emission spectra for wild type (a),
with L-histidine concentrations of 0, 0.14, 0.28, 0.56, 0.84,
1.12, 1.4, 1.68, and 4.48 µM are shown in curves
1-9, respectively. Changes in fluorescence of Y14H (b)
in the presence of L-histidine 0, 5, or 7.5 µM (which gave the same result as 10 µM) are shown in
curves 1-3, respectively.
Another way to determine whether the protein has
undergone a conformational change is by estimating the concentration of
liganded form in solution using closed form-specific mAbs. Such mAbs
capture the closed liganded form of wild type HisJ, and thus inhibit
histidine dissociation by trapping the liganded histidine. The effect
of 9D2 on histidine dissociation in both the wild type and Y14H was
tested. The binding proteins were first treated with excess labeled
histidine, then 9D2 was added, and they were dialyzed for 16 h; at this
time the final free histidine concentration was 30 nM, which
is the value of the wild type K. As
expected, in the absence of 9D2, binding of histidine by wild type and
Y14H reflects their respective K
values (Fig. 4, zero 9D2 concentration). The addition of 9D2 prior to
dialysis allows larger amounts of histidine to be retained (i.e. trapped) by either protein, which is consistent with the notion
that Y14H can assume the closed liganded form. The fact that bound
histidine can be trapped relatively effectively in Y14H despite its
67-fold poorer K
than the wild type
indicates that the protein undergoes the conformational change.
Figure 4:
Effect of 9D2 concentration on histidine
trapping by wild type and mutant proteins. Pure unliganded wild type
and mutant proteins (10 µg/ml each in MOPS, 10 mM; pH 7.0,
as indicated) were liganded with [H]histidine
(1.0 µM) and incubated with various concentrations of 9D2
for 1 h at room temperature, then dialyzed at 4 °C against
histidine-free buffer. At the end of the experiment, the
[
H]histidine concentration was 30 nM.
The amount of bound histidine was determined after dialysis for 18 h.
The results are expressed as the number of mole of histidine bound per
mole of HisJ.
Additional information about the nature of the closed liganded form
of Y14H can be provided by measuring the rate of reaction between 9D2
and the liganded form. The rate of reaction between liganded wild type
and 9D2 is relatively slow and thus can be measured (Wolf et
al., 1994). Fig. 5shows that the rate of 9D2 interaction
with liganded Y14H is much slower than with wild type, reaching the
wild type level upon much longer incubation times (i.e. within
30 min; data not shown). This result supports the notion that even
though the closed liganded form of Y14H can be assumed, it differs from
that of the wild type. These overall data confirm that Y14H undergoes a
conformational change upon ligand binding and that it does not assume
the correct closed liganded conformation.
Figure 5:
Rate of reaction of mAb with liganded
proteins. Wild type and mutant proteins (1.0 µg/ml each in MOPS, 10
mM, pH 7.0) were first mixed with
[H]histidine (1.0 µM), followed
immediately by the addition of a saturating amount of 9D2 (previously
determined to be 0.75 µg/ml). At the times indicated, aliquots (100
µl) were removed and filtered through a nitrocellulose membrane
(Lever, 1972b). The experimental point at time 0 was determined before
9D2 addition.
Does Y14H Assume the Closed Empty Form?
The
ability to trap and therefore quantitate HisJ in either of its closed
forms, empty or liganded, permits the analysis of whether the defect in
Y14H interferes with its ability to assume the closed empty form.
Preincubation of unliganded wild type protein with 9D2 inhibits binding
of subsequently added histidine because according to the scheme, the
wild type protein assumes the closed empty form even in the absence of
ligand and is sequestered by 9D2. Histidine binding activity was
measured after preincubating unliganded protein for various lengths of
time with 9D2, followed by the addition of a saturating concentration
of histidine. As previously shown (Wolf et al., 1994), 9D2
inhibits histidine binding by wild type by about 40% after
preincubation for 1 h (Fig. 6). However, 9D2 has no effect on
Y14H even after preincubation for 3.0 h, indicating that Y14H has not
been sequestered by 9D2 and therefore can be fully liganded upon
addition of excess histidine. Thus, Y14H does not assume the closed
empty form, or such a form is grossly abnormal.
Figure 6:
Rate of reaction of mAb with unliganded
proteins. Wild type and Y14H (10 µg/ml) were preincubated at room
temperature with 9D2 (3 µg/ml) for the indicated time intervals,
when [H]histidine (5
10
M) was added; 1.0 h later aliquots (300 µl) were
dialyzed at 4 °C against histidine free buffer. The final
[
H]histidine concentration was 30 nM.
Histidine binding is expressed relative to the value obtained when
histidine is added prior to the mAb. The 9D2 concentration is limiting
relative to the binding protein concentration; higher ratios of 9D2 to
HisJ and longer incubations will result in lower levels of binding by
the wild type (see Wolf et al., (1994) and Fig.
7a).
Does Y14H Bind Histidine Normally to the Open
Form?
Since Y14H can bind histidine and undergo the
conformational change, it appears that it cannot stabilize the closed
liganded form. If this were true, it should be possible to demonstrate
efficient binding of histidine to the open form of Y14H (step 2 in Fig. 1). This possibility can be tested by stabilizing the closed
liganded form as soon as it is formed through its sequestration with
9D2. Addition of 9D2 to Y14H in the presence of histidine
concentrations much lower than the K of
Y14H should result in stabilization of any liganded molecule being
formed; this would appear as a stimulation of histidine binding. In
contrast, 9D2 should inhibit histidine binding by the wild type because
unliganded molecules which assume the closed empty form would be
sequestered by 9D2 and made unavailable for further binding. Indeed, Fig. 7a shows that incubation of wild type for 1 h with
increasing concentrations of 9D2 inhibits binding of subsequently added
histidine, while histidine binding by Y14H is stimulated. Quantitation
of the binding of histidine to the open form of Y14H was measured by
varying the concentration of histidine in the presence of 9D2. Fig. 7b shows that at concentrations of histidine below
the measured K
value, as expected,
binding is poor in the absence of 9D2, and that the addition of 9D2
improves binding dramatically. Plotting these data according to
Lineweaver and Burk gives an apparent K
value of 0.2 µM, 10 times better than the K
value of 2 µM obtained by
equilibrium dialysis (). The apparent affinity obtained by
this method does not measure the true binding affinity for the open
form. This is due to the fact that the level of binding depends on the
length of exposure to 9D2: longer incubation times result in increased
levels of binding (data not shown). In any case, although Y14H at
equilibrium binds histidine very poorly at concentrations much lower
than its measured K
, large amounts of
liganded protein can be captured by stabilizing it with the mAb. The
relevance of this observation is discussed later.
Figure 7:
Effect of preincubation with 9D2 on
histidine binding by unliganded proteins. a, effect of varying
the concentration of 9D2. Unliganded wild type and mutant proteins (10
µg/ml each in MOPS, 10 mM, pH 7.0), were incubated with
various concentrations of 9D2 for 1 h at room temperature, then
dialyzed at 4 °C against buffer containing
[H]histidine (30 nM). b, effect
of varying the histidine concentration after preincubation with 9D2.
Pure unliganded Y14H (10 µg/ml in MOPS, 10 mM, pH 7.0) was
incubated in the absence (open squares) or presence (solid
squares) of 9D2 (3.0 µg/ml) for 1 h at room temperature, then
dialyzed at 4 °C against buffer containing the indicated
concentrations of
[
H]histidine.
S72P
S72P Does Not Undergo the Conformational
Change
S72P binds histidine with a K of 0.3 µM, indicating that some ability to
assume the liganded form(s) is retained () (Oh et
al., 1994b). However, the immunoblot analysis ()
indicates no reaction with either 7A2 or 9D2. ELISA experiments show
that 9D2 interacts with unliganded S72P, although very poorly, and that
the presence of histidine does not improve the interaction (Fig. 2, panel b). Panel d, shows no changes in
the interaction with varying concentrations of histidine. Thus, S72P
cannot assume the closed liganded form, or assumes a grossly deformed
one. The addition of 9D2 does not trap the bound ligand (Fig. 4).
Preincubation of the unliganded protein with increasing concentrations
of 9D2 does not stimulate histidine binding (Fig. 7a).
Therefore it appears that S72P binds histidine to the open form,
presumably with a K
of 0.3
µM, but cannot undergo a conformational change to a closed
liganded form that is recognized by 9D2.
(
)Therefore, since S72P does not assume a recognizable
closed liganded form, the K
value
measured by equilibrium dialysis appears to reflect binding to the open
form.
S92F
S92F Undergoes a Conformational Change Upon Ligand
Binding, but the Closed Form Is Unstable
S92F binds histidine
very poorly and interacts poorly with both closed form-specific mAbs () (Oh et al., 1994b). Quantitative ELISA
experiments confirm that unliganded S92F interacts with 9D2 poorly, and
that, similarly to the wild type, the interaction is considerably
improved by the presence of histidine (Fig. 2, panel b).
This indicates that S92F can indeed assume the closed conformation. The
extent of reaction is comparable to that of wild type suggesting that
the closed form is normal. However, in contrast to the wild type, when
the histidine concentration is varied, the half-maximal reaction with
9D2 occurs at about 10 µM (panel d), which
indicates that high histidine concentrations are needed to increase the
concentration of the closed form. Trapping by 9D2 of histidine bound by
S92F at high histidine concentration was tested. Fig. 4shows
that very little trapping occurs. Increasing the histidine and mAb
concentrations (to 13 µM and 0.8 µg/ml respectively)
enhanced significantly the amount of bound histidine trapped, although
it was only 12% of the total protein (data not shown). Fig. 7a shows that, in contrast to the results obtained
with Y14H, preincubation of unliganded S92F with increasing
concentrations of 9D2 does not stimulate binding in the presence of 30
nM histidine.(
)This is in agreement
with the conclusion that high histidine concentrations are needed to
shift the equilibrium toward the closed liganded form of S92F.
All these data together indicate that S92F binds histidine to the
open form, undergoes a conformational change, but that the equilibrium
is shifted toward the open liganded form.
G119R and T121A
G119R and T121A Undergo a Conformational Change upon
Ligand Binding, Assuming the Correct Closed Liganded Form, Which Is
Unstable; They Cannot Assume the Closed Empty Form
G119R and
T121A bind histidine poorly, but interact with 7A2 and 9D2 as well as
the wild type () (Oh et al., 1994b), indicating
that they can undergo the conformational change and assume a normal
closed liganded form. The ELISA experiments show that although the
interaction with 9D2 is good in the presence of histidine, it is very
poor in its absence (Fig. 2, panel c). The histidine
concentrations at which the half-maximal effect on the interaction with
G119R and T121A occurs are 3.3 and 4.0 µM, respectively (Fig. 2, panel d). Fig. 4shows that 9D2 can trap
the bound ligand in both proteins as effectively as in the wild type.
Moreover, the rate of the interaction between either protein and 9D2 is
as fast as with the wild type (Fig. 5), supporting the notion
that the closed liganded forms of G119R and T121A are similar to that
of the wild type. Thus, they appear to be defective in the
stabilization of the closed liganded form; however, once such a form is
taken, it appears normal. To examine whether they can assume the closed
empty form, the effect of preincubating unliganded G119R and T121A with
9D2 before exposure to histidine was tested. Histidine binding
increased with increasing concentrations of 9D2 (Fig. 7a). Thus, these proteins cannot assume the closed
empty form. Under these conditions G119R and T121A also bind histidine
at concentrations much lower than their respective measured K values, as was also shown for Y14H.
Other Mutant Proteins
The other HisJ mutant
proteins listed in appear to fall within categories that
match the types described above. The immunoblot analysis shows that
S69P, S70L, R77L, R77C, Y147A, and Y147D interact poorly with 7A2 and
9D2. The ELISA experiments show that in the absence of histidine all of
them interact with 9D2 poorly (data not shown) and histidine stimulates
this interaction to various extents (), indicating that
they undergo a conformational change upon ligand binding. 9D2 increases
histidine binding in all of them, indicating that they can assume the
closed liganded form. Thus, they behave like Y14H and presumably are
also defective in the stabilization of the closed form. They also
appear to assume a closed form that is different from that of the wild
type.
(
)such defective mutant proteins
must also be defective in transmembrane signaling. Thus, we are
suggesting that various functional domains of these receptors are
tightly interconnected. We first analyze the structural and functional
effects of the substitutions in HisJ, and then we discuss the
functional implications for overall transport.
Structural Analysis of the Mutant Proteins
The
underlying basis of the defective behavior of the mutant proteins can
be clarified by an analysis of the relevant interactions. I and Fig. 8summarize the structural
characteristics of the residues involved and of their substitutions.
Figure 8:
Schematic representation of the relevant
interactions in the ligand-binding site of HisJ. Schematic
representation of the nature and the positions of all the residues
under investigation and their interactions with the liganded histidine
and with relevant residues in the binding site. Covalent bonds and
presumed noncovalent interactions are designated by solid and dashed lines, respectively; Y14 and the imidazole ring of the
liganded histidine are depicted near each other and without a line to
reflect their tight stacking interaction (a detailed structural
analysis appears in Oh et al., (1994b) and in Yao et al. (1994)). W denotes a water
molecule.
Y14H
Tyr-14 (located in lobe I) has an important
function in ligand binding and stabilization of the closed form. Its
aromatic ring is stacked parallel to the imidazole ring of the liganded
histidine and, in the closed form, its hydroxyl group forms hydrogen
bonds with the backbone NH groups of Asp-161 and Leu-117 (both located
in lobe II) each via a water molecule. This stacking arrangement and
the hydrogen bonds stabilize the closed form. Since these lobe-lobe
interactions do not involve the ligand it is reasonable to suggest that
they would contribute to stabilizing also the closed empty form. The
replacement of Tyr-14 with histidine abolishes all of these bonds,
therefore impairing the stabilization of the closed forms, both
liganded and empty. Thus, the structural analysis agrees with the
biochemical analysis from which it was concluded that both the closed
empty and the closed liganded forms of Y14H are unstable. High
histidine concentrations shift the equilibrium toward the closed form
by increasing the concentration of the open liganded form,
J-H (Scheme in Fig. 1). We have no clear
explanation of why the closed liganded form of Y14H is altered. It
should be noted that the fact that 9D2 does not trap Y14H in the closed
empty form also excludes the possibility that closed form-specific mAbs
induce in general the conversion of the open empty form into the closed
empty form (Wolf et al., 1994).
S72P
Ser-72 is located in lobe I and has the
following five interactions with the ligand and with residues in lobe
II or in the hinge region: a hydrogen bond between the Ser-72 OH and
the NH group of the liganded histidine; a
hydrogen bond between the Ser-72 NH and the COO
group
of the liganded histidine; a hydrogen bond between the Ser-72 OH and
the Thr-121 OH via a water molecule; two hydrogen bonds between the
Ser-72 OH and three hinge residues, Gly-194, Phe-191, and Ala-90, via
two different water molecules ( Fig. 6in Oh et
al.(1993)). It should be noted that Ala-90 undergoes a 52°
change in
upon conformational change (the largest change in LAO
(Oh et al., 1993)). It can be predicted that the replacement
of Ser-72 with proline abolishes all of these bonds with the
consequence that the closed form is unstable and the ability of the
protein to undergo the conformational change is impaired. These
predictions are consistent with the biochemical analysis indicating
that S72P cannot assume the closed liganded form, or that such a from
is not recognizable.
S92F
Ser-92 is far from the ligand-binding site
and does not interact directly or indirectly via water molecules with
the ligand; it is located in the hinge and undergoes a 22° rotation
in the angle during the conformational change (Oh et
al., 1993). The replacement of Ser-92 with phenylalanine would
impair the ability of the protein to undergo such a conformational
change. The finding that S92F is defective in the ability to assume the
closed form is consistent with this structural analysis.
G119R and T121A
The backbone carbonyl group of
Gln-118 interacts with the amide nitrogen of Asp-53 and the Thr-121 OH
interacts via hydrogen bonds with Ser-72 OH, each through one water
molecule. These bonds should be involved in stabilizing the closed form
because Thr-121 and Gln-118 are located in lobe II and Ser-72 and
Asp-53 are located in lobe I. Since the Ramachandran /
angles (99°, -2°) of Gly-119 are not in the range which
is allowed for residues with side chains, the G119R substitution would
change the orientation of the Q118 neighbor. Gln-118 and Thr-121 are
essential in lobe-lobe interaction to lobe I. The T121A substitution
would abolish the hydrogen bond and thus impair the lobe-lobe
interaction. The Thr-121 NH also interacts with the COO
group of the liganded histidine, but the T121A substitution would
not change this interaction. In agreement with the biochemical
analysis, G119R and T121A can assume the open empty, open liganded, and
closed liganded forms, but not the closed empty form. G119R and T121A
are different from Y14H because they assume closed liganded forms that
are indistinguishable from that of the wild type.
Do the Lobes Undergo a Rigid Body Movement?
In
view of the notion that the conformational change consists of a rigid
body movement of one lobe relative to the other, as demonstrated by
crystallographic data (Oh et al., 1993, 1994a), the
enhancement of tryptophan fluorescence upon ligand binding needs
explanation. In addition, Trp-130 is buried (intrinsic solvent
accessibility <4%) and is located far from the ligand-binding site
(Oh et al., 1994b). Therefore, such an enhancement was
unexpected. A similar fluorescence enhancement has been observed in LAO
(data not shown), which contains a tryptophan residue in the same
position as HisJ; no significant change was observed in the immediate
environment of Trp-130 upon ligand binding (Oh et al., 1993).
However, it appears that the electronic environment of Trp-130 changes
upon ligand binding. Therefore, the resolution of the structures of LAO
in the open and closed forms must not be sensitive enough to detect
subtle differences in the immediate vicinity of Trp-130. Evidence
indicating changes in the environment of Trp-130 was also provided by a F nuclear magnetic resonance study of HisJ (Post et
al., 1984). The smaller fluorescence enhancement observed in Y14H
indicates that the environment of Trp-130 is affected by the mutation
in either the open or closed form.
Relevance to the Transport Mechanism
The structure
of the ligand-binding pocket of HisJ is well characterized from
crystallographic work; it is also supported by mutant analysis (this
work and Oh et al. (1994b)). However, little is known about
the domains that are involved in HisJ's interaction with the
membrane-bound complex and in transmembrane signaling. While it is
conceivable that all these domains are entirely independent of each
other, from the operational point of view they are more likely to be
connected functionally and to overlap physically. Therefore, alteration
of one of domain might affect the other(s). Since it is most likely
that the closed liganded form of binding proteins is the active form
for interaction and transmembrane signaling, it is important to
determine which mutations affect this form. Here we have shown for the
first time that defects in ligand binding can result in defective
closed liganded forms; therefore, interaction and/or transmembrane
signaling may also be altered. Physical interaction between the
receptor and the membrane-bound complex and transmembrane signaling can
be measured by chemical cross-linking and induction of ATP hydrolysis,
respectively (Prossnitz et al., 1988; Bishop et al.,
1989; Davidson et al., 1992). Preliminary results indeed
indicate that most of the mutants analyzed here also display lower
cross-linking and signaling activities.(
)Mutant
binding proteins in other systems may have similar properties (Adams et al., 1991; Martineau et al., 1990; Treptow and
Shuman, 1988; Zhang et al., 1992).
(
)We previously proposed that the interaction between a
conformation-specific mAb and the closed form of a binding protein may
be useful as a model for studying the interaction with the
membrane-bound complex (Wolf et al., 1994). Because we
demonstrate here that the interaction of several mutant proteins with
9D2 shifts the equilibrium toward the closed liganded form, we
speculate that the membrane-bound complex may also do so, even in the
case of the wild type. A consequence of this hypothesis would be that
for some mutants the K
for transport
would not necessarily be proportional to the K
for binding, as indeed appears to be the case ().
for
ligand binding). A possible scenario would be that the energy for
opening the lobes of the receptor while it is interacting with the
membrane-bound complex is derived directly from ATP hydrolysis, which
is known to supply the energy for overall transport (Ames et
al., 1989; Joshi et al., 1989; Prossnitz et al.,
1989; Dean et al., 1990).
sensor (Brown et al., 1993), resemble periplasmic
binding proteins and that these extracellular portions have been fused
into membrane-bound domains. Presumably they function similarly to
binding proteins to capture and supply the membrane-bound mechanism
with the appropriate ligand molecule, for translocation or signaling
purposes.
promoter
control. GA408, and GA432 harbor plasmids pFA247. hisJ5626 was
cloned from TA309 into pFA54, yielding plasmid pFA247 (in GA408).
Table: Summary of the properties of HisJ K mutant proteins
Table: Summary of the effects of substitutions on
HisJ structure and function
for histidine binding
is a property of all the mutant proteins, except for S70L; therefore, K
values are omitted from the predicted
effects in the third column. A partial analysis of mutant proteins
Y14H, S69P, R77L, S92F, T121, and Y147D was performed previously (Oh et al., 1994b).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.