©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure/Function Analysis of the Periplasmic Histidine-binding Protein
MUTATIONS DECREASING LIGAND BINDING ALTER THE PROPERTIES OF THE CONFORMATIONAL CHANGE AND OF THE CLOSED FORM (*)

Amnon Wolf (1), Eudean W. Shaw (1)(§), Byung-Ha Oh (3), Hendrik De Bondt (2)(¶), Anil K. Joshi (1)(**), Giovanna Ferro-Luzzi Ames (1)(§§)

From the (1)Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, the (2)Department of Chemistry and Structural Biology, Division of Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720 and the (3)Department of Life Science, Pohang University of Science and Technology, Hyoja-Dong San 31, Pohang, Kyungbuk 790-784, Korea

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 = 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.

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)()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).

The scheme shown in Fig. 1describes the working hypothesis we currently use for this purpose. The receptor alternates between four forms: closed empty (J), 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.


MATERIALS AND METHODS

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 Kfor 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).


RESULTS

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 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.

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 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.

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.


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 (2.810M 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 (510M) 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 Kvalue, 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.

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.


DISCUSSION

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),()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.

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.

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).

It appears that the liganded form interacts with the membrane-bound complex better than the unliganded form (as demonstrated by cross-linking experiments).()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 ().

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 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).

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 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.

  
Table: HisJ mutant proteins

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 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

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 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).



FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK12121 (to G. F.-L. A.) and AI30725 (to Sung-Hou Kim, which supported H. D. B. and B.-H. O.) and by a postdoctoral fellowship from the Damon Runyon-Walter Winchell Cancer Research Fund (to A. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.

Present address: Laboratory of Analytical Chemistry and Medicinal Physicochemistry, Faculty of Pharmacy KULeuven, E. Van Evenstraat 4, B-3000 Leuven, Belgium.

**
Present address: Children's Hospital Oakland Research Institute, 747 52nd St., Oakland, CA 94609.

§§
To whom correspondence and reprint requests should be addressed. Tel.: 510-642-1979; Fax: 510-643-7935.

The abbreviations used are: LAO, lysine-, arginine-, ornithine-binding protein; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; MOPS, 4-morpholinepropanesulfonic acid.

The possibility that S72P and S92F can assume the empty closed form cannot be tested because of their very poor affinity for both histidine and 9D2; extremely high concentrations of 9D2 would be required to perform this test.

In the presence of 6 µg/ml 9D2, histidine binding was higher than in the absence of histidine, but it was only 2% of the total S92F.

C. E. Liu and G. F.-L. Ames, unpublished data.

G. F.-L. Ames, K. Nikaido, and C. E. Liu, unpublished data.

G. F.-L. Ames and K. Nikaido, unpublished data.


ACKNOWLEDGEMENTS

We thank Jack F. Kirsch for the use of the fluorimeter and Carolyn Chi for performing the transport assays.


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