Both Lobes of the Soluble Receptor of the Periplasmic Histidine Permease, an ABC Transporter (Traffic ATPase), Interact with the Membrane-bound Complex
EFFECT OF DIFFERENT LIGANDS AND CONSEQUENCES FOR THE MECHANISM OF ACTION*

Cheng E. LiuDagger §, Pei-Qi LiuDagger , Amnon Wolf, Erick Lin, and Giovanna Ferro-Luzzi Amesparallel

From the Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The histidine permease of Salmonella typhimurium is an ABC transporter (traffic ATPase). The liganded soluble receptor, the histidine-binding protein HisJ, interacts with the membrane-bound complex HisQMP2 and stimulates its ATPase activity, which results in histidine translocation. In this study, we utilized HisJ proteins with mutations in either of the two lobes and wild type HisJ liganded with different substrates to show that each lobe carries an interaction site and that both lobes are involved in inducing (stimulating) the ATPase activity. We suggest that the spatial relationship between the lobes is one of the factors recognized by the membrane-bound complex in dictating the efficiency of the induction signal and of translocation. Several of the key residues involved have been identified. In addition, using constitutive ATPase mutants, we show that the binding protein provides some additional essential function(s) in translocation that is independent of the stimulation of ATP hydrolysis, and one possible mechanism is proposed, which includes the notion that liganded HisJ has different optimal conformations for signaling and for translocation.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The superfamily of ABC transporters (or traffic ATPases) (1, 2) is involved in the transport of a variety of compounds across membranes of both prokaryotes and eukaryotes. ABC transporters share extensive sequence homology and are composed of four structural elements: two integral membrane domains and two hydrophilic domains that contain a conserved nucleotide-binding motif (3, 4). In the prokaryotic systems, the various domains usually consist of separate polypeptides interacting with each other and forming a membrane-bound complex, whereas in eukaryotes, they are generally fused into a single polypeptide. Among the best studied prokaryotic systems are bacterial periplasmic permeases, which, in addition to the membrane-bound complex, also contain a soluble receptor (the periplasmic substrate-binding protein) that binds the substrate in the periplasm and delivers it to the membrane-bound complex for translocation (5).

The periplasmic histidine permease of Salmonella typhimurium and Escherichia coli is an ABC transporter that has been thoroughly characterized biochemically and genetically (6-11). This permease can serve as a good model system for understanding the mechanism of action of ABC transporters in general. The membrane-bound complex of the histidine permease, HisQMP2,1 comprises two integral membrane proteins, HisQ and HisM, and two copies of the nucleotide-binding subunit, HisP. HisQMP2 interacts with the soluble receptor, the histidine-binding protein (HisJ), and another closely related receptor, the lysine-, arginine-, ornithine-bindng protein (LAO) (70% identity with HisJ (12)). Both HisJ and LAO have been crystallized, and their structure has been determined. Similar to what has been found for the structure of other periplasmic receptors extensively studied (13), HisJ and LAO include two globular domains or lobes (lobe I and lobe II) that are far apart in the open unliganded form and close to each other in the closed liganded form. As determined by x-ray crystallography, the switch between the two states involves a large hinged-domain movement in which the two lobes act as rigid globular domains, changing their relative positions dramatically without resulting in significant structural changes within the lobes. The closed liganded form is stabilized by numerous interactions between the ligand and the side chain residues and the peptide backbone from both lobes and by several lobe-lobe interactions via water molecules (14-17). Liganded HisJ interacts with HisQMP2 and triggers a transmembrane signal that induces ATP hydrolysis by HisP (10, 11, 18). Preliminary investigations have shown that the liganded receptor displays an unexpected conformational plasticity that depends on the nature of the ligand, as was demonstrated using conformation-specific antibodies (8). Is this property a crucial factor with regard to the mode of interaction between the receptor and HisQMP2 and the subsequent response(s)?

In this study, we utilized HisJ proteins mutated in either of the two lobes and wild type HisJ liganded with different substrates to show, using biochemical tools, that each HisJ lobe carries an interaction site, that both lobes are involved in inducing the ATPase activity, and, specifically, that the spatial relationship between the lobes may be one of the factors dictating the efficiency of the induction signal and of translocation. Interestingly, there is a surprising lack of direct correlation between the induced levels of ATPase and the efficiency of translocation. Several of the HisJ residues involved in interaction have been identified, and we show that HisQMP2 can distinguish between the various liganded forms of HisJ and adjust accordingly the nature of the signal triggering ATP hydrolysis. Evidence is also provided that the soluble receptor supplies some essential function(s) in translocation that is independent of the stimulation of ATP hydrolysis, because translocation is very poor when ATP hydrolysis is constitutively and maximally expressed, unless HisJ is present.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Preparation of HisJ-- All binding proteins, wild type and mutant, were purified from S. typhimurium in the unliganded form by osmotic shock, ammonium sulfate precipitation, acid precipitation, carboxymethyl-cellulose chromatography, and DEAE-high pressure liquid chromatography, as described (19). Protein concentrations were determined using a modified Lowry protein assay (20).

Purification and Reconstitution of HisQMP2-- HisQMP2, wild type or mutant, was purified using metal affinity chromatography, making use of a polyhistidine extension engineered into the carboxyl terminus of HisP as described (21). It has been shown that the presence of the polyhistidine extension has no effect on the function of HisP (9). The purified complexes were reconstituted into proteoliposomes as described (10) and stored at -80 °C.

In Vitro Transport Assay-- Transport was measured in PLS (reconstituted proteoliposomes containing purified HisQMP2 with a carboxyl-terminal extension to HisP of 8 amino acids residues: Leu-Glu-His-His-His-His-His-His) as described (10) with 20 µM binding protein and 20 µM ligand (usually L-[3H]histidine) at 37 °C, unless stated differently. The assay was modified to incorporate an ATP-regenerating system that has been shown to improve the transport efficiency (10). The reconstituted PLS containing purified HisQMP2 complex were mixed with ATP, MgCl2, creatine phosphate, and creatine kinase (Boehringer Mannheim) to final concentrations of 15 mM, 15 mM, 20 mM, and 3-5 mg/ml, respectively; the mixture was frozen and thawed twice (rather than the customary five times because of the sensitivity of creatine kinase to freeze/thawing) before being subjected to the LiposoFastTM treatment. The resulting PLS were assayed immediately.

ATPase Assay-- ATPase activity was assayed as described (11). The initial rate of ATP hydrolysis was measured in the presence of 2 mM ATP and 10 mM MgSO4 at 37 °C. The HisJ-stimulated activity was corrected for the value for the intrinsic activity (in the absence of HisJ).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Both Lobes of HisJ Are Involved in the Interaction with HisQMP2

It has been speculated in the past, but not proven, that the two structural lobes of HisJ interact physically with separate sites on HisQMP2 (3, 22, 23). Here, we provide biochemical evidence in support of this notion, using as a measure of the efficiency of the interaction the in vitro ability of HisJ to stimulate (i) ATP hydrolysis and (ii) transport by HisQMP2. Both in vitro systems use PLS reconstituted with pure HisQMP2 and pure receptor, and the concentrations of all components and the energy source can be accurately controlled (10, 11). Transport is a functional assay of the interaction, whereas ATPase levels directly reflect the nature of the physical interaction between the receptor and the membrane-bound complex. The ATPase activity of HisQMP2 responds to receptor, unliganded and liganded, by being induced 2- and 10-fold, respectively (11). The functions of the two lobes were separated by studying the effect of various residue replacements in each of the lobes on the ability of HisJ to stimulate ATP hydrolysis and induce histidine translocation by HisQMP2. Residues located in solvent-exposed regions on the receptor surface were chosen for analysis because they would be most likely to be directly implicated in interaction and because the corresponding mutant HisJ proteins would be least likely to have an overall structure that is grossly deformed.

HisJ Mutations in Lobe I-- A loop structure (which includes residues 19-25) is evident in lobe I in the high resolution structure of LAO and HisJ (15). It is possible that this structure is involved in the interaction with HisQMP2 because of its extensive protrusion from lobe I and of its orientation. To test this hypothesis, the following mutations were introduced by in vitro mutagenesis into this region: deletion Delta Lys-20-Leu-26, eliminating residues Lys-20 to Leu-26 (hisJDelta 9163); single replacement K20E (hisJ9145); and double replacements G24A/E25K (hisJ9164/hisJ9165), K20E/E25K (hisJ9145/hisJ9165), and E18K/E25K (hisJ9166/hisJ9165). Except for E18K/E25K, the corresponding mutant HisJs were determined to have essentially normal binding affinity for histidine (Table I).2 Nevertheless, assay of the level of transport in vitro shows that most of the mutants display a level of transport that is different from that of the wild type, being both higher and lower (Fig. 1A; data summarized in Table I). Transport was performed at saturating histidine concentrations, thus eliminating the possibility that minor changes in histidine-binding activity would influence transport activity. Transport was also measured using an in vivo assay based on the ability of auxotrophs to grow on D-histidine as a source of histidine (25) and yielded essentially the same results (data not shown). Thus, although the protruding loop is not essential for ligand binding, it is involved in some aspect of transport. Presumably this area of lobe I interacts with the membrane-bound complex and thus may be also involved in the ATPase induction event. Therefore, the level of induction of ATPase activity was determined using saturating concentrations of L-histidine (Fig. 1B and Table I). Several of the loop-defective HisJs induce levels of ATPase activity that are different than that induced by the wild type and vary similarly to the respective transport activity levels. Deletion Delta Lys-20-Leu-26 and double mutant E18K/E25K display ATPase-inducing activities that are considerably lower than that of the wild type, which may account directly for their lower levels of transport. The affinity of these two mutant HisJs was also determined by using a variety of HisJ concentrations to induce the ATPase (data not shown); both have the same affinity for HisQMP2 as wild type HisJ. Therefore, the important distinction can be made that the decreased level of transport and ATP hydrolysis at saturating HisJ concentrations must reflect a defective signaling ability, rather than a defect in the physical interaction. For E18K/E25K, its poorer histidine-binding Kd value might additionally contribute to its poor transport activity. It is interesting to note that K20E/E25K has a higher transport and ATPase-inducing activities than the wild type HisJ, 1.7- and 1.4-fold, respectively. A higher level of transport is also displayed by G24A/E25K despite its normal level of ATP hydrolysis, indicating that the level of ATPase activity is not strictly or exclusively correlated with the level of transport, as will also be discussed later.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Effect of mutations in Lobe I


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of mutations in lobe I of HisJ. A, in vitro L-histidine transport was assayed in PLS reconstituted with purified HisQMP2 in the presence of various HisJs as indicated (20 µM) and 20 µM L-[3H]histidine at 37 °C. An ATP regenerating system was included in the PLS as described under "Materials and Methods." B, the initial rate of ATP hydrolysis was measured in PLS reconstituted with pure HisQMP2 as described under "Materials and Methods," in the presence of various HisJ as indicated (10 µM). The HisJ-stimulated activity has been corrected for the value of the intrinsic activity (in the absence of HisJ), which was <6% of the HisJ-stimulated activity, and is expressed as percentage activity relative to that of the wild type.

In conclusion, it appears that lobe I interacts with HisQMP2 (at least through the protruding loop), supplies at least part of the signal responsible for inducing the ATPase activity of HisQMP2, and is also involved in some aspect of the transport mechanism.

HisJ Mutations in Lobe II-- In the x-ray crystal structure of HisJ, residue Arg-154 in lobe II is surface exposed; it and the loop in lobe I are located on the same side of HisJ as the opening of the histidine-binding pocket (15). Earlier genetic studies had identified a mutation in hisJ, hisJ5625, that allows normal binding but is unable to support transport (24, 26). The DNA sequence of mutation hisJ5625 was determined to consist of a replacement of Arg-154 with a cysteine residue (R154C). To confirm the importance and properties of this residue, two additional replacements of Arg-154 (one neutral and one acidic) were created by in vitro mutagenesis: R154S and R154D. Both were shown to bind histidine as well as the wild type does and, like R154C, to be defective in transport (27). Therefore, Arg-154 is a possible candidate for being involved in an interaction with HisQMP2. Is it also involved in the signaling mechanism? If this were the case, substitution of Arg-154 should affect the levels of in vitro stimulation of the ATPase activity. Therefore, we purified these mutant HisJs and tested them for a variety of properties. First of all, they were tested by physicochemical techniques for not being grossly altered. They were shown to be indistinguishable from wild type HisJ with regard to their tryptophan fluorescence and their UV spectra (data not shown). Thus, in agreement with the fact that Arg-154 is on the surface and with their unchanged affinity for L-histidine, they are unlikely to have major structural alterations. Fig. 2A compares the ability of R154S and R154D to induce the ATPase activity with that of wild type HisJ. Essentially no ATPase was induced by R154S or R154D. The ability to transport was also measured (Fig. 2B); both are essentially unable to support transport. Table II summarizes these data together with results obtained for other lobe II mutants. As an additional indication of the importance of Arg-154 in interaction, evidence of physical interaction was also obtained by chemical cross-linking to HisQ using formaldehyde (23, 28). Table II shows that, in contrast to the wild type (and similarly to R154C), cross-linking is essentially absent in R154S and R154D. Thus, it appears that Arg-154 is directly involved in the interaction with HisQMP2.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of mutations in lobe II of HisJ. A, the initial rate of ATP hydrolysis was measured and expressed as described in Fig. 1. B, in vitro L-histidine transport was assayed and expressed as described in Fig. 1. Inset, in vitro L-histidine transport in the presence of 20 µM purified wild type HisJ or R154D or D149N was measured as described above except that about twice as much ATP-regenerating component was included.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Effect of mutations in Lobe II

In order to determine whether other residues are also part of the interaction site, Asp-149, which is very close to Arg-154 in the three-dimensional structure of HisJ, was replaced by in vitro mutagenesis with alanine or asparagine, and the biochemical properties of the respective mutant HisJs (D149A and D149N) were tested. Similarly to the R154 replacements, both have normal histidine-binding affinity (Table II), yet both are defective in histidine transport (Fig. 2B and Table II). They are also defective in stimulating ATPase activity, although not completely (Fig. 2A). In addition, both are defective in chemical cross-linking to HisQ, having approximately 40% of the cross-linking efficiency of the wild type (Table II). Thus, residue Asp-149 appears to be involved in the interaction.

The fact that several of the properties of D149A and D149N are only partially defective indicates that these mutant proteins are still capable of interacting with HisQMP2, although at a much reduced level. A method was developed to measure the affinity of HisJ for HisQMP2 by measuring the level of ATPase induction using varying concentrations of HisJ (10, 11). Using this method, D149A was shown to interact with HisQMP2 with an affinity essentially identical to that of wild type HisJ, whereas its maximal level of ATPase induction is 50% of that of the wild type. This result indicates that, similarly to the loop mutants in lobe I, D149A is specifically defective in signaling for ATP hydrolysis. Interestingly, when D149A is greatly overexpressed in strains that also produce wild type HisJ, it inhibits histidine transport in vivo (data not shown). Such a negative dominance characteristic supports the notion that D149A is still able to interact with the membrane-bound complex. Negative dominance was also observed for R154S, R154C, and R154D. Because physical interaction appears to be grossly altered in the Arg-154 replacement mutants, it is possible that the residual interaction observed with these proteins, as expressed by negative dominance, occurs through lobe I. In the case of R154S, it was also found that, despite its inhibition of transport by wild type HisJ, it does not inhibit ATP hydrolysis induced by liganded wild type HisJ (Fig. 3), a result similar to what has been found for wild type unliganded HisJ (11). Because unliganded HisJ has been shown to interact with HisQMP2 (23), a likely explanation is that the interaction between HisQMP2 and unliganded HisJ or liganded R154S is via lobe I. 


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3.   Dominant negative effects of HisJ mutant R154S on transport but not ATP hydrolysis by HisQMP2. A, in vitro L-histidine transport was assayed as described under "Materials and Methods," in the presence of wild type HisJ and mutant R154S at the indicated concentrations. B, the initial rate of ATP hydrolysis by purified HisQMP2 was assayed at 37 °C in the presence of 6 µM wild type HisJ and various concentrations of mutant HisJ (R154S) as indicated. Both transport and ATPase activity were expressed as percent activity relative to the activity obtained with wild type HisJ alone.

The alpha -helical patch on the surface where Arg-154 is located also includes Asp-144, which may also be involved in interaction (15). HisJ mutant D144N transports poorly D-histidine and azaserine (a histidine permease transport substrate). Its ability to be cross-linked to HisQ is also poor (Table II). Thus, it appears that Asp-144 is also part of the interaction site on HisJ. However, it is interesting to note that D144N binds and translocates L-histidine normally, and it can bind azaserine normally, although it cannot translocate it (data not shown). This behavior can be explained if the precise conformation of the closed liganded form were to vary with the nature of the ligand, resulting in efficient translocation if the ligand is L-histidine, but not if it is D-histidine or azaserine. The effect of ligand on conformation is discussed at length below.

In contrast to the above mentioned residues, Asp-156, which is very close to Arg-154 and Asp-149 in the three-dimensional structure, does not seem to be involved in the interaction. Replacement of Asp-156 with alanine (D156A) does not result in a significant change in properties, both with respect to ATPase activity, transport, and cross-linking ability (Fig. 2 and Table II). Thus, the region of lobe II where Asp-144, Asp-149, and Arg-154 are located is involved in stimulating the ATPase activity and the region of contact is both small and precisely defined, excluding the nearby residue Asp-156.

The Membrane-bound Complex Senses the Spatial Relationship between the Lobes, Which Depends on the Nature of the Ligand

The results presented above show that both HisJ lobes are involved in the interaction with HisQMP2. Considering that the loop in lobe I and residues Arg-154 and Asp-149 in lobe II are aligned on the same face of the liganded molecule, it is to be expected that HisQMP2 is capable of interacting with these elements simultaneously. HisQMP2 responds differently to unliganded and histidine-liganded HisJ by inducing different levels of ATPase (11) and by exhibiting different levels of cross-linking to HisJ; however, the individual surface structures of the two lobes, as is true of other binding proteins (29, 30), are essentially unchanged upon ligation (14). Therefore, it is likely that the relative position of the lobes is an important element in the interaction process that dictates the efficiency of ATP hydrolysis and transport. HisQMP2 presumably distinguishes between various receptor forms by "reading" the relative distance between the two lobes or their rotation angles. It has been shown (through the use of conformation-specific monoclonal antibodies) that HisJ assumes measurably different liganded conformations upon binding different ligands (8). It is possible that the various liganded forms would have variations in the relative positions between the two lobes and, as a consequence, stimulate different levels of ATPase. Fig. 4A shows that indeed, increasing concentrations of different ligands in the presence of a limiting HisJ concentration result in different levels of ATPase activity for each ligand, the relative ratios being 1.0 for L-histidine, 0.46 for L-arginine, and 0.17 for L-lysine. In a particularly striking example, HisJ that is fully liganded with D-histidine stimulates ATP hydrolysis by HisQMP2 as well as it does when it is liganded with L-histidine (data not shown), even though it has a much poorer affinity for D-histidine than for L-histidine (31). Because saturating ligand concentrations were reached, the ATPase differences do not reflect different ligand-binding affinities of HisJ. If an excess of liganded HisJ is used, thus saturating the membrane-bound complex with liganded receptor, the differences persist, indicating that differences in inducing ability are a property of the liganded forms (see Fig. 6; data not shown). The fact that the maximum levels of activity at saturating liganded receptor concentration are different excludes the possibility that the various closed liganded forms have identical conformations but reach different equilibria between the open liganded and closed liganded forms (i.e. different amounts of each liganded form), thus resulting in different levels of induction.3 Therefore, it is reasonable to conclude that the diverse levels of ATPase induction by various ligands are due to variations in the signaling ability of different liganded forms of HisJ, reflecting its different conformations and occurring independently of the respective binding affinities.


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of various ligands on the ability of HisJ to stimulate ATP hydrolysis and transport by HisQMP2. A, the ATPase activity of purified HisQMP2 was assayed at 37 °C in the presence of 20 µM wild type HisJ and various concentrations of L-histidine (solid circles), L-arginine (open circles), and L-lysine (open squares). B, in vitro L-histidine transport was assayed in PLS reconstituted from purified wild type HisQMP2 at 37 °C in the presence of 20 µM purified wild type HisJ and 20 µM L-[3H]histidine (open circles) or L-[3H]arginine (crosses), or 20 µM LAO and 20 µM L-[3H]arginine (closed diamonds).

Several alternative explanations for the different levels of ATPase induction by different ligands were considered and excluded. A possibility is that the various liganded forms of the receptor are identical and interact identically with HisQMP2, but ligands affect the ATPase activity by binding directly (or subsequently) with different affinities to a hypothetical site in HisQMP2, unrelated to the interaction site of the receptor. This possibility is excluded because the addition of free L-histidine and L-lysine (up to 20 mM) (11) or of L-arginine (data not shown) in the absence of HisJ does not induce the ATPase activity. Interestingly, L-arginine actually inhibits the intrinsic ATPase activity with a Ki of 10 mM (Fig. 5), which is an indication that a ligand-binding site is present and accessible in the membrane-bound complex in the absence of HisJ, although it is not involved in the ATPase induction process. The possibility that ligands affect ATPase activity through a hypothetical site in HisQMP2 that requires HisJ to be present was also excluded, because the addition of L-lysine to L-histidine-liganded HisJ in comparable concentrations did not decrease the level of ATPase stimulation from that characteristic of histidine to that characteristic of lysine (data not shown).4 A final possibility is that there are multiple independent interaction sites on HisQMP2, each separately responsible for interacting with different liganded HisJs. However, the relative ATPase activities in the presence of 12 µM HisJ are 1.0, 0.25, and 0.3 µmol/min/mg in the presence of 12 µM L-histidine, 2 mM L-lysine, and both 2 mM L-lysine and 12 µM L-histidine, respectively (from Fig. 6, at 12 µM HisJ), indicating that there is an overall single interaction site on HisQMP2, thus also excluding this possibility. Therefore, the different ATPase-stimulating abilities are due to different modes of interaction of each liganded receptor and, presumably, reflect varying relative spatial positions of the lobes.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 5.   L-Arginine inhibits the intrinsic ATPase activity of HisQMP2. The ATPase activity of purified HisQMP2 was assayed as described in Fig. 1 at 37 °C in the absence of binding protein and in the presence of various concentrations of L-arginine as indicated.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 6.   Excess free L-lysine does not affect the L-histidine-liganded HisJ-stimulated ATPase activity of HisQMP2. The ATPase activity of purified HisQMP2 was assayed at 37 °C as described in Fig. 1 in the presence of various concentrations of wild type HisJ as indicated in the abscissa and either 2 mM L-histidine (solid squares), 2 mM L-lysine (solid diamonds), or a mixture of 12 µM L-histidine and 2 mM L-lysine (open circles).

It is important to note that the different levels of ATPase activity displayed by HisJ liganded with different ligands does not necessarily result in correspondingly different levels of transport in vitro. As shown in Fig. 4B, 20 µM of HisJ transports L-histidine and L-arginine at the same rate, and even the closely related protein LAO transports L-arginine at the same rate under the same conditions. Therefore, the ATPase activity is not the only factor that affects the rate of transport.

The Interaction with HisJ Has Functions in Addition to Stimulating ATP Hydrolysis

Having established that both receptor lobes are involved in interaction and the subsequent functions of inducing the ATPase activity and translocating the ligand, we raised the question of whether these functions could be separated and possibly assigned to the individual lobes. Theoretically, one contact site would be sufficient to send the ATPase stimulation signal; therefore, the existence of two contact sites might reflect additional and/or separable functions of HisJ. The current model is that the function of HisJ is to send a signal that initiates ATP hydrolysis, which drives a conformational change leading to substrate translocation. In this case, ATP hydrolysis would be sufficient for translocation in the absence of HisJ. Here, we address this question by testing whether ligands can be efficiently translocated in the presence of ATP hydrolysis but in the absence of HisJ, using mutant HisQMP2 complexes that hydrolyze ATP at high rates in the absence of HisJ. If translocation is defective, a function separable from the induction of ATP hydrolysis should be postulated and the question asked as to which of the two lobes is involved.

Several mutations in hisP, which result in constitutive ATP hydrolysis (18), are known to allow transport in the absence of HisJ in vivo if the levels of the membrane-bound complex are elevated (32).5 The level of in vivo transport is poor, however, suggesting that an additional factor(s) is involved. A more careful analysis of this question is possible using the in vitro transport assay with PLS reconstituted with purified HisQMP2. HisQMP2 was purified from both the wild type and various constitutive hisP mutant strains and reconstituted into PLS. Table III shows that the intrinsic ATPase activity (i.e. in the absence of HisJ) is much higher in PLS containing HisP mutant proteins P172T and T205A than in wild type HisP, in agreement with previous data (18), and is in fact even higher than that of the wild type complex in the presence of HisJ. Therefore, if the only role of HisJ were the initiation of ATP hydrolysis, the level of constitutive ATPase would be sufficiently high in these mutants that they should translocate as efficiently as the complete wild type system, even in the absence of receptor. However, transport activity was as low as that of wild type HisQMP2 in the absence of HisJ. Therefore, ATP hydrolysis is insufficient to allow substrate translocation. In the presence of HisJ, both the ATPase activity of the wild type complex and its ability to transport are greatly stimulated (9- and 1000-fold, respectively). In contrast, whereas the ATPase activity of the two mutant complexes is stimulated only slightly,6 transport increases considerably: 55- and 32-fold for P172T and T205A, respectively. This result supports the notions that HisJ has one or more functions necessary for the translocation process7 in addition to initiating ATP hydrolysis and that there is no direct correlation between the activity levels of ATPase and of transport.

                              
View this table:
[in this window]
[in a new window]
 
Table III
ATPase and transport activities in signal-independent HisP mutants
The membrane-bound complex containing either wild type HisQMP2 or mutant HisQMP*2 with HisP mutations P172T or T205A were purified and reconstituted into PLS as described (21). Unliganded wild type and various mutant HisJs were purified by HPLC and their ability to stimulate ATP hydrolysis and in vitro L-histidine transport was assayed for the wild type HisQMP2 or mutant HisQMP*2 as described under "Materials and Methods."

Which of the HisJ lobes takes part in the additional function(s)? The effect of mutant HisJs that have retained at least a partial ability to interact with the membrane-bound complex was tested with respect to both the translocation and ATPase activities of HisP mutant proteins P172T and T205A. Lobe II mutant protein D149A, which is defective in interaction, was used to test whether it could restore translocation activity in HisP mutants P172T and T205A. Table III shows that it cannot, despite the fact that it allows the wild type to translocate, although poorly. The effect of D149A is indistinguishable from that of R154S, which interacts very poorly. Lobe II mutant D156A, which is outside of the interaction area, behaves like wild type HisJ in its ability to allow transport in the constitutive ATPase mutant complexes P172T and T205A. These data indicate that lobe II is involved in the presumed additional HisJ function(s) and that D156A is external to the region responsible for this function(s). The corresponding experiment with the available mutations in lobe I is not possible because all such mutants retain a considerable level of activity (Table I).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Various models for the mechanism of action of periplasmic permeases have been proposed in the past; they have been largely based on genetic and physiological analyses. We developed over recent years several biochemical and biophysical methodologies for the study of various aspects of the histidine permease. Several conclusions that can be extended to periplasmic permeases in general can be drawn by combining the results presented here with those from past studies from our and other laboratories: 1) the soluble receptor carries two sites for interaction with the membrane-bound complex, one on each lobe; 2) the two receptor sites are on the same side as the opening of the ligand-binding pocket; 3) the nature of the ligand affects the final shape of the liganded receptor; 4) the receptor, in its various liganded forms, varies its mode of stimulating the complex; 5) the membrane-bound complex recognizes the relative spatial organization of the two lobes, probably their distance from each other, and responds elevating different levels of ATPase activity; 6) there is no direct correlation between the affinities of the receptor for various ligands and the efficiencies of their translocation; 7) there is no direct relationship between ATPase and transport levels; 8) the receptor is involved in a function(s) in addition to stimulating the ATPase activity; and 9) the membrane-bound complex carries a ligand-recognition site. It is worthwhile to discuss various aspects of the above conclusions.

Making specific reference mostly to the histidine permease, it is clear that a unique interaction site on a single lobe of HisJ would have to change its surface structure in order to indicate its ligation status. However, comparison of the liganded and unliganded structures shows that there is no conformational change within each of the lobes upon binding of substrate (14, 15). It is more likely that the relative positions of the two lobes supply significant information to HisQMP2 by assuming a variety of overall conformations upon ligand binding, reflecting the presence and the nature of a ligand.8 Such distance variations are detected in vitro by biochemical and structural assays; they must convey information that is recognized by the membrane-bound complex, which responds with specific levels of ATP hydrolysis and transport efficiency. The involvement of both lobes of the maltose-binding protein in interaction has also been proposed using genetics analysis (34), and differences in various liganded forms were detected using functional assays in solution, suggesting that different ligands might result in different degrees of lobe closure (35). Thus, varying the response according to the nature of the transported substrate may be a general feature of periplasmic permeases.

The ligand appears to modulate the signaling mode indirectly, by varying the nature of the interaction, resulting in an unpredictable relationship between the affinity of the ligand for the receptor and the efficiency of transport. An analysis of mutant HisJs with altered affinities for histidine and various levels of translocation also shows that there is no correlation between the Km for transport and the Kd for ligand binding, which is contrary to what has been commonly assumed (5, 36). Table IV, in which mutant HisJs are listed in the order of decreasing affinity for L-histidine (Kd), together with their respective Km values for transport, illustrates this lack of correlation, as seen from the respective Kd/Km ratios. Presumably the various HisJs listed in Table IV assume different liganded conformations depending on their respective mutations, thus accounting for the lack of direct correlation with the transport Km values. Resolution of the crystal structure of these liganded forms would provide ultimate support for this notion. In conclusion, both the ligand-binding specificity and the final conformation of the liganded receptor, with the distance between the two lobes being especially important, are likely to exert an important effect in modulating the extent of permease performance.

                              
View this table:
[in this window]
[in a new window]
 
Table IV
Affinity of L-histidine for binding to HisJ and for transport

The findings that the exact conformation of the liganded receptor depends on the nature of the ligand, that the induced ATPase levels are not directly related to the affinities of the respective ligands for the receptor, and that HisJ provides an additional function, beyond that of inducing ATP hydrolysis, is important for understanding the mechanism of action of these permeases. A possible model for periplasmic transport would assign to the liganded receptor the function of a valve or a one-way gate imposing the unidirectional flow of ligand (from outside to inside) (see also Ref. 37). It is proposed that an essential aspect of such a one-way mechanism is to release the ligand within a closed chamber formed by the two lobes of the receptor interacting with the periplasmic face of the membrane-bound complex. This step would result in concentrating the released ligand in the millimolar to molar range, depending on the actual size of this presumed chamber. If formation of a translocation channel in HisQMP2 were part of the translocation mechanism, as has already been hypothesized (37), the released ligand would have gained access in high concentration to the mouth of this channel, such that it would be able to diffuse down its concentration gradient from a small restricted area to a larger space, the cytosol. In this model, inducing the receptor to release its ligand into a closed chamber by opening its lobes is an essential aspect of the translocation process. We speculate that i) the two receptor lobes provide anchoring sites (as opposed to being "signaling" sites only) for the receptor on the periplasmic surface of HisQMP2; ii) when energy is applied (by ATP hydrolysis induced by interaction with the optimally placed lobes of the liganded receptor), a channel is formed within HisQMP2; iii) the formation of the channel forces open the lobes of the receptor, causing the release of the bound ligand to a high concentration into a closed chamber; iv) an optimal conformation of the lobes of the liganded receptor is required for ligand release; v) the ligand moves through the channel down its concentration gradient; and vi) the channel closes spontaneously (or using additional ATP hydrolysis energy). Thus, this model takes into consideration why ATP hydrolysis per se is insufficient to complete the act of translocation. This is in contrast to models in which the interaction of the binding protein is reserved for activating ATP hydrolysis and consequently exposing an occluded binding site in the membrane complex (e.g. Ref. 33). The model also suggests a possible explanation for the lack of correlation between induced ATPase levels and transport activities: the liganded HisJ conformation that is optimal for signaling and inducing ATP hydrolysis is different from that in which the lobes are forced open to release the ligand.

The presence of a ligand-binding site within the membrane-bound complex of periplasmic permeases has been proposed several times in the past (10, 18, 38, 39). It appears that such a site is accessible in the absence of HisJ because signal-independent ATPase constitutive mutants can translocate ligand in the absence of the receptor as long as the ligand concentration is high (32). In the case of the histidine permease, additional evidence for such a site has been provided (Ref. 10 and Fig. 5). In the absence of the receptor, the concentration of ligand at the mouth of the presumed channel would not be sufficiently high to create a concentration gradient, thus explaining the poor translocating ability of receptor-independent mutants. Therefore, the additional essential function of the soluble receptor may be that of delivering ligands in high concentration to such a site, rather than, or in addition to, exposing it. This site might also be involved in regulating the speed of ligand movement within the presumed channel by binding ligands with discriminating affinities.

The hypothesis that the loop in lobe I and a patch in lobe II form the interaction site with HisQMP2 (15) is strongly supported by the data presented here. A specific speculation can be made concerning the function of Arg-154 and Asp-149, two lobe II residues that are known to be involved in interaction and that show dramatic changes upon interaction. Consistent with their involvement in an essential function, these two residues are conserved in HisJ and LAO. The respective crystal structures show that Arg-154 is totally exposed to the solvent, protruding from the protein and free of hydrogen bonds in the unliganded form (14, 15); the side chain of Asp-149 is partially exposed to the solvent. When the protein is liganded, the side chain of Asp-149 forms a hydrogen bond with the side chain of Arg-154, thus locking the side chain of Arg-154 at a fixed angle. Such a fixed orientation of the Arg-154 side chain might be a critical and specific feature of the interaction of HisJ with the membrane-bound complex. In support of this notion, substitution of Arg-154 with cysteine, serine, or aspartic acid abolishes transport activity, and substitution of Asp-149 with alanine or asparagine strongly decreases transport.

It is interesting that despite the close similarity between prokaryotic and eukaryotic ABC transporters, there is no evidence, as of now, for the presence of a soluble receptor in the eukaryotic transporters. As discussed previously (37), an important function of the soluble receptor in prokaryotes might be that of trapping and concentrating scarce substrates at the mouth of the translocating channel. In this respect, a remarkable evolutionary relationship has been found between periplasmic transport systems and the metabotropic glutamate receptor (40), and between the transport systems and the parathyroid Ca2+-sensing receptor (41). These eukaryotic receptors have been found to contain large extracellular substrate-binding domains that are homologous to periplasmic receptors. Indeed, on the basis of the x-ray structure of LAO, a model has been proposed for the glutamate-binding domain of the ionotropic glutamate receptor, which has been presumed to trap the signaling molecule and initiate a signal transduction mechanism that may be analogous to that of bacterial periplasmic permeases (40). Therefore, understanding the molecular mechanism of interaction and signaling between the receptor and the membrane-bound complex in these prokaryotic systems may provide useful evidence toward understanding the mechanism of action of related eukaryotic membrane receptors.

    ACKNOWLEDGEMENTS

We thank Kishiko Nikaido, who performed the cross-linking experiments, and Dr. David Kreimer for helpful discussions.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK12121 (to G. F.-L. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger These authors contributed equally to this work.

§ Present address: Chiron Corp., 4560 Horton St., Emeryville, CA 94608.

Present address: Peptor Ltd., Kiryat Weizmann, Rehovot, Israel 76326.

parallel To whom correspondence should be addressed. Tel.: 510-642-1979; Fax: 510-643-7935; E-mail: giovanna{at}uclink4.berkeley.edu.

The abbreviations used are: HisQMP2, membrane-bound complex containing HisQ, HisM, and HisP; LAO, lysine-, arginine-, ornithine-binding protein; PLS, proteoliposomes.

2 It has been shown that Kd variations of at least 1 order of magnitude are necessary to affect transport (24).

3 Recent physicochemical studies have also shown that the various open liganded and their respective closed liganded forms reach the same equilibrium (D. I. Kreimer and F. G.-L. Ames, manuscript in preparation).

4 This experiment can be performed only with this combination of ligands because L-lysine cannot replace HisJ-liganded L-histidine undeer these conditions because its Kd value is 10,000-fold higher than that of L-histidine.

5 Mutations allowing transport in the absence of the receptor and causing constitutive ATPase activity have also been characterized for the maltose permease. In contrast to those described for the histidine permease, the maltose permease mutations are located in the integral membrane components and carry multiple mutations (33).

6 These mutant HisP proteins have a maximum stimulated hydrolyzing ability of about 4 µmol/min/mg of HisQMP2, corresponding to a turnover rate of about 16 s-1.

7 These in vitro results match previously obtained in vivo results that showed that the presence of HisJ greatly increases the transport activity of these mutants (26, 32).

8 It is possible that additional structural information is supplied within each lobe upon ligation, because it cannot be excluded that there are conformational changes within the individual lobes that are not detected in the crystal structure.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

  1. Ames, G. F.-L., Mimura, C., and Shyamala, V. (1990) FEMS Microbiol. Rev. 75, 429-446[CrossRef]
  2. Hyde, S. C., Emsley, P., Hartshorn, M. J., Mimmack, M. M., Gileadi, U., Pearce, S. R., Gallagher, M. P., Gill, D. R., Hubbard, R. E., and Higgins, C. F. (1990) Nature 346, 362-365[CrossRef][Medline] [Order article via Infotrieve]
  3. Doige, C. A., and Ames, G. F.-L. (1993) Annu. Rev. Microbiol. 47, 291-319[CrossRef][Medline] [Order article via Infotrieve]
  4. Higgins, C. F. (1992) Annu. Rev. Cell Biol. 8, 67-113[CrossRef]
  5. Boos, W., and Lucht, J. M. (1996) in Escherichia coli and Salmonella: Cellular and Molecular Biology (Neidhardt, F. C., ed), pp. 1175-1209, American Society for Microbiology, Washington, D. C.
  6. Ames, G. F.-L. (1986) Annu. Rev. Biochem. 55, 397-425[CrossRef][Medline] [Order article via Infotrieve]
  7. Baichwal, V., Liu, D., and Ames, G. F.-L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 620-624[Abstract]
  8. Wolf, A., Lee, K. C., Kirsch, J. F., and Ames, G. F.-L. (1996) J. Biol. Chem. 271, 21243-21250[Abstract/Free Full Text]
  9. Nikaido, K., Liu, P.-Q., and Ames, G. F.-L. (1997) J. Biol. Chem. 272, 27745-27752[Abstract/Free Full Text]
  10. Liu, C. E., and Ames, G. F.-L. (1997) J. Biol. Chem. 272, 859-866[Abstract/Free Full Text]
  11. Liu, C. E., Liu, P.-Q., and Ames, G. F.-L. (1997) J. Biol. Chem. 272, 21883-21891[Abstract/Free Full Text]
  12. Higgins, C. F., and Ames, G. F.-L. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 6038-6042[Abstract]
  13. Quiocho, F., and Ledvina, P. S. (1996) Mol. Microbiol. 20, 17-25[Medline] [Order article via Infotrieve]
  14. Oh, B.-H., Kang, C.-H., De Bondt, H., Kim, S.-H., Nikaido, K., Joshi, A., and Ames, G. F.-L. (1994) J. Biol. Chem. 269, 4135-4143[Abstract/Free Full Text]
  15. Oh, B.-H., Pandit, J., Kang, C.-H., Nikaido, K., Gokcen, S., Ames, G. F.-L., and Kim, S.-H. (1993) J. Biol. Chem. 268, 11348-11353[Abstract/Free Full Text]
  16. Yao, N., Trakhanov, S., and Quiocho, F. A. (1994) Biochemistry 33, 4769-4777[Medline] [Order article via Infotrieve]
  17. Kang, C.-H., Shin, W.-C., Yamagata, Y., Gokcen, S., Ames, G. F.-L., and Kim, S.-H. (1991) J. Biol. Chem. 266, 23893-23899[Abstract/Free Full Text]
  18. Petronilli, V., and Ames, G. F.-L. (1991) J. Biol. Chem. 266, 16293-16296[Abstract/Free Full Text]
  19. Nikaido, K., and Ames, G. F.-L. (1992) J. Biol. Chem. 267, 20706-20712[Abstract/Free Full Text]
  20. Peterson, G. L. (1977) Anal. Biochem. 83, 346-356[Medline] [Order article via Infotrieve]
  21. Liu, C. E. (1996) Mechanism of Action of the Periplasmic Histidine Permease in Salmonella typhimurium.Ph.D. thesis, University of California, Berkeley
  22. Wolf, A., Shaw, E. W., Oh, B.-H., De Bondt, H., Joshi, A. K., and Ames, G., F.-L. (1995) J. Biol. Chem. 270, 16097-16106[Abstract/Free Full Text]
  23. Ames, G. F.-L., Liu, C. E., Joshi, A. K., and Nikaido, K. (1996) J. Biol. Chem. 271, 14264-14270[Abstract/Free Full Text]
  24. Kustu, S. G., and Ames, G. F.-L. (1974) J. Biol. Chem. 249, 6976-6983[Abstract/Free Full Text]
  25. Ames, G. F.-L., Noel, K. D., Taber, H., Spudich, E. N., Nikaido, K., Afong, J., and Ardeshir, F. (1977) J. Bacteriol. 129, 1289-1297[Medline] [Order article via Infotrieve]
  26. Ames, G. F.-L., and Spudich, E. N. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 1877-1881[Abstract]
  27. Prossnitz, E. (1989) In Vitro Reconstitution of the Histidine Transport System of Salmonella typhimurium.Ph.D. thesis, University of California, Berkeley
  28. Prossnitz, E., Nikaido, K., Ulbrich, S. J., and Ames, G. F.-L. (1988) J. Biol. Chem. 263, 17917-17920[Abstract/Free Full Text]
  29. Quiocho, F. A., Wilson, D. K., and Vyas, N. K. (1989) Nature 340, 404-407[CrossRef][Medline] [Order article via Infotrieve]
  30. Vyas, M. N., Vyas, N. K., and Quiocho, F. A. (1994) Biochemistry 33, 4762-4768[Medline] [Order article via Infotrieve]
  31. Ames, G. F.-L., and Lever, J. E. (1972) J. Biol. Chem. 247, 4309-4316[Abstract/Free Full Text]
  32. Speiser, D. M., and Ames, G. F.-L. (1991) J. Bacteriol. 173, 1444-1451[Medline] [Order article via Infotrieve]
  33. Covitz, K.-M. Y., Panagiotidis, C. H., Hor, L.-I., Reyes, M., Treptow, N. A., and Shuman, H. A. (1994) EMBO J. 13, 1752-1759[Abstract]
  34. Hor, L. I., and Shuman, H. A. (1993) J. Mol. Biol. 233, 659-670[CrossRef][Medline] [Order article via Infotrieve]
  35. Hall, J. A., Ganesan, A. K., Chen, J., and Nikaido, H. (1997) J. Biol. Chem. 272, 17615-17622[Abstract/Free Full Text]
  36. Furlong, C. E. (1987) in Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (Neidhardt, F. C., ed), pp. 768-796, American Society for Microbiology, Washington, D. C.
  37. Ames, G. F.-L., and Lecar, H. (1992) FASEB J. 6, 2660-2666[Abstract/Free Full Text]
  38. Higgins, C. F., Haag, P. D., Nikaido, K., Ardeshir, F., Garcia, G., and Ames, G. F.-L. (1982) Nature 298, 723-727[Medline] [Order article via Infotrieve]
  39. Treptow, N. A., and Shuman, H. A. (1985) J. Bacteriol. 163, 654-660[Medline] [Order article via Infotrieve]
  40. O'Hara, P. J., Sheppard, P. O., Thøgersen, H., Venezia, D., Haldeman, B. A., McGrane, V., Houmed, K. M., Thomsen, C., Gilbert, T. L., and Mulvihill, E. R. (1993) Neuron 11, 41-52[Medline] [Order article via Infotrieve]
  41. Brown, E. M., Gamba, G., Riccardi, D., Lombardi, M., Butters, R., Kifor, O., Sun, A., Hedlger, M. A., Lytton, J., and Herbert, S. C. (1993) Nature 366, 575-580[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.