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Originally published In Press as doi:10.1074/jbc.M600754200 on July 28, 2006

J. Biol. Chem., Vol. 281, Issue 39, 29213-29220, September 29, 2006
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Identification of the Cadaverine Recognition Site on the Cadaverine-Lysine Antiporter CadB*

Waraporn Soksawatmaekhin{ddagger}, Takeshi Uemura{ddagger}, Natsuko Fukiwake{ddagger}, Keiko Kashiwagi§, and Kazuei Igarashi{ddagger}1

From the {ddagger}Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan and the §Faculty of Pharmaceutical Sciences, Chiba Institute of Science, 15-8 Shiomi-cho, Choshi, Chiba 288-0025, Japan

Received for publication, January 25, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Amino acid residues involved in cadaverine uptake and cadaverine-lysine antiporter activity were identified by site-directed mutagenesis of the CadB protein. It was found that Tyr73, Tyr89, Tyr90, Glu204, Tyr235, Asp303, and Tyr423 were strongly involved in both uptake and excretion and that Tyr55, Glu76, Tyr246, Tyr310, Cys370, and Glu377 were moderately involved in both activities. Mutations of Trp43, Tyr57, Tyr107, Tyr366, and Tyr368 mainly affected uptake activity, and Trp41, Tyr174, Asp185, and Glu408 had weak effects on uptake. The decrease in the activities of the mutants was reflected by an increase in the Km value. Mutation of Arg299 mainly affected excretion, suggesting that Arg299 is involved in the recognition of the carboxyl group of lysine. These results indicate that amino acid residues involved in both uptake and excretion, or solely in excretion, are located in the cytoplasmic loops and the cytoplasmic side of transmembrane segments, whereas residues involved in uptake were located in the periplasmic loops and the transmembrane segments. The SH group of Cys370 seemed to be important for uptake and excretion, because both were inhibited by the existence of Cys125, Cys389, or Cys394 together with Cys370. The relative topology of 12 transmembrane segments was determined by inserting cysteine residues at various sites and measuring the degree of inhibition of transport through crosslinking with Cys370. The results suggest that a hydrophilic cavity is formed by the transmembrane segments II, III, IV, VI, VII, X, XI, and XII.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Polyamines (putrescine, spermidine, and spermine) play important roles in cell proliferation and differentiation (1-3), and cellular polyamine content is regulated by biosynthesis, degradation, and transport (4-6). With regard to transport, the properties of three polyamine transport systems were characterized in Escherichia coli (7-9). They include spermidine-preferential and putrescine-specific uptake systems, which belong to the family of ATP-binding cassette transporters, and a protein, PotE, involved in the excretion of putrescine by a putrescine-ornithine antiporter activity. Furthermore, it has been reported that cadaverine and aminopropylcadaverine function as compensatory polyamines for cell growth (10), and CadB, a cadaverine-lysine antiporter, is strongly involved in cell growth at acidic pH like PotE (11, 12). Analogous to the speF-potE operon (13), cadB is one component of the cadBA operon, in which cadA encodes lysine decarboxylase (14, 15). Expression of the cadBA operon is regulated by a positive regulator CadC, which is encoded by adjacent cadC gene (14, 15), and is induced by acidic pH and lysine (11). The cadBA and speF-potE operons contribute to an increase in pH of the extracellular medium through excretion of cadaverine and putrescine, the consumption of a proton, and a supply of carbon dioxide during the decarboxylation reaction (12, 16) so that expression of these two operons is important for cell growth at acidic pH. It is also shown that expression of the speF-potE operon is increased when the cadBA operon is not induced (12). Other antiport systems for basic amino acids and their amine products (histidine/histamine and arginine/agmatine), coupled with decarboxylation, have been reported to generate a proton motive force in Lactobacillus buchneri and E. coli (17, 18). At neutral pH, both CadB and PotE have cadaverine and putrescine uptake activity and contribute to cell growth of a polyamine-requiring mutant MA261 (12, 13). Although we have recently studied the function of CadB in detail (12), the structure of CadB is not well understood. When homology between the amino acid sequences of CadB and PotE was compared, high sequence similarity was observed (30.7% overall identity) (12). We have previously identified amino acid residues involved in putrescine uptake and excretion by PotE (8). In this study, we have identified a likely cadaverine recognition site on CadB based on the information concerning putrescine recognition by PotE.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid and Site-directed Mutagenesis of cadB Gene—Plasmid pMWcadB was prepared using pMW119 (Nippon Gene Co.) as described previously (12). Site-directed mutagenesis of cadB gene was carried out with the QuikChange site-directed mutagenesis kit (Stratagene). Mutations were confirmed by DNA sequencing using a Seq 4 x 4 personal sequencing system (Amersham Biosciences) or CEQ8000 DNA genetic analysis system (Beckman Coulter). A list of oligonucleotide primers for mutagenesis has not been included but is available from the authors upon request. pUCcadB was constructed by inserting 1.5 kbp BamHI and EcoRI fragments of pMWcadB into the same restriction sites of pUC119 (Takara Shuzo Co.).

Culture Conditions—E. coli JM109 (recA1 supE44 endA1 gyrA96 relA1 thi {Delta} (lac-proAB)/F' (traD36 proAB+ lacIq lacZ{Delta}M15)) (19) containing pMWcadB was cultured in a 19-amino acid supplemented medium containing 0.4% glucose for uptake by intact cells or 1% glycerol for preparation of inside-out membrane vesicles (20). Ampicillin (100 µg/ml) and/or kanamycin (50 µg/ml) were added to the medium, if necessary. Where indicated, 0.2 mM 2-mercaptoethanol was added to the medium.

Cadaverine Uptake by Intact Cells—E. coli JM109/pMW119 or JM109/pMWcadB cultured until A540 = 0.5 in the presence of 0.5 mM isopropyl beta-D-thiogalactopyranoside (IPTG)2 was suspended in buffer I containing 0.4% glucose, 62 mM potassium phosphate, pH 7.0, 1.7 mM sodium citrate, 7.6 mM (NH4)2SO4, and 0.41 mM MgSO4 to yield a protein concentration of 0.1 mg/ml. The cell suspension (0.48 ml) was preincubated at 30 °C for 5 min, and the reaction was started by the addition of 0.02 ml of 0.25 mM [14C]cadaverine (370 MBq/mmol, Sigma). Thus, the concentration of [14C]cadaverine in the reaction mixture was 10 µM. After incubation at 30 °C for 30 s to 3 min, the cells were collected on cellulose acetate filters (0.45 µm; Advantec Toyo Co.), and the filters were washed three times with a total of 9 ml of buffer I. To determine the Km values of cadaverine for cadaverine uptake activity of cadB mutants, 10-200 µM cadaverine was used as a substrate. The radioactivity on the filters was measured with a liquid scintillation spectrometer. Protein content was determined by the method of Lowry et al. (21).

Preparation of Inside-out Membrane Vesicles and Cadaverine Uptake by the Vesicles—Cadaverine-lysine antiport activity by CadB was estimated by measuring cadaverine uptake by inside-out membrane vesicles prepared according to the method of Houng et al. (22). E. coli JM109/pMWcadB cells were cultured until A540 = 0.5 in the presence of 0.5 mM IPTG. Inside-out membrane vesicles were prepared by French press treatment (10,000 p.s.i.) of the cells suspended in a buffer containing 100 mM potassium phosphate buffer, pH 6.6, 10 mM EDTA in the presence of 2.5 mM lysine (23). After removal of unbroken cells and cell debris, membrane vesicles were collected by ultracentrifugation (170,000 x g, 1 h). Membrane vesicles were washed with a buffer containing 10 mM Tris-HCl, pH 7.5, 0.14 M KCl, 2 mM 2-mercaptoethanol, and 10% glycerol, collected by ultracentrifugation, and suspended in the same buffer at the protein concentration of 5-10 mg/ml. The reaction mixture (0.1 ml) for the uptake by inside-out membrane vesicles contained 10 mM Tris-HCl, pH 8.0, 0.14 M KCl, 100 µM [14C]cadaverine (1.48 GBq/mmol), and 100 µg of inside-out membrane vesicle protein. After incubation at 22 °C for 10 s to 1 min, membrane vesicles were collected on cellulose nitrate filters (0.45 µm; Advantec Toyo Co.) and washed twice with a total of 9 ml of washing buffer (10 mM Tris-HCl, pH 8.0, and 0.14 M KCl). To determine the Km values of cadaverine for cadaverine-lysine antiport activity of cadB mutants, 100-2000 µM cadaverine was used as a substrate. Radioactivity on the filters was measured with a liquid scintillation spectrometer.

Western Blot Analysis of CadB Protein—Rabbit antibody for the CadB protein was prepared according to the method of Posnett et al. (24) using the multiple antigenic peptide, KVY-GEVDSNGIPKK, which correspond to amino acids 308-321 of the CadB protein. For Western blot analysis of CadB, inside-out membrane vesicles (30 µg protein) were separated by SDS-poly-acrylamide gel electrophoresis on a 12% acrylamide gel and transferred to a polyvinylidene fluoride membrane (Immobilon P, Millipore). CadB protein was detected with ProtoBlot Western blot AP System (Promega), except that 0.2% Triton X-100 was used instead of 0.05% Tween 20 (25).

Reactivity of [14C]NEM ([ethyl-1-14C]N-Ethylmaleimide) with CadB Protein—E. coli JM109/pUCcadB cells were cultured until A540 = 0.5 in a 19-amino acid supplemented medium containing 1% glycerol and 0.5 mM IPTG with or without 0.2 mM 2-mecaptoethanol. Right-side-out membrane vesicles were prepared as described previously (26). Reactivity of [14C]NEM with CadB was tested according to the method of Sahin-Tóth et al. (27). Right-side-out membrane vesicles (200 µg protein) were suspended with 50 mM sodium phosphate, pH 7.5, in a final volume of 50 µl, and 10 µl of 2.4 mM [14C]NEM (2.1 GBq/mmol, American Radiolabeled Chemicals Inc.) was added. Incubation was carried out at 22 °C for 10 min. Reactions were terminated by adding 1.8 µl of 1 M dithiothreitol (final: 30 mM), and the vesicles were solubilized by adding 15 µl of 10% dodecyl beta-D-maltoside. CadB was then precipitated with 50 µl of anti-CadB serum and 20 µg of PANSORBIN® cells (Calbiochem). Cells were washed three times with 50 mM sodium phosphate, pH 7.5, containing 0.1 M NaCl and 0.02% dodecyl beta-D-maltoside. Cells were suspended in 40 µl of SDS sample buffer (25 mM Tris-HCl, pH 6.8, 5% glycerol, 1% sodium dodecyl sulfate, 0.05% bromphenol blue) and boiled for 4 min. After centrifugation, a 30-µl aliquot was subjected to SDS-poly-acrylamide gel electrophoresis on an 8.5% acrylamide gel. Incorporation of [14C]NEM into CadB was visualized by BAS2000 II imaging analyzer (Fuji Film).

Analysis of Hydropathy and Sequence Homology of Proteins—Average hydropathy profiles of the proteins were obtained according to the method of Kyte and Doolittle (28) with a hydrophilicity/hydrophobicity plot (GENETYX-MAC Version 10, GENETYX Co.). A model of the secondary structure of proteins was then constructed according to these profiles. Sequence homology of the protein was analyzed by the CLUSTALW V1.83 program (29).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Amino Acid Residues Involved in Uptake and Excretion of Cadaverine—It is known that polyamines are recognized by proteins through interaction with acidic and aromatic amino acid residues (7, 8, 30, 31). There are 9 aspartic acid and 7 glutamic acid residues in CadB. These residues were individually mutated to asparagine and glutamine, respectively. Cadaverine uptake activity was measured using E. coli JM109 transformed with pMW encoding wild type or mutated CadB. Cadaverine excretion (i.e. cadaverine-lysine antiporter activity) was measured by [14C]cadaverine uptake using lysine-loaded inside-out membrane vesicles prepared from E. coli JM109 transformed with pMW encoding wild type or mutated CadB. As shown in Fig. 1, both uptake and excretion were greatly decreased with the mutants E204Q and D303N and moderately with E76Q and E377Q, and only uptake was moderately decreased with D185N and E408Q. There are 11 tryptophan and 13 tyrosine residues in CadB. These residues were mutated to leucine and the activities were measured. Both uptake and excretion were strongly decreased with the mutants Y73L, Y89L, Y90L, Y235L, and Y423L and moderately with Y55L, Y246L and Y310L, and only uptake was greatly decreased with W43L, Y57L, Y107L, Y366L, and Y368L and moderately with W41L and Y174L (Fig. 1).


Figure 1
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  		FIGURE 1.
Effect of Asp, Glu, Trp, and Tyr mutations on the cadaverine uptake and excretion activities of CadB. Assays for cadaverine uptake and excretion were performed under standard conditions. 100% activity of cadaverine uptake and excretion was 3.85 and 0.14 nmol/min/mg protein, respectively. Values are the mean ± S.D. of three samples. Activities of E. coli having pMW119 vector instead of pMWcadB or pMW mutated cadB were shown in the column labeled None. Amino acid residues indicated by white letters are involved in both activities, and residues in rectangles are involved in the uptake of cadaverine only.

 


Figure 2
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  		FIGURE 2.
Effect of Cys mutations on the cadaverine uptake and excretion activities of CadB. Assays were performed as described in the legend of Fig. 1 Values are the means ± S.D. of three samples. The amino acid residue (Cys370) indicated by the white letter is involved in both activities.

 


Figure 3
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  		FIGURE 3.
Necessity of Cys370 residue for the uptake and excretion activities of CadB. Assays were performed as described in the legend of Fig. 1. Values are the means ± S.D. of three samples. The amino acid residue (Cys370) indicated by the white letter is involved in both activities.

 
In our previous studies of PotE, we found that cysteine residues were involved in the recognition of putrescine by PotE (8). There are 8 cysteine residues in CadB. Each cysteine was mutated to serine, and uptake and excretion were measured. As shown in Fig. 2, both uptake and excretion were decreased significantly with the mutant C370S, and then with C397S, but mutations C12S, C125S, C196S, C282S, C389S, and C394S had no effect or only modest effects. To confirm that Cys370 is important for CadB activities, all 8 cysteine residues were replaced by serine and both uptake and excretion were measured. As shown in Fig. 3, the activities of the {Delta}C mutant were decreased greatly, and they were restored only with the mutant {Delta}C C370, which has one cysteine residue at position 370 but not with the mutants {Delta}C C12, C125, C196, C282, C389, C394, and C397. The results confirmed that the SH group of Cys370 is involved in both uptake and excretion.

The largest effects on both uptake and excretion of cadaverine were seen with the CadB mutants E204Q, D303N, Y73L, Y89L, Y90L, Y235L, and Y423L (Fig. 1). To confirm the importance of these amino acid residues, Glu204 was replaced by Asp and Asp303 by Glu. As shown in Fig. 4A, replacement of Glu204 and Asp303 by Asp and Glu did not restore the activities of CadB, indicating that Glu204 and Asp303 are critical for both activities. Similarly, Tyr73, Tyr89, Tyr90, Tyr235, and Tyr423 were replaced by Phe and Trp. Replacement of Tyr73, Tyr89, and Tyr235 by Phe and Trp did not restore the activities of CadB. Replacement of Tyr90 by Phe, but not by Trp, and replacement of Tyr423 by Phe and Trp slightly restored the activities of CadB. These results also indicate that these five tyrosine residues are critical for both activities of CadB. Expression of mutated CadB proteins was examined by Western blot analysis using 30 µg of protein of inside-out membrane vesicles. Comparable amounts of mutated CadB proteins were expressed on the membranes, indicating that the mutations do not affect expression of CadB (Fig. 4B).

The Km values for uptake and excretion of cadaverine by mutants were then determined by Lineweaver-Burk plots. As shown in Table 1, the Km value for uptake of cadaverine by wild-type CadB was 20.8 µM. In mutants that caused a reduction in activity, the Km value for cadaverine was increased, and the change in Km paralleled the decrease in cadaverine uptake activity shown in Figs. 1 and 2. As a control, the Km values were measured with W269L and D436N mutants, whose activities were almost the same as that of wild type. They were nearly equal to the Km value of wild type. These results indicate that all of the key mutants are at amino acid residues involved in the recognition of cadaverine. The Km value for excretion of cadaverine by CadB was 390 µM (Table 1). In CadB mutants that caused a decrease in excretion, the Km value for cadaverine was increased, in parallel with the decrease in cadaverine excretion shown in Figs. 1 and 2. Although the change in the Km value for uptake of cadaverine was greater than the change in the Km value for excretion of cadaverine for any given mutant except the mutants Y73L and Y423, the change in the Km value for uptake was parallel with that for excretion.


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  		TABLE 1
The Km value of mutated CadB for cadaverine uptake and excretion

 


Figure 4
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  		FIGURE 4.
Cadaverine uptake and excretion activities of Glu204, Asp303, Tyr73, Tyr89, Tyr90, Tyr235 and Tyr423 mutants of CadB (A) and their expression (B). A, assays were performed as described in the legend of Fig. 1. Amino acid residues shown in the horizontal axis are the replaced amino acid residues. Values are the mean ± S.D. of three samples. B, Western blot analysis was performed using 30 µg of protein of the inside-out membrane vesicles.

 
For the related protein PotE, a putrescine-ornithine anti-porter, residue Lys301 in C5 (cytoplasmic loop 5) is involved in recognition of the carboxyl group of ornithine (8). Thus, we determined whether a basic amino acid(s) in the cytoplasmic loops is involved in recognition of the carboxyl group of lysine. As shown in Fig. 5, excretion but not uptake of cadaverine was markedly reduced in R299A in C5, whereas both uptake and excretion activities were reduced to a similar, small extent in all other mutants. The results suggest that Arg299 is involved in the recognition of lysine.

Location of Amino Acid Residues Involved in Uptake and Excretion—Average hydropathy profiles of CadB indicated that CadB consists of 12 helices like PotE. Sequence homology between CadA and PotE was 30.7% (12). To confirm the high sequence similarity between CadB and PotE, the multiple sequence alignments were analyzed by the CLUSTALW program (29). The score between PotE and CadB was 27.3, but the scores between PotE and other proteins were less than 16. Thus, the secondary structure of CadB was constructed as shown in Fig. 6, which is similar to that of PotE. Amino acids important for both uptake and excretion were located in the transmembrane helices II, III, VI, VII, X, and XII or in the cytoplasmic region between transmembrane helices II and III (C2), VI and VII (C4), VIII and IX (C5), and X and XI (C6). Amino acid residues in the transmembrane helices are also located in the cytoplasmic side rather than the periplasmic side. In contrast to PotE, the Km value for cadaverine uptake by CadB is high (20.8 µM). Since numerous amino acid residues are involved in uptake of cadaverine, the participation of any individual residue in recognition of cadaverine may be weak. Those amino acids are mainly located in the transmembrane helices and the periplasmic loops and may be slightly separated from the substrate binding site. However, it is expected that those amino acid are located close to each other in the tertiary structure of CadB.

Transmembrane Helix Packing of CadB—It has been reported that a hydrophilic cavity of lactose permease having twelve transmembrane helices is formed between eight transmembrane helices: I, II, IV, V, VII, VIII, X, and XI (32). The situation is similar in the multidrug efflux transporter AcrB (33) and oxalate transporter OxlT (34). The hydrophobic cavity in each of these two transporters also consisted of 8 transmembrane helices. As for CadB, six transmembrane helices have functional amino acid residues: helices II, III, VI, VII, X, and XII. Thus, we determined whether two additional helices also contribute to a hydrophilic cavity in CadB. For this purpose, one more serine residue was converted back to the original cysteine in the {Delta}C C370 mutant, and uptake and excretion were measured. As shown in Fig. 7A, uptake and excretion were greatly inhibited with {Delta}C C370/C125, C370/C389, and C370/C394, but not with {Delta}C C370/C12, C370/C196, C370/C282, and C370/C397. The results suggest that inhibition of the transport is due to S-S bond formation between two cysteine residues Cys370 and Cys125, Cys389, orCys394. The activity of these mutants {Delta}C C370/C125, C370/C389, and C370/C394 was recovered when these mutants were cultured in the medium containing 0.2 mM 2-mercaptoethanol, and the activity was measured in the presence of 2 mM 2-mercaptoethanol (Fig. 7A). The activity of other mutants {Delta}C C370/C12, C370/C196, C370/C282, and C370/C397 did not change significantly by these treatments. The results suggest that inhibition of the transport activity of {Delta}C C370/C125, C370/C389, and C370/C394 is through S-S bond formation between two cysteine residues. The S-S bond formation between C370 and C125, C389, or C394 in CadB was confirmed by reactivity with [14C]NEM (Fig. 7B). [14C]NEM reacted with CadB ({Delta}C C370/C125, C370/C389, and C370/C394) very weakly compared with CadB ({Delta}C C370) when cells were cultured in the absence of 2-mercaptoethanol, while it reacted strongly with these CadB proteins when cells were cultured in the presence of 0.2 mM 2-mercaptoethanol. Thus, it is most probable that helix IV having Cys125 and helix XI having Cys389 and Cys394 form a hydrophilic cavity in CadB together with helices II, III, VI, VII, X, and XII. Although Cys397 is also located on helix XI, the activities were not inhibited significantly with {Delta}C C370/C397, suggesting that Cys397 is sterically distant from Cys370.


Figure 5
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  		FIGURE 5.
Effect of Arg and Lys mutations on the cadaverine uptake and excretion activities of CadB. Arg and Lys located in the cytoplasmic loops were mutated to Ala. Assays were performed as described in the legend of Fig. 1. Values are the mean ± S.D. of three samples. The amino acid residues in rectangles are involved in lysine recognition.

 


Figure 6
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  		FIGURE 6.
Amino acid residues involved in the activities of CadB. A model of the secondary structure of proteins was constructed according to the average hydropathy profiles obtained with a Hydrophilicity/Hydrophobicity Plot (GENETYX-MAC Version 10). Putative transmembrane segments are shown in large boxes. Amino acid residues involved in cadaverine uptake activity and both uptake and excretion activities are classified by symbols shown in the figure. The large and small circles indicate strong and moderate involvement in the activities, respectively.

 
As stated above, it was shown that Cys12 in helix I and Cys282 in helix VIII did not make S-S bond with Cys370 in helix X (see Fig. 6). The results suggest that helices I and VIII do not contribute to the hydrophilic cavity. To test whether helices V and IX do not form a hydrophilic cavity, Val159 and Ile163 in helix V and Ala327 and Leu333 in helix IX were converted to cysteine in the {Delta}C C370 mutant. As shown in Fig. 8, the uptake and excretion by the CadB having mutation in helix V ({Delta}C C370/C159 and {Delta}C C370/C163) did not decrease significantly and transport by mutation in helix IX ({Delta}C C370/C327 and {Delta}C C370/C333) was weakly inhibited (about 50% inhibition). Since helix IX is close to helix X, it may be possible for Cys370 in helix X to interact partly with Cys327 and Cys333 in helix IX. The results suggest that helices V and IX do not contribute to a hydrophilic cavity. It was also confirmed that helix VI contributes to a hydrophilic cavity because mutations in helix VI ({Delta}C C370/C201 and {Delta}C C370/C203) greatly decreased uptake and excretion by CadB. As a control, the activities were measured using the mutants in which cysteine residues were inserted into cytoplasmic loops (C1 to C7) of the {Delta}C C370 mutant. As shown in Fig. 8, the activities of the C2 to C7 mutants were greatly inhibited except those of the C1 mutant. When these mutants were cultured in medium containing 0.2 mM 2-mercaptoethanol, and the activity was measured in the presence of 2 mM 2-mer-captoethanol, the activity of the mutants recovered to that of the mutant {Delta}C C370 (data not shown). The results indicate that cysteine in the cytoplasmic loops except C1 loop can make S-S bond with Cys370. The Cys4 in C1 may be distant from Cys370 in helix X. Single cysteine mutants, in which one amino acid of wild type CadB was mutated to cysteine such as CadB A4C and CadB V201C, did not decrease the uptake and excretion activities (data not shown).


Figure 7
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  		FIGURE 7.
Inhibition of the activities of the {Delta}C C370 mutant by other Cys residues (A) and reactivity of [14C]NEM with the mutants (B). A, upper figure: assays were performed as described in the legend of Fig. 1. 100% activity of cadaverine uptake and excretion was 2.30 and 0.11 nmol/min/mg of protein, respectively. Values are the means ± S.D. of three samples. Lower figure: cadaverine uptake activity of mutants was measured using cells cultured in the presence and absence of 0.2 mM 2-mercaptoethanol. The uptake activity of the cells cultured with 0.2 mM 2-mercaptoethanol was measured in the presence of 2 mM 2-mercaptoethanol. 100% activity was 2.51 nmol/min/mg of protein. Values are the means ± S.D. of three samples. B, reactivity of [14C]NEM with CadB mutants was tested as described under "Experimental Procedures." Experiments were repeated three times and the results were reproducible.

 


Figure 8
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  		FIGURE 8.
Change of the activities of the {Delta}C S370C mutant by the insertion of one more cysteine residue. One amino acid residue shown in the figure was converted to Cys, and assays were performed as described in the legend of Fig. 7A. Values are the means ± S.D. of three samples.

 


Figure 9
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  		FIGURE 9.
Model of cadaverine recognition site on CadB. Amino acid residues involved in both uptake and excretion activities are shown in red circles, and amino acid residues involved in cadaverine uptake activity shown in green circles. Large and small circles are strongly and weakly involved in the activities, respectively. Cys on the pink circle has an inhibitory effect through S-S bond formation with Cys370.

 
Based on these results, a model was constructed of the hydrophilic cavity of CadB which recognizes cadaverine (Fig. 9). The hydrophilic cavity probably consists of eight, rather than six, transmembrane helices. It is shown that five tyrosines, one glutamic acid, and one aspartic acid residues of CadB are strongly involved in the recognition of cadaverine. Thus, both hydrophobic and hydrophilic interactions between cadaverine and CadB are necessary for specific recognition of cadaverine. Since cadaverine is diaminopentane and putrescine is diaminobutane, it may be reasonable that more tyrosine residues of CadB are involved in recognition of cadaverine by CadB compared with the recognition of putrescine by PotE (8). Outward-facing and inward-facing conformations (32) probably change the relative position of amino acid residues shown in red and green circles in the hydrophilic cavity. In the outward-facing conformation, amino acid residues shown in the green circle become closer to cadaverine and contribute to the uptake of cadaverine.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have recently studied the physiological functions of CadB (12). CadB, a cadaverine transport protein, together with lysine decarboxylase which is a product of cadA, contributes to form a proton motive force at acidic pH, neutralization of the medium and supply of CO2. In this study, we identified amino acid residues which recognize cadaverine. Amino acid residues involved in the recognition of cadaverine were Glu, Asp, Tyr, Trp, and Cys. Residues involved in both uptake at neutral pH and excretion at acidic pH were mainly localized in the cytoplasmic loops (C2, C4, C5, and C6) and the cytoplasmic side of transmembrane segments. Furthermore, Arg299 involved in the excretion activity of cadaverine was also localized in C5. However, amino acid residues involved only in uptake were located in the periplasmic loops (P3 and P6) and the transmembrane segments. This is in contrast to PotE where residues involved only in uptake were also localized in the cytoplasmic loops and the cytoplasmic side of transmembrane segments (8). The conformational change of CadB during uptake and excretion of cadaverine may be greater than that of PotE during uptake and excretion of putrescine. Furthermore, many amino acid residues are involved in the recognition of cadaverine by CadB compared with recognition of putrescine by PotE (8). Since the hydrophobic moiety separating the two amino groups is larger in cadaverine than in putrescine, more aromatic amino acids, especially tyrosine, may be necessary to recognize cadaverine.

It is also noted that the SH-group of Cys370 seemed to be important for both uptake and excretion by CadB. The SH-group of Cys370 may be necessary for recognition of the NH2-group of cadaverine. As for the uptake of cadaverine at neutral pH, it is suggested that H+ may be cotransported with cadaverine (12). The SH-group of Cys370 may also be involved in the recognition of H+ during the influx of H+ and cadaverine. It is shown that Cys397 is slightly involved in the both activities of CadB (see Fig. 2). The Cys397, together with Cys370, may be involved in the recognition of the NH2-group of cadaverine or H+ in case of the influx of cadaverine.

It has been reported that the hydrophilic cavity of lactose permease (32), multidrug efflux transporter AcrB (33), and oxalate transporter OxlT (34) consisted of 8 transmembrane helices. The hydrophilic cavity of these proteins are an approximate 2-fold symmetry as for the NH2- and COOH-terminal six-helix domains. We have also constructed the model consisting of 8 transmembrane helices, which have functional amino acids as well as cysteines involved in the activities of CadB. However, the NH2- and COOH-terminal six-helix domains of CadB did not have an approximate 2-fold symmetry (see Fig. 9) like lactose permease, AcrB and OxlT. This is also the case of PotE. Since the function of CadB is different at acidic and neutral pH, i.e. CadB has two catalytic activities, the topology in the NH2- and COOH-terminal six-helix domains may be changed in the group of these proteins like CadB and PotE. Experiments are in progress to determine the crystal structure of CadB.


   FOOTNOTES
 
* This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan; the Sankyo Foundation of Life Science, Japan; and by the Tokyo Biochemical Research Foundation, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 81-43-226-2871; Fax: 81-43-226-2873; E-mail: iga16077{at}p.chiba-u.ac.jp.

2 The abbreviations used are: IPTG, isopropyl beta-D-thiogalactopyranoside; NEM, N-ethylmaleimide. Back


   ACKNOWLEDGMENTS
 
We thank Dr. K. Williams for his help in preparing the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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