Spermidine-preferential uptake system in Escherichia coli. Identification of amino acids involved in polyamine binding in PotD protein.

Spermidine-binding sites on PotD protein, a substrate-binding protein in periplasm, in the spermidine-preferential uptake system in Escherichia coli were studied by measuring polyamine transport activities of right-side-out membrane vesicles with mutated PotD proteins prepared by site-directed mutagenesis of the potD gene and by measuring polyamine binding activities of these mutated PotD proteins. Polyamine transport activities of the mutated PotD proteins paralleled their polyamine binding activities. It was found that Trp-34, Thr-35, Glu-36, Tyr-37, Ser-83, Tyr-85, Asp-168, Glu-171, Trp-229, Trp-255, Asp-257, Tyr-293, and Gln-327 of PotD protein were involved in the binding to spermidine. When spermidine uptake activities were measured in intact cells expressing the mutated PotD proteins, it was found that Glu-171, Trp-255, and Asp-257 were more strongly involved in the binding of spermidine to PotD protein than the other amino acids listed above. The dissociation constants of spermidine for the mutated PotD proteins at Glu-171, Trp-255, and Asp-257 increased greatly in comparison with those for the other mutated PotD proteins. Since these three amino acids clearly interact with the diaminopropane moiety of spermidine, the results are in accordance with the finding that PotD protein has a higher affinity for spermidine than for putrescine. Putrescine was found to bind at the position of the diaminobutane moiety of spermidine.

Spermidine-binding sites on PotD protein, a substratebinding protein in periplasm, in the spermidine-preferential uptake system in Escherichia coli were studied by measuring polyamine transport activities of right-sideout membrane vesicles with mutated PotD proteins prepared by site-directed mutagenesis of the potD gene and by measuring polyamine binding activities of these mutated PotD proteins. Polyamine transport activities of the mutated PotD proteins paralleled their polyamine binding activities. It was found that Trp-34, Thr-35, Glu-36, Tyr-37, Ser-83, Tyr-85, Asp-168, Glu-171, Trp-229, Trp-255, Asp-257, Tyr-293, and Gln-327 of PotD protein were involved in the binding to spermidine. When spermidine uptake activities were measured in intact cells expressing the mutated PotD proteins, it was found that Glu-171, Trp-255, and Asp-257 were more strongly involved in the binding of spermidine to PotD protein than the other amino acids listed above. The dissociation constants of spermidine for the mutated PotD proteins at Glu-171, Trp-255, and Asp-257 increased greatly in comparison with those for the other mutated PotD proteins. Since these three amino acids clearly interact with the diaminopropane moiety of spermidine, the results are in accordance with the finding that PotD protein has a higher affinity for spermidine than for putrescine. Putrescine was found to bind at the position of the diaminobutane moiety of spermidine.
The polyamine content in cells, which plays important roles in cell proliferation and differentiation (1,2), is regulated by polyamine biosynthesis, degradation, and transport. As for the latter, we obtained and characterized three clones of polyamine transport genes (pPT104, pPT79, and pPT71) in Escherichia coli (3). The system encoded by pPT104 is the spermidinepreferential uptake system, and that by pPT79 is the putrescine-specific uptake system. Furthermore, these two systems are periplasmic transport systems (4,5), each consisting of four kinds of proteins: the pPT104 clone encodes PotA, PotB, PotC, and PotD proteins, and the pPT79 clone encodes PotF, PotG, PotH, and PotI proteins, judging from the deduced amino acid sequences of their nucleotide sequences (6,7). PotD and PotF proteins are periplasmic substrate-binding proteins, and PotA and PotG proteins are membrane-associated proteins with a nucleotide-binding site. PotB and PotC proteins and PotH and PotI proteins are transmembrane proteins that probably form channels for spermidine and putrescine, respectively. Their amino acid sequences in the corresponding proteins are similar to each other. In contrast, the putrescine transport system encoded by pPT71 consists of one membrane protein (PotE protein) with 12 transmembrane segments (8) and is active in the excretion of putrescine from cells through putrescine-ornithine antiporter activity (9). We also found that spermidine uptake by membrane vesicles is strongly dependent on PotD protein, and the uptake by intact cells is completely dependent on ATP through its binding to PotA protein (10). Furthermore, PotA protein was shown to have ATPase activity, and its association with membranes is strengthened by the existence of channel-forming PotB and PotC proteins (11).
Recently, we determined the crystal structure of PotD protein in a complex with spermidine at 2.5-Å resolution (12). It was revealed that four acidic and five aromatic amino acid residues in PotD protein interact with spermidine (13). In this study, we tried to identify the amino acid residues in PotD protein that are involved in the binding of spermidine by using mutated PotD protein produced by site-directed mutagenesis of the potD gene. We found that Glu-171, Trp-255, and Asp-257, among 13 amino acids involved in the interaction with spermidine, are particularly important in the recognition.
Mutagenesis of potD Gene-To prepare potD mutants, a 1.7-kilobase EcoRI-HindIII fragment of pUCpotD was inserted into the same site of M13mp19 (20). Site-directed mutagenesis was carried out by the method of Sayers et al. (21) with a Sculptor TM in vitro mutagenesis system (Amersham Corp.), using the oligonucleotides shown in Table I. The mutated DNA fragments were isolated from the replicative form of M13 and religated into the same site of pUCpotD. Mutations were confirmed by DNA sequencing (22) using the M13 phage system (20) with commercial and synthesized primers.
Polyamine Uptake by Right-side-out Membrane Vesicles-Right-sideout membrane vesicles were prepared from E. coli DR112/pPT86 as described previously (23). Periplasmic proteins were obtained from E. * This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan and by the Hayashi Memorial Foundation for Female Natural Scientists of 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EMBL Data Bank with accession number(s) M64519.
coli TG1 containing pUCpotD-or pUC-mutated potD according to the method of Oliver and Beckwith (24). PotD protein occupied ϳ60% of the total periplasmic proteins, and they were used for the PotD protein source. The reaction mixture (0.1 ml; containing 20 mM potassium phosphate buffer (pH 6.6), 50 mM HEPES/KOH (pH 7.6), 5 mM MgSO 4 , 20 mM D-lactate (lithium salt), 100 g of membrane vesicle protein, 70 g of PotD or mutated PotD protein, and 20 M [ 14 C]putrescine (4.07 GBq/mmol) or 4 M [ 14 C]spermidine (4.07 GBq/mmol)) was incubated at 37°C for 10 min. Membrane vesicles were collected on membrane filters (cellulose acetate, 0.45 m; Advantec Toyo), and the radioactivity was counted with a liquid scintillation spectrometer. Protein content was measured by the method of Lowry et al. (25).
Assay for Polyamine Binding to PotD Protein-The reaction mixture (0.1 ml; containing 10 mM Tris-HCl (pH 7.5), 30 mM KCl, and a combination of 10 g of PotD protein and 4 M [ 14 C]spermidine (2.03 GBq/ mmol) or 100 g of PotD protein and 35 M [ 14 C]putrescine (4.07 GBq/mmol)) was incubated at 30°C for 5 min. PotD protein was collected on membrane filters (cellulose nitrate, 0.45 m; Advantec Toyo), and the radioactivity was counted with a liquid scintillation spectrometer. The dissociation constant (K d ) and the number of binding sites (B max ) of spermidine for PotD protein were calculated from a Scatchard plot (26) by changing the substrate concentrations from 1 to 50 M.
Spermidine Uptake by Intact Cells-E. coli MA261 potD::Km containing pMWpotD-or pMW-mutated potD was grown in medium A until A 540 reached 0.3. The assay for spermidine uptake was performed as described previously (23), except that 1 M (4.07 GBq/mmol) or 10 M (370 MBq/mmol) [ 14 C]spermidine was used as substrate.

Spermidine and Putrescine Uptake Activities of Right-sideout Membrane Vesicles and Mutated PotD Protein-Recently,
we determined the tertiary structure of PotD protein in a complex with spermidine by x-ray crystallographic analysis (13).  1 that may be involved in the spermidine binding. In addition, it has been reported that Tyr-86 and Arg-170 might also participate in the spermidine binding from the prediction of the structural similarity between PotD protein and maltose-binding protein (27). Therefore, we prepared PotD proteins mutated at the 15 amino acids listed above by site-directed mutagenesis of the potD gene (Table I) and analyzed the activities of the mutated PotD proteins by three different assay methods.
First, spermidine and putrescine uptake activities were measured using right-side-out membrane vesicles and PotD or mutated PotD protein. Right-side-out membrane vesicles were prepared from E. coli DR112/pPT86, in which relatively large amounts of PotA, PotB, and PotC proteins are synthesized (6). Periplasmic protein prepared from E. coli TG1/pUCpotD was used as the source of PotD and mutated PotD proteins. Spermidine uptake activities decreased greatly with mutated PotD proteins (W255L 2 and D257N) in which a closely located amino acid or one interacting with the secondary amine of spermidine is modified (Fig. 1B). The activity also decreased with all mutated PotD proteins (E36Q, T35A, Y37A, Y293A, W229L, W34L, E171Q, S83A, D168N, and Q327A) whose amino acids were strongly suggested to be involved in the binding of spermidine by x-ray analysis. Spermidine uptake activities with other mutated PotD proteins (Y86A and R170L) did not change significantly (data not shown).
Putrescine uptake activity greatly decreased with certain mutated PotD proteins (E36Q, Y37A, Y293A, W34L, and D257N) in which an amino acid interacting with the diaminobutane moiety of spermidine is modified (Fig. 1B). The activity did not significantly decrease with other mutated PotD proteins (E171Q, Y85A, and D168N) in which an amino acid interacting with the aminopropyl moiety of spermidine is modified. The mutated PotD protein W255L slightly decreased the activity. The results indicate that putrescine occupies the position corresponding to the binding site of the diaminobutane moiety of spermidine in PotD protein. It should also be noted that the substitution of Thr-35, which was involved in the binding of spermidine, was not connected with the interaction with putrescine.
Spermidine and Putrescine Binding to Mutated PotD Protein-Polyamine binding to PotD protein was measured using 4 M spermidine or 35 M putrescine as substrate. As shown in Fig. 1C, the amounts of spermidine bound to the mutated PotD proteins almost paralleled the spermidine uptake activities of the proteins except for the E36Q mutated protein. The release of spermidine from this mutated protein may be slower than 1 Numbers of amino acids start from methionine for initiation. Thus, the 23 amino acids corresponding to the signal peptide are actually released from PotD protein. 2 The mutated PotD protein W255L contains leucine instead of tryptophan at position 255.

FIG. 1. Polyamine uptake by right-side-out membrane vesicles and mutated PotD protein and polyamine binding to mutated PotD protein.
A, the 13 amino acids of PotD protein expected to be involved in the interaction with spermidine. B, polyamine uptake. Control activities (100%) with normal PotD protein for spermidine (f) and putrescine ( ) uptake were 24.6 and 4.8 pmol/min/mg of protein, respectively. C, polyamine binding. Control activities (100%) with normal PotD protein for spermidine (f) and putrescine ( ) binding were 7.31 and 0.42 nmol/mg of protein, respectively. Each value is the average of duplicate determinations. that from the others. Putrescine binding was measured using a 10 times greater amount of mutated PotD protein since the affinity for putrescine (K d ϭ 100 M) is much lower than that for spermidine (K d ϭ 3.2 M) (7). The amounts of putrescine bound to the mutated PotD proteins also almost paralleled the putrescine uptake activities of the proteins. Since the W255L mutated protein significantly decreased both the putrescine uptake and binding activities, Trp-255 may also be involved in the interaction with the primary amine of putrescine, which corresponds to the secondary amine of spermidine.
Spermidine Uptake Activities of Intact Cells Containing Mutated PotD Proteins-Spermidine uptake activities were measured with E. coli MA261 potD::Km containing pMW-mutated potD, in which the expression of PotD or mutated PotD protein was about four times greater than that in wild-type MA261 cells. The activity with intact cells was measured with 1 or 10 M spermidine, as the K m value for spermidine is 0.1 M (3); it was observed to be much greater than that with right-side-out membrane vesicles and PotD protein. One reason for this was that the possibility of PotD protein interaction with the channel-forming PotB and PotC proteins was increased by the existence of the outer membrane. When the activity was measured in intact cells, the difference among the activities of the mutated PotD proteins became more pronounced. As shown in Table II, Asp-257 was the most important amino acid for recognition of spermidine, with Trp-255 and Glu-171 also being strongly involved. Other mutated PotD proteins slightly decreased the spermidine uptake activity, but the mutated PotD protein S83A did not. Replacement of Asp-257 by Glu resulted in uptake activity at 70% of normal PotD protein, indicating that a negative charge at position 257 is important (data not shown).
The dissociation constants (K d ) and the number of binding sites (B max ) of spermidine for the mutated PotD proteins were then measured. Since the B max value (1 mol/PotD protein) did not change significantly, the change in binding affinity was mainly due to the change in the K d values. As shown in Table  II, the K d value of spermidine for normal PotD protein was estimated to be 3.7 M. However, the K d values of spermidine for the three mutated proteins D257N, W255L, and E171Q increased greatly, paralleling the decrease in spermidine uptake activities of intact cells. We previously reported that the concentration of spermidine-PotD protein in the periplasm would be ϳ3.1 M if the spermidine concentration was 0.1 M under standard conditions (10).

DISCUSSION
The spermidine-preferential uptake system belongs to the ATP-binding cassette superfamily (4,5). The tertiary structures of several periplasmic substrate-binding proteins in the ATP-binding cassette superfamily have been characterized by x-ray crystal structural analysis (28 -31). The structural similarity among substrate-binding proteins is striking. There are two globular domains connected by short peptide segments. Between the domains lies a large cleft that binds the substrate. Furthermore, structure/function analysis of histidine-binding protein was performed with several mutated histidine-binding proteins (32,33). In the case of PotD protein, it was suggested by x-ray crystal structural analysis (13) that 13 amino acids may be involved in the interaction with spermidine.
In this study, we tried to identify the amino acids that are involved in the binding of spermidine by measuring spermidine uptake activities of right-side-out membrane vesicles and in-  tact cells with mutated PotD proteins as well as spermidine binding activities of mutated PotD proteins. We found that amino acids, especially Glu-171, Trp-255, and Asp-257, involved in the interaction with the diaminopropane moiety of spermidine are more crucial in the binding of spermidine to PotD protein than those involved in the interaction with the aminobutyl moiety of spermidine. Putrescine was found to bind at the position of the diaminobutane moiety of spermidine. These results explain why spermidine has a higher affinity for PotD protein than putrescine. Among the above three amino acids, Asp-257, which interacts with the secondary amine of spermidine, is most strongly involved in the binding to spermidine. In this connection, it should be noted that the secondary amine of spermidine or spermine most effectively contributes to the interaction with tRNA (34).
The putrescine-specific uptake system encoded by pPT79 also belongs to the ATP-binding cassette superfamily (7), and PotF protein is a substrate-binding protein that exists in periplasm. The amino acid sequences of PotD and PotF proteins exhibit 35% homology, indicating that the two proteins have similar three-dimensional structures (Fig. 3). However, Tyr-85, Trp-255, and Gln-327, which are involved in the interaction with the aminopropyl moiety of spermidine, are missing at the equivalent positions in PotF protein. On the other hand, Trp-34, Tyr-37, Trp-229, Asp-257, and Tyr-293, which recognize the diaminobutane moiety of spermidine, exist at the equivalent positions in PotF protein. In PotF protein, Thr-35 and Glu-36, which also recognize the diaminobutane moiety of spermidine, were replaced by Ser and Asp, respectively. The affinity of PotF protein for putrescine was stronger than that of PotD protein (7,10). Since the side chains of Ser and Asp are smaller than those of Thr and Glu, putrescine may be accommodated easily into the cleft between the N-and C-terminal domains of PotF protein. In this regard, it should be noted that Thr-35 of PotD protein was involved in the recognition of spermidine, but not putrescine.