(Received for publication, January 22, 1996; and in revised form, March 1, 1996 )
From the
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.
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 spermidine-preferential 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.
Figure 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 () and putrescine (&cjs2090;) 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 (
) and putrescine (&cjs2090;) binding were 7.31 and
0.42 nmol/mg of protein, respectively. Each value is the average of
duplicate determinations.
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 ()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.
The dissociation constants (K) and the number
of binding sites (B
) of spermidine for the
mutated PotD proteins were then measured. Since the B
value (1 mol/PotD protein) did not change significantly, the
change in binding affinity was mainly due to the change in the K
values. As shown in Table 2, the K
value of spermidine for normal PotD protein was
estimated to be 3.7 µM. However, the K
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
3.1 µM if the spermidine concentration was 0.1
µM under standard conditions (10) .
The amino acids involved in the recognition of spermidine are summarized in Fig. 2. There were four acidic amino acids, three tyrosines, three tryptophans, and one molecule each of serine, threonine, and glutamine, and they were classified into three groups: most strongly involved, Asp-257, Glu-171, and Trp-255; moderately involved, Asp-168, Trp-34, Trp-229, Tyr-85, and Tyr-293; and weakly involved, Glu-36, Tyr-37, Thr-35, Ser-83, and Gln-327.
Figure 2: Spermidine-binding site of PotD protein. Shaded boxes, most strongly involved amino acid; shaded ovals, moderately involved amino acid; white boxes, weakly involved amino acid.
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, 29, 30, 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 intact 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.
Figure 3: Comparison of amino acid sequences of PotD and PotF proteins. Asterisks indicate the amino acids involved in the interaction with spermidine; shaded boxes indicate the amino acids involved in the differential interaction with spermidine. Identical amino acids are indicated by colons, and equivalent amino acids by periods.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) M64519[GenBank].