From the Biomolecular Engineering Research Institute,
6-2-3 Furuedai, Suita, Osaka, 565-0874 and ¶ Faculty of
Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku,
Chiba 263-8522, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PotF protein is a periplasmic substrate-binding protein of the putrescine transport system in Escherichia coli. We have determined the crystal structure of PotF protein in complex with the substrate at 2.3-Å resolution. The PotF molecule has dimensions of 54 × 42 × 30 Å and consists of two similar globular domains. The PotF structure is reminiscent of other periplasmic receptors with a highest structural homology to another polyamine-binding protein, PotD. Putrescine is tightly bound in the deep cleft between the two domains of PotF through 12 hydrogen bonds and 36 van der Waals interactions. The comparison of the PotF structure with that of PotD provides the insight into the differences in the specificity between the two proteins. The PotF structure, in combination with the mutational analysis, revealed the residues crucial for putrescine binding (Trp-37, Ser-85, Glu-185, Trp-244, Asp-247, and Asp-278) and the importance of water molecules for putrescine recognition.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Natural polyamines (putrescine, spermidine, and spermine) are ubiquitous in almost all prokaryotic and eukaryotic cells. These small aliphatic cations are protonated at a physiological pH and, thus, in the cell they easily bind to nucleic acids. Through these interactions, polyamines are known to be involved in the biosynthesis of nucleic acids and proteins and to mediate the cell growth and proliferation (1, 2). Furthermore, spermidine was found to donate a portion of its molecule for the enzymatic biosynthesis of hypusine, a unique amino acid that plays a crucial role in cell proliferation (3). In the past few years, polyamines have been shown to modulate and to block a number of K+ channels and glutamate receptors, thereby controlling the resting membrane potential and the excitability of various cells (4, 5). Studies of the factors that regulate the cellular polyamine content, therefore, are important for both basic science and medicine.
The intracellular polyamine content is controlled through the polyamine metabolism and by the uptake/excretion activities of polyamine transport systems in a concerted manner. Thus, the inhibitors of both pathways would be the potential drugs in the therapy of cancer in which the polyamines play a pathogenic role (6). Although the mechanisms of polyamine biosynthesis/degradation have been extensively studied at the molecular level, little is known about polyamine transport. No genes responsible for the transport of polyamines in mammalian cells have been isolated (6). However, three polyamine transport systems in Escherichia coli were recently characterized (7). One of these systems possesses putrescine uptake and excretion activities and consists of one transmembrane protein, PotE (8). The other two consist of four proteins each and have only the uptake activity, with specificity to the different polyamines. The PotA, PotB, PotC, and PotD proteins constitute the spermidine/putrescine transport system, with strong preference to spermidine (9, 10), whereas the PotF, PotG, PotH, and PotI proteins belong to the putrescine-specific transport machinery (11). In these two systems, PotF (PotD) is the primary putrescine (spermidine/putrescine) receptor in the periplasmic space. PotH and PotI (PotB, PotC) are the transmembrane components, which form the channel for the polyamines in the membrane. PotG (PotA) is the membrane-associated, ATP-binding protein that provides the energy for polyamine uptake.
PotF (PotD) belongs to the family of periplasmic binding proteins involved in active transport and chemotaxis in Gram-negative bacteria (12). A dozen high resolution x-ray structures of periplasmic receptors, with specificity to carbohydrates (13-20), sulfate (21), phosphate (22), different amino acids (23-29), and di- and oligopeptides (30, 31), have been determined in the past few years. Despite the lack of significant sequence homology, all of these proteins share a similar fold and structural topology. They consist of two globular domains with a narrow interdomain groove, which constitutes the substrate binding site. Recently, the periplasmic receptors were classified in two groups, on the basis of their three-dimensional structures. The group I proteins have three interdomain linker segments, whereas the group II proteins have only two (12).
The crystal structure of the PotD protein, which has high affinity to spermidine (Kd = 3.2 µM) and lower affinity to putrescine (Kd = 100 µM), was solved in complex with spermidine (32, 33). The atomic model and the mutational analysis (34) of the protein residues in the vicinity of the substrate binding site revealed the mechanism of spermidine recognition by the protein. However, the reason why spermidine and putrescine bind to PotD with different affinities remained obscure. The PotF and PotD proteins have 35% homology in their amino acid sequences. In addition, most of the residues involved in spermidine recognition by the PotD protein are conserved between the two proteins. Nevertheless, the PotF protein possesses a high binding affinity to only putrescine (Kd = 2.0 µM) and does not bind other polyamines. To elucidate the mechanism of substrate binding and specificity of the PotF protein, we have determined the crystal structure of PotF in complex with putrescine at a 2.3-Å resolution. The mutational analysis of PotF combined with the comparison of the PotF and PotD structures provides the insight into the mechanism by which the PotF protein discriminates between the closely related putrescine and spermidine molecules.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bacterial Strains, Plasmids, and Culture Conditions-- The spermidine uptake- and polyamine biosynthesis-deficient mutant E. coli KK313 (7) and its putrescine uptake-deficient mutant KK313potF::Km (11) were grown in medium A in the absence of polyamines as described previously (35). Plasmids pPT79 (containing the potFGHI genes), pPT104 (containing the potABCD genes), pPT79.3 (containing the potGHI genes), and pUCpotF were prepared as described previously (7, 11). Plasmid pMWpotF was prepared by inserting the 1.7-kilobase EcoRI-HindIII fragment of pUCpotF into the same restriction site of pMW119 (36). Transformation of E. coli cells with plasmids was carried out according to the method of Maniatis et al. (37). Appropriate antibiotics (30 µg/ml chloramphenicol, 100 µg/ml ampicillin, and 50 µg/ml kanamycin) were added during the culture of E. coli.
Structure Determination-- PotF was crystallized, and two Pt derivatives were prepared as described previously (38). The PotF crystals belong to the space group P21212, with unit cell dimensions a = 269.4 Å, b = 82.33 Å, and c = 93.74 Å. There are four PotF molecules in the asymmetric unit. Attempts to solve the PotF structure using molecular replacement and the PotD molecule as a search model failed. The PotF structure was solved by the multiple isomorphous replacement method, in combination with 4-fold noncrystallographic symmetry averaging (see Table I). Two crystals of the HgI4 derivative were prepared by soaking native crystals for 2 and 4 days in solutions containing 2 mM and 0.5 mM HgI4, respectively. The heavy atom sites for all four derivatives were refined with the MLPHARE program (39).1 The final figure of merit at 3-Å resolution was 0.64. The multiple isomorphous replacement electron density map was improved by solvent flattening, and a rough partial poly(A)LA model was built for each of the four molecules in the asymmetric unit using the O program (40). This partial model provided an initial matrix for the noncrystallographic symmetry-related PotF molecules. The noncrystallographic symmetry averaging, in combination with solvent flattening and histogram matching (1000 cycles), was applied using the DM program (41). Almost all of the side chains and the main chain oxygens were clearly seen in the final electron density map. The atomic model was easily built in this map for one PotF molecule, using the O program (40).
Refinement-- The PotF structure was refined using the slow cooling protocol in X-PLOR (42) with the manual rebuilding of the model after each refinement step (Table I). The refinement converged to an R-factor = 19.2% (R-free = 26.0%). The final (2Fo-Fc) map was of high quality, with clear electron densities for the putrescine molecules bound to each of the four PotF molecules. The average B-factor was 38-Å2 for all the protein atoms.
|
Mutagenesis of the PotF Gene-- To prepare potF mutants, the 1.7-kilobase EcoRI-HindIII fragment of pUCpotF was inserted into the same site of M13mp19 (43). Site-directed mutagenesis was carried out by the method of Sayers et al. (44) with the SculptorTM in vitro mutagenesis system (Amersham Pharmacia Biotech). The mutated DNA fragments were isolated from the replicative form of M13 and were religated into the same site of pUCpotF. Some mutants were prepared using the polymerase chain reaction (45) with the QuikChangeTM site-directed mutagenesis kit (Stratagene). Mutations were confirmed by DNA sequencing (46) with commercial and synthesized primers. The sequences of the oligonucleotides used for the mutagenesis are shown in Table II.
|
Assay for Putrescine Binding to the PotF
Protein--
Periplasmic proteins were obtained from E. coli JM105 (supE endA sbcB15 hsdR rpsL thi
(lac-proAB)) (47) containing either pUCpotF or pUC
mutated potF, according to the method of Oliver and Beckwith
(48). The PotF protein represented approximately 50% of the total
periplasmic protein content, and this was used as the PotF protein
source (see Fig. 3A). The reaction mixture (0.1 ml)
containing 10 mM Tris-HCl (pH 7.5), 30 mM KCl,
10 µg of PotF protein, and 4 µM
[14C]putrescine (2.24 GBq/mmol) was incubated at 30 °C
for 5 min. The PotF protein was collected on membrane filters
(cellulose nitrate, 0.45 µm; Advantec Toyo), and the radioactivity
was counted with a liquid scintillation spectrometer. The protein
content was measured by the method of Lowry et al. (49).
Putrescine Uptake by Intact Cells-- E. coli KK313potF::Km containing pPT79.3 and pMWpotF (or pMW mutated potF) was grown in medium A until the A540 reached 0.3. The assay for putrescine uptake was performed as described previously (50) using 10 µM [14C]putrescine (370 MBq/mmol) as the substrate.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Structure Description--
PotF consists of 370 amino acids (41 kDa), including a 26-amino acid signal peptide, which is cleaved after
biosynthesis and was not included in the sample used for
crystallization. The PotF crystal structure lacks three amino acids,
two N-terminal and one C-terminal, which are disordered in the crystal.
The PotF molecule has an ellipsoidal shape, with approximate dimensions of 54 × 42 × 30 Å, and belongs to the /
type of
proteins. It consists of two structurally similar globular domains.
Each domain is composed of a central, five-stranded mixed
-sheet
flanked on both sides by six and eight
-helices in the N- and
C-domains, respectively (Fig. 1). Both
domains consist of two distinct amino acid segments. Residues 29-133
and 279-332 constitute the N-terminal domain, whereas residues
139-273 and 347-369 form the C-terminal domain. Thus, PotF has three
interdomain linker segments and belongs to group I of the periplasmic
receptors according to the classification (12). Linkers 1 (residues
134-138) and 2 (residues 274-278) form a two-stranded antiparallel
-sheet at the center of the molecule, which makes up the floor of
the interdomain cleft. Linker 3 (residues 333-346) is located on the
molecular surface and consists of two
-helices joined by a short
loop. One disulfide bridge is formed between Cys-175 and Cys-239,
which stabilizes the conformation of the C-domain.
|
Putrescine Binding Site-- The putrescine binding site is located in the deep cleft (26 × 6 × 10 Å) at the interface between the two PotF domains. The putrescine is almost completely engulfed in the cleft and makes multiple polar and van der Waals interactions with the PotF residues (Fig. 2). The PotF residues from both domains are involved in putrescine binding. Two putrescine amino groups are recognized through hydrogen bonds with adjacent acidic residues and main chain carbonyl oxygens. In total, there are six direct and six water-mediated hydrogen bonds between the putrescine amino nitrogens and the PotF residues. The N1 putrescine atom at the entrance of the binding cavity is hydrogen bonded to Ser-38, Asp-39, and Asp-247 and interacts with Ser-38, Asp-247, and Ser-226 through the triad of water molecules, which is conserved in all four PotF molecules (Fig. 2). The N2 atom of putrescine is completely buried in the cleft and makes strong hydrogen bonds with the carboxyl oxygens of Asp-278 and with a water molecule (W4, Fig. 2), which in turn is tightly bound to the adjacent Ser-85 and Glu-185. This water molecule (average B-factor = 30 Å2) is conserved among the four independent PotF. The affinity of PotF to putrescine is enhanced through the 36 nonspecific van der Waals interactions between the putrescine atoms and the five hydrophobic PotF residues (Fig. 2). The putrescine backbone lies inside the hydrophobic cylinder, with an approximate radius of 3.6 Å formed by the PotF side chains of Trp-37, Tyr-40, and Tyr-314 from the N-domain, Trp-244 from the C-domain, and Phe-276 in the linker region.
|
Mutational Analysis-- The crystal structure of the PotF protein in complex with putrescine revealed the protein residues involved in putrescine binding. To elucidate the functional roles of these residues in putrescine binding, mutant proteins were prepared by site-directed mutagenesis, and their putrescine affinities and uptake activities were measured (Fig. 3). Among the mutated residues, 13 corresponded to those of the spermidine binding site in the homologous PotD protein, whereas Asp-247 lacked an analogue in PotD (Fig. 4).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Comparison of PotF with PotD and Other Periplasmic-binding
Proteins--
The two domain architecture and the topology of each
domain of PotF resemble the structures of other periplasmic receptors (12). Among these, the PotF structure was most similar to its closest
homologue, PotD (35% sequence identity), which in turn was shown to be
similar to the maltodextrin-binding protein in the ligand-bound form
(32). The r.m.s.d. between all C atoms of PotF and PotD
was only 1.5 Å (Fig. 5). At the same
time the N-domains of the two proteins exhibited better structural homology (r.m.s.d. = 1.1 Å) than their C-domains (r.m.s.d. =1.7 Å),
as was also found in other periplasmic binding proteins. In contrast to
PotD, PotF contains one disulfide bridge, formed between Cys-175 and
Cys-239. These two cysteines are located at the beginning of two
adjacent
-strands in the central
-pleated sheet of the C-domain
and thus may stabilize the conformation of this
-sheet.
|
Polyamine Binding by PotF and PotD--
PotD can bind not only
spermidine but also putrescine with lower affinity. After superposition
of the PotF and PotD molecules, the putrescine bound to PotF coincides
well with the diaminobutane moiety of the spermidine bound to PotD
(Fig. 5). Among the 13 amino acid residues that were shown to be
important for spermidine binding by PotD (Fig. 4B), seven
residues (Trp-37, Tyr-40, Ser-85, Glu-185, Trp-244, Asp-278, and
Tyr-314) are absolutely conserved in the PotF sequence, and their side
chains overlap well in the three-dimensional structures (r.m.s.d. = 0.8 Å). All of these residues are crucial for the putrescine binding and
uptake activities of PotF (Figs. 3 and 4A). Surprisingly,
the mutations of Ser-85 and Glu-185, which do not directly contact with
putrescine, substantially impaired the PotF activities. On the other
hand, they interact with the putrescine amino group through the water
molecule W4 (Fig. 2 and 4A). The presence of this water
molecule is important as it makes a barrier for putrescine in the
binding cleft. The displacement of the water W4 would facilitate the
penetration of putrescine deeper into the cleft and would disturb the
interactions of the substrate with the major amino acid residues
involved in putrescine recognition. One feasible role of Ser-85 and
Glu-185 is to fix the position of the water W4, and thus, to stabilize the volume and the shape of the binding cavity. The carboxyl oxygens of
Glu-185 form strong hydrogen bonds (3.0 and 3.6 Å) with the N1 atom of Trp-244, which stacks on the substrate.
Therefore, a second possible role of Glu-185 is to maintain the
favorable orientation of the Trp-244 side chain. Among 36 van der Waals contacts between putrescine and the PotF aromatic side chains, 21 interactions are made with Trp-37 and Trp-244, which is in good
agreement with the mutational analysis (Fig. 3).
Implications for Substrate Specificity-- Why does PotF bind only putrescine? Judging by the structure of PotD, the most logical explanation is that PotF recruits some bulky side chain that protrudes deeply into the binding cavity and prevents the spermidine binding by steric hindrance with the spermidine aminopropyl moiety. The alignment of the PotF and PotD sequences shows that the Gln-327 side chain in PotD, which is involved in binding to the amino group of the aminopropyl portion of spermidine, is replaced by the longer side chain of Lys-349. On the basis of the PotD structure, the PotF model was constructed in which Lys-349 blocks the binding of spermidine to PotF (33). However, this model disagrees with the PotF crystal structure. The structural alignment of the PotF and PotD sequences shows that the actual counterpart of Gln-327 (PotD) in PotF is Leu-348, the side chain of which is far from the binding site, due to the difference in the local conformations between PotD and PotF.
In PotF, the position of the N1 atom of putrescine is strictly fixed, whereas in PotD, the N1 atom of spermidine is more flexible, as discussed above. To understand whether this difference may prevent spermidine binding to PotF, we made a docking model of the spermidine molecule with a fixed position of its N1 amino group. There was no conformation that lacked steric hindrance with some protein residues in the PotF binding site. Thus, we presume that the substrate selectivity of PotF is primary dominated by the unique hydrogen bond network with the polyamine N1 amino group, so that polyamines larger than putrescine are not able to fit to the shape of the PotF binding cavity. In conclusion, the crystal structures of the PotF and PotD proteins provide the first insight into the mechanism of specific binding and discrimination between different polyamines by biological macromolecules. The structural results, in combination with the mutational analysis, revealed that polyamine recognition could be achieved by the cooperation of multiple polar and hydrophobic interactions. The arrangement and the chemical properties of amino acids in the PotF (PotD) binding site may be used as a template for studies of other polyamine-binding proteins. One encouraging example is that a sequence comparison with PotD revealed the residues crucial for polyamine binding in a glutamate receptor (52-54). ![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. Y. Maeda and K. Imada for the opportunity to use the x-ray facilities at the International Institute for Advanced Research (Kyoto, Japan) and for their assistance in the data collection. We thank Dr. Y. Kimura for providing us with computer facilities.
![]() |
FOOTNOTES |
---|
* 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.
§ To whom correspondence may be addressed: International Institute for Advanced Research, Matsushita Electric Industrial Co., Ltd., 3-4 Hikaridai, Seika, Soraku, Kyoto, 619-0222, Japan. Tel.: 81-774-98-2543; Fax: 81-774-98-2575.
To whom correspondence may be addressed. Tel.: 81-43-290-2897;
Fax: 81-43-290-2900.
1 This program was modified by D. G. Vassylyev.
2 The abbreviation used is: r.m.s.d., root mean square deviation.
3 The mutated PotF protein W37L contains leucine instead of tryptophan at position 37 from the initiator methionine.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|