(Received for publication, October 31, 1995; and in revised form, January 26, 1996)
From the
PotD protein is a periplasmic binding protein and the primary
receptor of the polyamine transport system, which regulates the
polyamine content in Escherichia coli. The crystal structure
of PotD in complex with spermidine has been solved at 2.5-Å
resolution. The PotD protein consists of two domains with an
alternating -
-
topology. The polyamine binding site is
in a central cleft lying in the interface between the domains. In the
cleft, four acidic residues recognize the three positively charged
nitrogen atoms of spermidine, while five aromatic side chains anchor
the methylene backbone by van der Waals interactions. The overall fold
of PotD is similar to that of other periplasmic binding proteins, and
in particular to the maltodextrin-binding protein from E.
coli, despite the fact that sequence identity is as low as 20%.
The comparison of the PotD structure with the two maltodextrin-binding
protein structures, determined in the presence and absence of the
substrate, suggests that spermidine binding rearranges the relative
orientation of the PotD domains to create a more compact structure.
Polyamines, such as putrescine, spermidine, and spermine, are
ubiquitous in all living organisms. They are involved in a wide variety
of biological reactions, including nucleic acid and protein
synthesis(1, 2) . These compounds exist as linear
molecules with two (putrescine;
NH-(CH
)
-NH
),
three (spermidine;
NH
-(CH
)
-NH
-(CH
)
-NH
),
and four (spermine;
NH
-(CH
)
-NH
-(CH
)
-NH
-(CH
)
-NH
)
positively charged nitrogen atoms. It is a crucial subject in cell
biology to elucidate the detailed mechanisms of polyamine biosynthesis
and transport by which the cellular polyamine contents are controlled.
Although the biosynthetic pathways for polyamines have been studied
extensively, the transport mechanism remains
obscure(1, 2) .
The polyamine transport genes in Escherichia coli have been cloned and
characterized(3, 4, 5, 6, 7) .
The proteins encoded by pPT104 constitute the spermidine-preferential
uptake system, which belongs to a periplasmic transport
system(8, 9) . This spermidine transport machinery
consists of four protein subunits, PotA, -B, -C, and -D. The PotA (M 43,000) protein, which is bound to the inner
surface of the cytoplasmic membrane, is a strong candidate for an
ATP-hydrolyzing, energy-generating factor. In fact, the PotA protein
contains a consensus nucleotide-binding sequence, and exhibits ATPase
activity(10) . Both the PotB (M
31,000)
and PotC (M
29,000) proteins have six
transmembrane spanning segments linked by hydrophilic peptides with
variable lengths, and hence they are assumed to jointly form a channel
for spermidine and putrescine. The PotD protein is a periplasmic
binding protein and consists of 348 amino acids, corresponding to a
molecular mass of 39 kDa. Although it binds both spermidine and
putrescine, spermidine is preferred(6) .
The crystal structures of several periplasmic binding proteins specific for substrates, such as amino acids (lysine/arginine/ornithine(11, 12) , leucine/isoleucine/valine(13) , and leucine(14) ), oligopeptide (15) , tetrahedral oxyanions (sulfate (16) and phosphate(17) ), and saccharides (arabinose(18) , galactose/glucose(19) , ribose(20) , and maltodextrin(21, 22) ) have been solved by x-ray analysis. These binding proteins, which have remarkably broad specificities, share a similar main chain fold, although they lack significant sequence similarities. Furthermore, they consist of two similar domains, which show an ``opening-closing'' movement likened to a Venus flytrap, depending upon substrate binding(22) .
The polyamines are unique substrates for a periplasmic binding protein, and their specific interactions with cognate binding proteins have never been studied in terms of three-dimensional structure. Therefore, a crystallographic study of the PotD protein at an atomic resolution was performed to elucidate the detailed mechanism of its specific substrate recognition and the characteristics of the main chain folding. In this paper, we report the molecular structure of the PotD-spermidine complex determined at 2.5-Å resolution by x-ray analysis.
The major heavy atom sites of KPtCl
and Pb(NO
)
derivatives prepared by
soaking were determined from their difference Patterson maps. The
initial analysis of the x-ray data showed that the structure factors
for the reflections with the odd h indices were much smaller
than those with the even h indices (<F(2n + 1 , k, l)> = 0.5*<F (2n, k, l)>) in a 6-Å
resolution shell. This fact, along with the analysis of the heavy atom
sites in the derivatives, confirmed that there are two dimers of the
protein in the asymmetric unit, connected by an almost precise
translational symmetry with of the a-axis of the
crystal(24) . The heavy atom parameters were refined with the
programs PROTEIN (25) and MLPHARE (26) against the
3.0-Å resolution data, including anomalous data from all
derivatives. The latter program provided the mean figure of merit of
0.63. Solvent flattening (27) and noncrystallographic symmetry
averaging techniques (28) were applied to improve the phases.
The 2-fold molecular averaging, using only reflections with h = 2n, was successful in substantially improving
the map. However, this map was still insufficient to achieve a complete
chain tracing. The structure determination statistics for the MIR
phasing are summarized in Table 1.
Figure 1:
Structure
of the PotD. A, primary sequence of the PotD protein with the
secondary structure elements. The amino acid sequence has been deduced
from the base sequence of the potD gene(6) . Residues
involved in ligand binding are indicated by an asterisk. The underlines indicate a disordered region (Asp and
Asp
) in the crystal. A conserved sequence motif between
PotD and MBP is indicated by <-Motif-> and spans
residues 46-54 of PotD(35) . B, ribbon model of
the PotD monomer, drawn with the program MOLSCRIPT(44) . The N
domain lies at the bottom, and the C domain is at the top. The
-helices are green, the
-strands
are blue, the coils and loops are yellow, and the
motif region is sky blue. The spermidine molecule is bound to
the central cleft between the two domains. The binding site for the
spermidine molecule is marked by the side chains of the four acidic
residues.
The PotD
molecule has an ellipsoidal shape with dimensions of 30 40
55 Å. It consists of two distinct domains divided by a
deep cleft. Each domain is formed by two noncontiguous polypeptide
segments. Nevertheless, the two domains are very similar in the
arrangements of their secondary structure elements. The first domain (N
domain; residues 26-131 and 257-302) consists of five
-strands and six
-helices. The other domain (C domain;
residues 132-256 and 303-348), with a larger size, contains
five
-strands and seven
-helices. The
-sheet within each
domain is flanked by several
-helices on both sides. The
polypeptide chain crosses over three times between the two domains,
which noncovalently interact with each other by an extensive interface.
The three crossing segments and the interface form a deep cleft with
approximate dimensions of 20 Å long, 5 Å wide, and 14
Å deep (Fig. 1B). The PotD protein, with its many
-
-
repeats, is classified as an
/
type. Of the
amino acids, 40 and 18% are located in the
-helices and the
-sheets, respectively. The remaining amino acids (42%) belong to
loops and coils. There is no substantial difference among the backbone
structures of the four independent molecules in the asymmetric unit.
Their root mean square (r.m.s.) (
)deviations for the
superimposed C
atoms are as low as 0.40 Å. However, when the
C
atoms of the N and C domains are superimposed separately among
the corresponding domains in the asymmetric unit, the C domain shows a
larger r.m.s. deviation value (0.42 Å) than the N domain (0.33
Å).
Figure 2: Dimeric structure of PotD. Ribbon drawing of a dimer. Each monomer in a dimer is related by a noncrystallographic 2-fold axis. The spermidine molecule is shown by a red ball-and-stick model, and the consensus motif is indicated by blue balls. The dimer is primarily stabilized by a network of hydrogen bonds and salt bridges.
Figure 3:
Electron density showing the bound
spermidine. Stereo view of the electron density for spermidine
in the binding pocket and its environment. The electron density map was
calculated with the coefficients Fo
-
Fc
, and the phases from the refined protein and the
solvent atoms with the spermidine molecule are omitted. The contour
levels are at 3.5
. The residues of the N domain are green, the C domain is blue, and the substrate is red. The conformation of the bound spermidine and its
environment is shown in Fig. 4.
Figure 4: Interactions of spermidine with protein atoms. A, a stereo picture showing hydrogen bonds, salt bridges, and van der Waals interactions. Hydrogen bonds and salt bridges are indicated by dotted lines. The spermidine molecule is shown by the shadowed ball-and-stick model. B, schematic diagram of hydrogen bonding and van der Waals interactions with spermidine.
The substrate binding site is
located at the middle of the cleft between the two domains. This site
forms a hydrophobic box, which is composed of four aromatic side
chains, Trp, Tyr
, Trp
, and
Tyr
in the N domain and Trp
in the C
domain. These aromatic side chains anchor the methylene backbone of the
spermidine molecule through van der Waals interactions. The methylene
bonds of spermidine are sandwiched between the aromatic side chains of
Trp
and Trp
, which are arranged in parallel (Fig. 4A). The side chain of Trp
,
oriented perpendicular to the previous side chains, covers the
spermidine like a lid over the cleft.
Another important feature in
the binding site is that four acidic residues, Glu,
Asp
, Glu
, and Asp
, recognize
the charged nitrogen atoms of spermidine through numerous ionic
interactions (Fig. 4B). The conformations of these
aromatic and acidic residues are conserved well among the four
molecules in an asymmetric unit. One terminal amino group of propyl
amine moiety in the spermidine forms the salt bridges with the carboxyl
side chains of Asp
and Glu
, and the
hydrogen bonds with the side chain of Gln
and
Tyr
. The secondary amino group in the middle is recognized
through the side chain of Asp
, and the other terminal
amino group forms a salt bridge with the side chain of Glu
and a hydrogen bond with the side chain of Thr
.
These aromatic and acidic side chain atoms embed the spermidine
molecule in the cleft so as to prevent no solvent access.
Figure 5:
Topological diagrams of PotD and MBP. The
-helices are represented as lettered cylinders, and the
-strands are indicated by arrows with numbers.
Common secondary structure elements between PotD and MBP are shadowed. The active residues of the two binding proteins are
shown as solid lines.
Another notable similarity between the two structures is that a
conserved sequence motif is found in both PotD and MBP(35) .
This conserved sequence motif spans residues 46-54 (FTKETGIKV) of
PotD, which corresponds to the loop between 1 and
B, and
residues 53-61 (FEKDTGIKV) of MBP (Fig. 1). These
sequences exhibit a remarkably similar conformation between the two
molecules, as proved by the very small r.m.s. deviation value of 0.40
Å for the nine C
positions.
Figure 6:
Comparison of PotD and MBP. The PotD
molecule (yellow) is superimposed on the MBP molecule (blue) by matching only the C atoms within the N domain.
The open form of MBP is shown on the right, and the closed
form is on the left. This view is rotated by 90° about the vertical axis in Fig. 1B.
The PotD protein can
bind putrescine as well as spermidine, although the affinity of
putrescine is much lower than that of spermidine. The dissociation
constants (K) for spermidine and putrescine are
3.2 µM and 100 µM, respectively(23) .
These K
values reflect the spermidine-preferential
recognition for the primary receptor of the polyamine transport system.
The shorter putrescine molecule could possibly make ionic interactions
with the acidic residues of Glu
and Asp
, and
form van der Waals interactions with the aromatic residues
Trp
, Tyr
, Trp
,
Trp
, and Tyr
. These interactions may
stabilize the closed conformation. However, the smaller number of
interactions, as compared with those with spermidine, would decrease
the stability of the closed form.
In spite of many relevant reports(38, 39, 40, 41, 42, 43) , mutational analyses have not clearly identified the interface of periplasmic binding proteins with their membrane protein components yet. However, it should be noted that the consensus sequence lies in the N domain, which shows a higher similarity of PotD and MBP in terms of the three-dimensional structure. Furthermore, in most of the periplasmic binding proteins including PotD and MBP (Fig. 5), the folding topology of the N domain is more conserved than the C domain(22) . Therefore, it is likely that the interface of PotD with the membrane components is located in the N domain rather than the C domain.