Interactions of a periplasmic binding protein with a tetradentate siderophore mimic

Article abstract of DOI:10.1002/anie.201300751

Iron-bound structure: The ferric complex of a tetradentate siderophore mimic was synthesized and co-crystallized with the periplasmic binding protein CeuE of Campylobacter jejuni. In addition to electrostatic and hydrogen-bonding interactions between the binding pocket and the substrate, the structure showed direct coordination of two amino acid side chains to the Fe^III center (orange, see figure). Copyright

Full text of DOI:10.1002/anie.201300751

Angewandte
Chemie
DOI: 10.1002/anie.201300751
Siderophores
Interactions of a Periplasmic Binding Protein with a Tetradentate
Siderophore Mimic**
Daniel J. Raines, Olga V. Moroz, Keith S. Wilson, and Anne-K. Duhme-Klair*
III
To ensure the uptake of essential Fe ions, microorganisms
have developed iron chelators known as siderophores, of
catecholate siderophores 2 and 4 are fragments of their
hexadentate counterparts protochelin and enterobactin,
respectively. It is still unclear if “secondary” siderophores,
such as 2 and 4, are produced as siderophores in their own
right or whether they are biosynthetic precursors or degra-
[1–4]
which over 500 examples have now been documented.
Whilst the most powerful siderophores are hexadentate
ligands and form coordinatively saturated octahedral 1:1
complexes with Fe ions, others are ligands of lower denticity
but still bind Fe ions and aid their transport into the
Examples include the bidentate 2,3-dihy-
droxybenzoylserine and citrate, which is likely to act as
a tridentate ligand. In addition, tetradentate siderophores
from a variety of microorganisms have been identified,
including those of the bis(hydroxamate) type, such as
III
[12–14]
dation products of their hexadentate parent compounds.
III
However, the use of low denticity siderophores by pathogenic
[1,5–7]
[15]
bacterial cell.
bacteria has been documented.
Further examples of
[
8]
bis(catecholamide) siderophores are summarized in the
Supporting Information (Scheme S1). In contrast to the well
characterized iron uptake mediated by hexadentate side-
rophores, the exact biological roles of tetradentate side-
rophores and their interactions with ferric siderophore trans-
port proteins have yet to be established.
[9]
[
10]
rhodotorulic acid
(1), and the bis(catecholamide) type,
[
5]
such as azotochelin (2) or the hydrolysis product of enter-
obactin (3), N,N’-bis(2,3-dihydroxybenzoyl)-O-seryl
(4; Scheme 1). Whilst 1 is structurally related to
hexadentate hydroxamate siderophores derived from d-N-
Owing to the reduced number of chelating groups, the
[
6,11]
III
serine
Fe complexes of tetradentate siderophores have a lower
thermodynamic and kinetic stability than those of their
[
1,2,4]
[1,2]
hydroxy ornithine, such as ferrichrome and ferricrocin,
hexadentate counterparts,
a disadvantage in environments
III
with low Fe ion concentrations. However, under less
challenging conditions low-denticity siderophores, which are
likely to have a lower biosynthetic cost, may confer a com-
petitive advantage to the bacteria that are able use them. In
addition to faster Fe dissociation kinetics, the Fe com-
plexes of tetradentate siderophores possess more positive
III
III
[1,2]
III
redox potentials, which can be beneficial to Fe ion uptake
mechanisms that rely on reductive iron release.
Along with the evolution of diverse siderophore struc-
tures, microorganisms have had to adapt their receptor and
transport proteins to accommodate the uptake of the resulting
III
[16]
Fe complexes.
Interestingly, most bacteria do not rely
solely on their own siderophores for iron acquisition but also
acquire siderophores from competing species by producing
suitable receptor proteins. The Gram-negative, food-borne
pathogen Campylobacter jejuni relies on such exogenous
siderophores (xenosiderophores) for iron uptake, in partic-
ular the hexadentate enterobactin and possibly its hydrolysis
Scheme 1. Molecular formulas of H -rhodotorulic acid (1), H -azoto-
2
5
chelin (2), H -enterobactin (3), H -N,N’-bis(2,3-dihydroxybenzoyl)-O-
6
6
[
17]
products also.
Enterobactin is produced by commensal
seryl serine (4), and H -4-LICAM (5).
4
intestinal bacteria. In C. jejuni, ferric enterobactin is recog-
nized by the outer membrane receptors CfrA and CfrB and
transported into the periplasm, where it is captured by the
periplasmic binding protein (PBP) CeuE. The resulting
complex interacts with the inner membrane transporter to
[
*] D. J. Raines, Dr. O. V. Moroz, Prof. K. S. Wilson,
Dr. A.-K. Duhme-Klair
Department of Chemistry, University of York
Heslington, York, YO10 5DD (UK)
E-mail: anne.duhme-klair@york.ac.uk
[18,19]
enter the cytoplasm of the cell.
Previously, we co-crystallized CeuE with the Fe complex
of the enterobactin mimic MECAM (Figure 1).
III
[
**] We thank the UK Biotechnology and Biological Sciences Research
Council (BBSRC) for financial support, the Diamond Light Source
for access to beamlines i03 and i24, Dr. J. Turkenburg, S. Hart for
assistance with data collection, and Dr. A. Leech for help with
circular dichroism spectroscopy.
6À
[20]
This
structure revealed that the CeuE binding pocket contains
three positively charged arginine residues, which balance the
3
À
threefold negative charge of the [Fe(catecholate)3] unit and
donate hydrogen bonds to oxygen donors that are coordi-
nated to the iron center. Two additional hydrogen bonds are
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201300751.
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1
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.
Angewandte
Communications
[
27]
bind a variety of siderophores. We therefore investigated if
the next component of the relevant iron import system, the
PBP CeuE, shows similar ligand promiscuity. Because
C. jejuni needs to acquire its essential iron from a wide
variety of sources, ranging from animal hosts, poultry, milk,
and drinking water to the colonized human host, this
pathogen would benefit greatly from a flexible iron uptake
system. Given that C. jejuni depends on xenosiderophores
[
6]
and considering that enterobactin is prone to hydrolysis, we
[28]
focused our attention on H -4-LICAM (5), a mimic of the
4
tetradentate enterobactin fragment N,N’-bis(2,3-dihydroxy-
benzoyl)-O-seryl serine (4; Scheme 1).
Herein we report the structures of both apo-CeuE and of
À
the CeuE-[Fe(4-LICAM)] complex (PDB codes: 3zkw and
3
zk3, experimental details provided in the Supporting Infor-
mation). The fold of apo-CeuE is that of a typical type III
PBP (cluster A-II), with two domains linked by a long a-
[
29,30]
helix.
In the 4-LICAM complex, the ligand is bound in
III
Figure 1. Molecular formula of H -MECAM with Fe coordinating
6
a shallow binding pocket formed by the two domains.
Superposition of the apo and the complex structure shows
that the binding of the ferric complex of 4-LICAM triggers
only minor structural changes (Supporting Information, Fig-
ure S1).
III
donor atoms highlighted in gray (left) and crystal structure of [{Fe -
6
À
(
MECAM)}2] in complex with two CeuE molecules (right; PDB code:
chu; figure produced using CCP4mg ). Ligand atoms: C gray, O red,
N blue, Fe orange, electrostatic surface representation of the binding
pocket with green cylinders for carbon atoms of the binding amino
acid residues.
[
26]
2
À
CeuE-[Fe(4-LICAM)] is the first co-crystal structure
that provides insights into the interactions of a PBP with
4
À
a tetradentate siderophore (Figure 2). The 4-LICAM ligand
chelates a single iron center in a tetradentate fashion with the
catecholate oxygens interacting with the positively charged
side chains of Arg118, Arg205, and Arg249. The two
formed, one between a tyrosine residue and a coordinated
catecholate oxygen and one between a lysine residue and the
carbonyl oxygen of one of the three catecholamide units.
6
À
Unexpectedly, the MECAM was found to bridge between
III
two Fe centers and two CeuE monomers.
Co-crystal structures of two related ferric siderophore
PBPs have since been reported, FeuA from Bacillus subtilis in
[
21]
complex with ferric bacillibactin,
enterobactin and
[
22]
MECAM, and ViuP from Vibrio cholerae in complex with
ferric vibriobactin (Scheme 2). These proteins bind mono-
[
23]
Scheme 2. Molecular formulas of H -vibriobactin and H -petrobactin
6
6
III
with Fe coordinating donor atoms highlighted in gray.
III
nuclear, coordinatively saturated Fe complexes of hexaden-
tate siderophores. In addition, CeuE shares structural sim-
ilarities with the B. subtilis protein YclQ, which binds the
mixed citrate-bis(3,4-catecholamide) siderophore, petrobac-
III
À
Figure 2. Top) Crystal structure of the CeuE-[Fe (4-LICAM)] complex.
[24]
tin (Scheme 2),
siderophore binding protein of V. cholerae, VctP.
and the second periplasmic catecholate-
4À
III
CeuE=blue ribbon, 4-LICAM chelating a single Fe ion (orange).
Bottom) Binding pocket (blue ribbon backbone with green cylinders
for carbon atoms of the binding amino acid residues), 4-LICAM , and
[
25]
4À
It has been suggested that one of the outer-membrane
siderophore receptors of C. jejuni, CfrA, may have evolved to
III
Fe (orange). Images created using CCP4mg.
2
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Chemie
III
III
À
remaining coordination sites of the octahedral Fe center are
The structure of the CeuE-[Fe (4-LICAM)] complex
occupied by a nitrogen atom and an oxygen donor provided
has the metal center bound in a L-configuration, as seen in
6
À [20]
by nearby His227 and Tyr288, respectively. The FeÀO bond
the co-crystal structures of (CeuE) -[{Fe(MECAM)} ] ,
2 2
3
À
3À
lengths involving the catecholate groups range from 1.9 to
FeuA-[Fe(MECAM)] , FeuA-[Fe(enterobactin)] , and
3
À [21,22]
2
.3 ꢀ and are consistent with those previously found in both
FeuA-[Fe(bacillibactin)] .
To confirm that the L-con-
the crystal structures of ferric bis(2,3-catecholamide) com-
plexes and co-crystal structures of ferric tris(2,3-catechola-
figuration is retained in solution, the circular dichroism (CD)
spectrum of CeuE-[Fe(4-LICAM)] was recorded in the
wavelength range of the ligand-to-metal charge transfer
(LMCT) band at around 500 nm (Figure 4). The negative
[
31]
À
[
20–23]
mides) with their respective binding proteins.
The FeÀO
and FeÀN bond distances for the tyrosine and histidine
interactions are 1.9 ꢀ and 2.3 ꢀ, respectively and agree well
with those in the crystal structure of the inorganic ion
transporter cFbpA from C. jejuni, in which an Fe ion is
coordinated by four tyrosines and one histidine residue.
III
[
32]
4
À
4
-LICAM binds with a 1:1:1 Fe/siderophore/protein
stoichiometry, in contrast to the unusual bridged dimer seen
6
À
with MECAM , in part reflecting the linker between the
iron-binding groups being too short for bridging. Similar
mononuclear binding is observed in the structures of FeuA in
complex with ferric bacillibactin, enterobactin, and
[
22]
[23]
MECAM,
and ViuP with ferric vibriobactin.
The
III
oxygen donor of Tyr288, directly coordinated to the Fe
center in CeuE-[Fe(4-LICAM)] , is positioned 3.5 ꢀ away
from the metal center in (CeuE) -[{Fe(MECAM)} ] , where
À
6
À
Figure 4. CD spectra of CeuE with ferric 4-LICAM bound (black) and
ferric 4-LICAM in the absence of protein (gray); concentrations 68 mm
in 20 mm TRIS buffer, 10 mm NaCl, 0.6% DMSO, pH 8.
2
2
it instead donates a hydrogen bond to one of the deproton-
ated catecholate oxygen donors of the ligand. His227, the
second Fe protein ligand, is disordered, forming part of
a flexible loop in both the apo-CeuE and (CeuE) -[{Fe-
band with a minimum at 400 nm and the positive band with
2
6
À
(
MECAM)}2] structures.
a maximum at 595 nm are indicative of the L-configura-
[
33]
Two of the other structurally characterized ferric side-
tion. In the absence of CeuE, the ferric 4-LICAM complex
shows no CD, confirming that the protein selectively binds the
complex in the L-configuration from the racemic mixture.
rophore PBPs, YclQ and VctP, have Tyr and His side chains
located in positions similar to those found in CeuE-[Fe(4-
À
LICAM)] (Figure 3; Figure S3). YclQ binds the ferric
In summary, the tetradentate siderophore mimic H -4-
4
III
LICAM was synthesized, coordinated to Fe and co-crystal-
lized with the periplasmic binding protein CeuE of C. jejuni.
In addition to the expected electrostatic and hydrogen-
bonding interactions between the binding pocket of CeuE
III
À
and [Fe (4-LICAM)] , the structure revealed the direct
III
coordination of two amino acid side chains to the Fe center.
By displaying this previously unobserved binding mode,
CeuE is the first siderophore binding protein to provide
insights into the recognition and capture of both tetradentate
4
À
and hexadentate siderophores, as exemplified by 4-LICAM
6
À
and MECAM . It is remarkable that despite its flexibility
with regard to ligand denticity, CeuE retains selectivity for the
L-configuration at the Fe center. His and Tyr residues are
III
Figure 3. Overlay of the substrate binding pockets of CeuE (gray), YclQ
conserved in a number of PBPs including YclQ and VctP. It
would therefore be interesting to establish if these proteins
can undergo similar structural changes to adapt to the binding
of lower denticity siderophores. Such adaptability would be
consistent with the observation that one PBP often serves
a whole group of outer-membrane receptors, for example
FepB of E. coli serves a range of ferric catecholamide
(
red; PDB code: 3gfv; His disordered over two sites) and VctP (green;
III
PDB code: 3tef). Tyr and His side chains involved in Fe coordination
in CeuE-[Fe(4-LICAM)] are shown as cylinders with the carbon atoms
À
colored the same as their ribbons.
bis(catecholate) complex formed by 3,4-dihydroxybenzoic
[
24]
[1]
acid, whilst VctP is proposed to bind both [Fe(enterobac-
receptors, including Fiu, Cir, and FepA.
3
À
2À
tin)] and [Fe(H-vibriobactin)] by being able to accom-
[25]
modate different numbers of negative charges. We propose
that these two amino acid residues are part of a mechanism
that enables certain PBPs to capture more than one type of
ferric siderophore.
Received: January 28, 2013
Published online: && &&, &&&&
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Communications
Keywords: iron uptake · metalloproteins ·
periplasmic binding proteins · protein structures ·
tetradentate siderophores
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Angew. Chem. Int. Ed. 2013, 52, 1 – 5
These are not the final page numbers!

Angewandte
Chemie
Communications
Siderophores
Iron-bound structure: The ferric complex
of a tetradentate siderophore mimic was
synthesized and co-crystallized with the
periplasmic binding protein CeuE of
Campylobacter jejuni. In addition to elec-
trostatic and hydrogen-bonding interac-
tions between the binding pocket and the
substrate, the structure showed direct
coordination of two amino acid side
D. J. Raines, O. V. Moroz, K. S. Wilson,
A.-K. Duhme-Klair*
&&&& — &&&&
Interactions of a Periplasmic Binding
Protein with a Tetradentate Siderophore
Mimic
III
chains to the Fe center (orange, see
figure).
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!