doi:10.1016/j.jmb.2010.09.005 J. Mol. Biol.

(2010) 404, 220–231

Contents lists available at www.sciencedirect.com

Journal of Molecular Biology

j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b

Two Stacked Heme Molecules in the Binding Pocket

of the Periplasmic Heme-Binding Protein HmuT from
Yersinia pestis
Daniel Mattle†, Antra Zeltina†, Jae-Sung Woo†, Birke A. Goetz
and Kaspar P. Locher⁎
Institute of Molecular Biology and Biophysics, ETH Zürich, HPK D17, Schafmattstrasse 20, CH-8093 Zürich, Switzerland

Received 18 April 2010; The periplasmic binding protein HmuT from Yersinia pestis (YpHmuT) is a
received in revised form component of the heme uptake locus hmu and delivers bound hemin to the
1 September 2010; inner-membrane-localized, ATP-binding cassette (ABC) transporter HmuUV
accepted 3 September 2010 for translocation into the cytoplasm. The mechanism of this process, heme
Available online transport across the inner membrane of pathogenic bacteria, is currently
1 October 2010 insufficiently understood at the molecular level. Here we describe the crystal
structures of the substrate-free and heme-bound states of YpHmuT, revealing
Edited by G. Schulz two lobes with a central binding cleft. Superposition of the apo and holo states
reveals a minor tilting motion of the lobes surrounding concomitant with
Keywords: heme binding. Unexpectedly, YpHmuT binds two stacked hemes in a central
heme binding; binding cleft that is larger than those of the homologous periplasmic heme-
iron uptake; binding proteins ShuT and PhuT, both of which bind only one heme. The
heme transport protein; hemes bound to YpHmuT are coordinated via a tyrosine side chain that
type III periplasmic binding contacts the Fe atom of one heme and a histidine that contacts the Fe atom of
protein; the other heme. The coordinating histidine is only conserved in a subset of
protein crystallography periplasmic heme binding proteins suggesting that its presence predicts the
ability to bind two heme molecules simultaneously. The structural data are
supported by spectroscopic binding studies performed in solution, where up
to two hemes can bind to YpHmuT. Isothermal titration calorimetry suggests
that the two hemes are bound in discrete, sequential steps and with
dissociation constants (KD) of ∼0.29 and ∼29 nM, which is similar to the
affinities observed in other bacterial substrate binding proteins. Our findings
suggest that the cognate ABC transporter HmuUV may simultaneously
translocate two hemes per reaction cycle.
© 2010 Elsevier Ltd. All rights reserved.

*Corresponding author. E-mail address:

locher@mol.biol.ethz.ch.
Present address: D. Mattle, Department of Molecular
Biology, University of Aarhus, Gustaf Wieds Vej 10C,
DK-8000 Aarhus C, Denmark.
† These authors contributed equally to this work.
Abbreviations used: ABC, ATP-binding cassette; hmu,
heme uptake system; DMSO, dimethyl sulfoxide; ITC,
isothermal titration calorimetry; OD600, optical density at
600 nm; OM, outer membrane; PBP, periplasmic binding
protein; SBP, substrate-binding protein; SeMet,
selenomethionine.
Introduction
The survival of pathogenic bacteria such as
Yersinia pestis in mammalian host organisms
depends on their successful acquisition of iron, an
essential micronutrient involved in a multitude of
biological processes.1 The availability of free iron in
host organisms is low, and bacteria have therefore
evolved various iron uptake systems.2 While some
systems include the biosynthesis of iron-binding
molecules (siderophores), others facilitate the

0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.
Structure of bacterial heme binding protein HmuT
uptake of heme directly acquired from the host.3
Pathogenic bacteria that are Gram-negative have an
outer membrane (OM) that is impermeable to heme
or siderophores. It contains transport proteins that
depend on the function of the energy-providing
TonB complex located in the inner membrane
(TonB-dependent receptors). Once the substrate
(heme or siderophore) reaches the periplasmic
space, it is captured by a specific substrate-binding
protein (SBP) and delivered to the external surface of
an ATP-binding cassette (ABC) transporter located
in the inner membrane.4
The ABC transporter facilitating heme uptake in
Y. pestis is the HmuUV-T system.5 It belongs to the
type II ABC importers, 6 with the membrane-
spanning HmuU and the ATP-binding HmuV
forming the transport complex, while HmuT is the
periplasmic SBP. There is at present no published
crystal structure of a heme ABC importer, although
it is expected to be similar in fold to the vitamin B12
transporter BtuCD.7,8 By contrast, two OM trans-
porters for heme, the TonB-dependent HasR from
Serratia marcescens 9 and ShuA from Shigella
dysenteriae,10 have been structurally characterized,
as have the soluble part of the heme-binding
lipoprotein IsdE from Staphylococcus aureus
(SaIsdE),11 the periplasmic binding proteins (PBPs)
ShuT from S. dysenteriae (SdShuT), and PhuT from
Pseudomonas aeruginosa (PaPhuT).12 In the context of
our studies of the Y. pestis heme ABC importer
HmuUV-T, we have determined the structure of the
PBP YpHmuT, which has the same fold as other type
III binding proteins. However, unlike its homolo-
gues SdShuT and PaPhuT, YpHmuT simultaneously
binds two stacked hemes in a substrate-binding cleft
that is larger than those of the homologues. This is
the first report, to our knowledge, of a SBP capturing
two substrates simultaneously and also the first
report of such heme stacking within a single heme
transport protein. We describe the apo and the holo
structures of YpHmuT and spectroscopic as well as
isothermal titration calorimetry (ITC) data that
demonstrate the 2:1 binding stoichiometry in solu-
tion. Our findings open new perspectives for
understanding the ATP-to-substrate stoichiometry
of ABC transporters. At the same time, they suggest
that depending on the environment, an adaptation
of the SBP to acquire more than one substrate may
provide an evolutionary advantage.
Results
YpHmuT is a type III PBP
The crystal structures of apo and holo forms of
YpHmuT were determined at 1.5 and 2.5 Å
resolution, respectively. Details of the purification
221
and crystallization procedures are given in Materials
and Methods, and a summary of the data collection
and refinement statistics is given in Table 1. For both
the apo and the holo crystals, the asymmetric units
contained two molecules of YpHmuT, with minor
differences between them at the present resolution
(Table 2). The heme-bound protein was crystallized
after addition of dimethyl sulfoxide (DMSO)-
dissolved hemin at a molar ratio of 2:1 before
concentration of the protein. If larger amounts of
hemin were used, it was found in the filtrate of
the concentrator.
YpHmuT features two structurally similar
domains (lobes) with low sequence similarity. Both
lobes contain central β-sheets surrounded by short
α-helices (Fig. 1a). The two lobes are connected by a
single α-helix (“backbone helix”). This structural fold
is typical for type III PBPs, including the vitamin B12
binding protein BtuF,13,14 the ferrichrome binding
protein FhuD,15 and the heme binding proteins
SaIsdE, SdShuT, and PaPhuT. Of the cognate ABC
transporters, only the structure of the B12 transporter
BtuCD has been determined. The superposition of
YpHmuT with EcBtuF (Fig. 1b) therefore not only
demonstrates the similarities of the protein folds but
also reveals that YpHmuT features two conserved,
surface-exposed glutamates (E77 and E206), one in
each lobe. Similarly positioned glutamates are
present in EcBtuF, where they have been predicted13
and later demonstrated8 to have an important role in
docking the SBP to the cognate ABC transporter
BtuCD. The α-helical section of the backbone helix,
which is thought to rigidify type III SBPs, is ∼ 10 Å
shorter in YpHmuT when compared to EcBtuF,
SdShuT, PaPhuT, or EcFhuD.
Heme-binding pocket of YpHmuT
Unexpectedly, two stacked heme molecules are
bound in the central cleft between the N and C lobes
of YpHmuT. The hemes are stacked with their
propionate groups pointing in opposite directions,
resulting in “internal” and “external” propionate
groups. The unbiased, experimental electron density
clearly defined the two heme moieties (not shown).
Figure 2 shows a simulated annealing Fo − Fc map
after omitting both hemes and the side chains of five
residues making close contacts with the hemes. The
maps demonstrate the presence of the two hemes
and define the interactions of YpHmuT with them.
To our knowledge, this is the first time that two
substrate molecules are observed in the binding
pocket of a SBP. In particular, the structurally
characterized heme-binding proteins SaIsdE,
SdShuT, and PaPhuT contain only one heme in
their binding pockets.
The comparison with the periplasmic SdShuT and
PaPhuT proteins reveals differences in YpHmuT
that are relevant to heme binding (Fig. 3). (i) The
222 Structure of bacterial heme binding protein HmuT

Table 1. Data collection and refinement statistics

HmuT crystal Heme-bound SeMet Heme-bound native Heme-free native
Data collection
Space group P6122 P6122 P21
Wavelength (Å) 0.9184 0.9796 0.9794 0.9788 0.9191
Unit cell parameters
a, b, c (Å) 89.2, 89.2, 288.9 89.2, 89.2, 288.9 61.7, 66.7, 62.2
α, β, γ (°) 90.0, 90.0, 120.0 90.0, 90.0, 120.0 90.0, 110.7, 90.0
Resolution (Å) 30–2.8 30–2.8 30–2.8 30–2.5 30–1.48
30–2.8 30–2.8 30–2.8 30–2.5 30–1.48
Observed reflections 213,686 (41,754) 148,205 (28,247) 295,784 (56,489) 936,171 (70,805) 537,900 (37,506)
Unique reflections 31,453 (5858) 31,455 (5876) 31,616 (5897) 24,545 (1763) 78,325 (5865)
Rmeas (%) 6.9 (27.8) 6.1 (29.4) 6.6 (43.7) 5.5 (60.7) 8.9 (52.8)
Average I/σ 18.9 (6.4) 17.43 (5.13) 22.0 (4.9) 47.8 (7.9) 14.2 (4.3)
Completeness (%) 99.3 (99.3) 99.3 (99.6) 99.8 (100.0) 99.9 (100.0) 98.9 (97.0)
Redundancy 6.8 (7.1) 4.7 (4.8) 9.4 (9.6) 38.1 (40.2) 6.9 (6.6)
Wilson B-factor (isotropic, Å2)a 71.2 15.6

Refinement
Rwork 0.196 (0.248) 0.152 (0.181)
Rfreeb 0.238 (0.295) 0.181 (0.218)
No. of protein residues 508 506
No. of water molecules 45 537
No. of iron atoms 4 —
No. of bromide ions — 7
Average B-factor (Å2) 81.5 14.3
r.m.s.d.
Bond lengths (Å) 0.005 0.009
Bond angles (°) 0.998 1.195
Values for the outermost resolution shells are given in parentheses.
a
Calculated by XDS.
b
Test sets: 6.4% and 7.0% of reflections for heme-bound and heme-free HmuT, respectively.

diheme binding pocket of YpHmuT is larger than to the heme-bound Fe atoms are different. While all
the heme-binding pockets of the homologues. The three periplasmic heme-binding proteins contain a
distance between Y71 and R228 (residues involved conserved tyrosine residue whose hydroxyl group
in heme binding) of PaPhuT is 6.2 Å, as measured forms a bond with the Fe atom, only YpHmuT
from the hydroxyl group of Y71 to the ɛ-nitrogen of features a histidine residue (H167) that binds the Fe
R228. A similar distance is measured in SdShuT atom of the second heme molecule (Fig. 3a). A similar
(5.9 Å between the hydroxyl group of Y94 to the histidine residue is absent in SdShuT and PaPhuT
methyl group of A196). By contrast, the distance (Fig. 3b). An arginine residue packs against the distal
between the hydroxyl group of Y70 and the side of the single heme in PaPhuT, whereas an
ɛ-nitrogen of H167 of YpHmuT is 8.4 Å, making alanine and two leucines fulfill this role in SdShuT. A
room for the second heme molecule. (ii) The contacts BLAST search and sequence alignments reveal that
the tyrosine residue from the N lobe (Y70 in
YpHmuT) is strictly conserved (Fig. 3c), but heme-
Table 2. Root-mean-square distances of YpHmuT specific SBPs can be grouped by whether the
r.m.s.d. (Å) histidine residue contacting the Fe atom of the
second heme (H167 in YpHmuT) is present or not
Full chain N lobe C lobe
Substrate (residues (residues (residues (Fig. 3d). We propose that the presence of such a
Superposition binding Chain 27–278) 27–114) 156–278) histidine in other homologous SBPs predicts whether
Reference Apo A 0.92 0.26 0.57
two hemes might be bound, whereas its absence
Moving Apo B would suggest a stoichiometry of one heme per
Reference Holo A 0.43 0.24 0.38 protein. Phylogenetic analysis did not reveal an
Moving Holo B obvious physiological relationship between species
Reference Apo A 1.31 0.22 0.74 that express HmuT homologues with histidines in
Moving Holo A
Reference Apo A 1.55 0.23 0.88 the binding pockets.
Moving Holo B In addition to the axial ligands to the central Fe
Reference Apo B 1.81 0.33 0.93 atoms, YpHmuT also provides other contacts to
Moving Holo A bound heme: There are three hydrogen bonds from
Reference Apo B 2.03 0.28 1.09
Moving Holo B
the protein to one of the internal propionate groups
of the Tyr-coordinated heme (Fig. 2), including two
Structure of bacterial heme binding protein HmuT 223

(a) backbone helix

C
N

E77

E206

(b)

C C
N N
C C
N N

Fig. 1. Overall structure of the periplasmic heme-binding protein YpHmuT. (a) Ribbon representation with the two
lobes (N and C lobes in different shades of green) connected via a backbone α-helix. The substrates (heme molecules) are
shown as yellow and cyan sticks in the binding cleft. The side chains of conserved surface glutamates are shown as sticks
and labeled. (b) Stereo view of superimposed YpHmuT (green) and EcBtuF [magenta, Protein Data Bank (PDB) code
1N2Z] highlighting the similarity in fold.

bonds formed by amino groups of the backbone and
one by the hydroxyl group of Y200. By contrast, the
second internal propionate group forms no hydro-
gen bonds but van der Waals interactions with R199
and Y200. The histidine-coordinated heme points its
propionate groups away from YpHmuT, where they
form no contact with the protein. There are
numerous additional van der Waals interactions
between the protein and the heme moieties provided
by the side chains of T179, Y200, M165, M255, and
L258.
Although the presence of two substrates bound to
YpHmuT was unexpected, stacking of heme mole-
cules bound to proteins has previously been
observed. In particular, the heme-degrading MhuD
protein reveals a similar stacking of two heme
moieties,16 but the edges featuring the propionate
groups are not pointing in opposite directions as in
YpHmuT, but are rotated by 90° around an axis
perpendicular to the tetrapyrrole plane. Another
difference is that only one heme-bound Fe atom is
directly contacted by a side chain (a histidine) in
MhuD, whereas the other Fe atom is bound to a Cl−
ion. Heme stacking has also been observed in the
structure of the OM lipoprotein ChaN from Cam-
pylobacter jeuni,17 which features a partially exposed
heme molecule bound to its surface, with the
hydroxyl group of a tyrosine residue bound to the
Fe atom. In the crystal, ChaN forms dimers that
reveal a similar heme stacking as in YpHmuT,
although the stoichiometry is one heme per ChaN
monomer.
The binding pocket of the substrate-free state of
YpHmuT is partially disordered. The electron
density map is discontinuous in molecule A
between residues 166 and 171 (not shown), which
224 Structure of bacterial heme binding protein HmuT

Fig. 2. Stereo view of electron density maps of diheme binding pocket of YpHmuT. An Fo − Fc simulated annealing
omit map, contoured at 3σ, is shown in purple after omitting both heme moieties and reducing the five side chains in
closest contact with the hemes (H167, Y70, Y200, R72, M165) to alanines. In total, 114 atoms were omitted for the
calculation of the omit map, including the two Fe atoms at the center of the heme moieties. The gray electron density
is the final 2Fo − Fc electron density map, contoured at 1σ. Relevant side chains are labeled. Dashed lines in cyan
indicate the three hydrogen bonds from one of the internal propionate groups with the protein. The other three
propionate groups make no hydrogen bonds.

includes H167 that binds heme (see above). The
equivalent section of molecule B is ordered due to
lattice contacts. This indicates a high degree of
flexibility of the main-chain residues 166–171, which
only become well ordered upon binding of heme
moieties.
Spectroscopic and ITC data support the
observed binding stoichiometry
To assess whether the presence of two heme
molecules bound to YpHmuT may be an artifact
related to crystallization, we performed titration
experiments using soluble YpHmuT by recording
UV/Vis spectra between 300 and 700 nm. Heme,
but not the protein, has absorption peaks in this
range, and the interactions of tyrosine residues with
the central Fe atom have previously been used to
study heme binding.16,18,19 We used two distinct
experimental setups: In the first setup, soluble
YpHmuT was present in a cuvette, and heme was
added in increments before spectra were recorded.
For these heme titrations, we used previously
described conditions that produce monomeric
heme by preparing stocks either in DMSO or in
NaOH.16,18 We also equilibrated each sample for
5 min before recording spectra to allow for
equilibration.16 Figure 4a and b show the results of
heme titrations. Unlike MhuD (which also binds two
hemes), YpHmuT has two strong and distinct
interactions of side chains (Y70 and H167) with the
Fe atoms of the heme moieties. We observe not only
a binding ratio of two hemes per YpHmuT but also
distinct absorption maxima for the two binding
events, one at ∼ 400 nm and another at ∼ 375 nm.
Because of the distinct spectral properties of the two
protein-bound hemes and the low absorbance of
hemin, a quantitative evaluation of these titrations
to obtain KD values is difficult from these data,
although the results suggest that both dissociation
constants are in the submicromolar range.
In a second setup, we kept the heme concentration
constant and varied that of YpHmuT. To prevent
aggregation of heme, we used the nonionic deter-
gent N-dodecyl-β-D-maltopyranoside, as the rather
hydrophobic heme moiety tends to cluster or bind to
hydrophobic surfaces. In the absence of YpHmuT,
heme has a Soret maximum around 404 nm (Fig. 4c).
This maximum shifts to 375 nm upon addition of
YpHmuT up to a ratio of two hemes per YpHmuT
(red curve). Additional YpHmuT shifts the Soret
maximum back to 399 nm (blue curve), suggestive of
a 1:1 ratio of heme to protein at these conditions.
This supports the notion that YpHmuT can bind one
or two hemes in its binding pocket, depending on
the concentration. When plotting the absorbance at
404 nm against the concentration of YpHmuT (Fig.
4d), we observed a pronounced kink at two,
Structure of bacterial heme binding protein HmuT 225

Fig. 3. Heme binding in different SBPs. (a) Binding pocket of YpHmuT with two stacked hemes. Y70 interacts with the
Fe atom of one heme, while H167 contacts that of the second heme. (b) Binding pocket of the heme-binding protein
PaPhuT (PDB code 2R79), which binds only one heme. (c and d) Sequence alignments of heme binding proteins around
Y70 (c) and H167 (d). The top five sequences contain a conserved His residue in the C lobe and are shaded light green,
indicating that they may bind two hemes as shown in (a). The bottom five sequences contain only the strictly conserved
Tyr residue in the N lobe and are shaded orange, suggesting that they bind only one heme as shown in (b). The overall
sequence identities of different heme PBPs compared to YpHmuT are 55% (Klebsiella pneumoniae), 41% (Burkholderia
pseudomallei), 34% (Vibrio fischeri), 32% (P. piscicida), 35% (S. dysenteriae), 34% (Bartonella bacilliformis), 33% (P. aeruginosa),
31% (Shewanella oneidensis), and 30% (Idiomarina loihiensis).

suggesting that heme binding to YpHmuT is
saturable at a ratio of two hemes per YpHmuT.
To determine dissociation constants (KD), we
carried out ITC experiments, where hemin was
added to YpHmuT equilibrated in the experimen-
tal chamber. The results (Fig. 5) demonstrate two
discrete heme-binding steps, the first with a
subnanomolar affinity (KD of ∼ 0.29 nM) and the
second with a lower but still rather high affinity
(KD of ∼ 29 nM). It should be pointed out that for a
single binding event, ITC can typically determine
dissociation constants between ∼ 20 nM and
20 μM,20 suggesting that a subnanomolar KD is
usually outside of the experimental reach of the
method. However, because there is second heme-
binding event in YpHmuT that is coupled to the
first and has a KD within the useful range of ITC,
the observed data can be successfully fit assuming
a two-step, sequential binding process (Fig. 5c),
which yields quantitative information about the
first binding event (higher affinity) as well. To
evaluate the reliability of the curve fitting, we
performed simulations of the binding data using
various combinations of dissociation constants
(Supplementary Fig. 1). We noticed that the data
can also be reasonably fit with other combinations
of dissociation constants, as long as (i) two
sequential binding events are assumed, (ii) the
higher KD (second heme binding, lower affinity)
has a value of no larger than 30 nM, and (iii) the
two dissociation constants differ by a factor of 100.
This suggests that the affinity of YpHmuT for
heme could, in principle, be even higher than
suggested by fitting of the ITC data. The results of
the ITC studies are, nevertheless, in agreement
with the spectroscopic studies and suggest that
YpHmuT does indeed bind two heme moieties
and with affinities in a physiologically relevant
range and similar to those of other binding
proteins.21
226 Structure of bacterial heme binding protein HmuT

Fig. 4. Heme binding by YpHmuT observed by UV/VIS spectroscopy and titration. In (a) and (b), the results of hemin
titrations are shown. YpHmuT (5 μM; no protein in the reference) was present in the cuvette, and NaOH-monomerized
hemin was added in small increments, after which the solution was mixed and allowed to equilibrate for 5 min.16 (a)
Difference spectra (reference subtracted from sample) for selected heme concentrations (colors as indicated) reveal two
distinct maxima, one at 373 nm and another at 404 nm. (b) Change in absorbance at 373 nm versus heme concentration
indicates saturable heme binding to YpHmuT, with a maximum just above a heme/protein ratio of 2. The experiment was
done in duplicate, and error bars are indicated. In (c) and (d), the results of YpHmuT titration are shown. The
concentration of hemin in the sample was 20 μM, whereas that of YpHmuT was varied. (c) Spectra are shown at selected
YpHmuT concentrations. (d) Change in absorbance at 404 nm versus YpHmuT concentration reveals a pronounced kink at
2 heme:YpHmuT. The experiment was done in duplicate, and error bars are indicated.

A tilting motion is observed upon binding
Superpositions of apo and holo YpHmuT mole-
cules were performed and r.m.s.d. values were
calculated for all combinations and lobes (Table 2).
The apo state was found to have a slightly higher
flexibility compared to the heme-bound state as
indicated by higher r.m.s.d. values between the
noncrystallographic symmetry mates. Heme bind-
ing to YpHmuT leads to a conformational change
that was analyzed by the protein motion analysis
software DynDom.22 This revealed two domains, a
fixed domain (residues 28–155 and 260–276) and a
moving domain (residues 156–259). The moving
domain tilted by ∼ 12° around an axis that encloses
an angle of ∼ 40° with backbone helix (Fig. 6).
Compared with the opening–closing of lobes
observed in type I and II binding proteins (e.g., the
maltose-binding protein), the motion associated
with heme binding is smaller and does not include
a kinking of the backbone helix.
Discussion
Conformational changes in SBPs
SBPs undergo substantial conformational
changes while acquiring substrates, docking to
their cognate ABC transporters, and releasing their
cargo. The comparison of the apo and holo states
of YpHmuT reveals the conformational changes
concomitant with heme binding. YpHmuT is a
type III binding protein similar to the B12-binding
EcBtuF that has been structurally characterized in
the apo and holo states.13,14 Type III SBPs are
characterized by a rigidifying backbone helix,
Structure of bacterial heme binding protein HmuT
Fig. 5. ITC study of heme binding to YpHmuT. (a)
Hemin (0.5 mM) was added to a solution of 20 μM
YpHmuT in steps and at 30 °C to a final molar ratio of
∼ 3.2. (b) The data were analyzed using Origin software
and revealed two distinct binding steps. (c) the KD values
were computed from the curve fittings, and the thermo-
dynamic parameters are indicated.
which is thought to prevent an opening–closing
motion of a Venus flytrap, a mechanism used to
describe type I and II SBPs including the maltose-
Fig. 6. Conformational change in YpHmuT upon heme binding. Stereo view of substrate-free (red) and heme-bound
(green) YpHmuT after the N lobes were superimposed. For the heme-bound state, only the C lobe is shown for clarity. The
arrow indicates the tilting/pivoting of the C lobe upon heme binding.
227
binding protein MalE. 23–27 The change from
substrate-free to heme-bound YpHmuT consists
of only a minor tilting or pivoting of one lobe
relative to the other (Fig. 6). However, a more
prominent conformational change is expected
upon delivery of the cargo into the translocation
pathway of the cognate ABC transporter. Al-
though there is no structure of the YpHmuUV-T
transporter complex, that of the related B 12
transporter complex EcBtuCD-F has been
determined.8 BtuF was found to undergo a lobe
opening only when in complex with BtuCD, but
not in solution. The BtuCD-F structure also
revealed that the conserved glutamates of BtuF
interact with patches of conserved arginine resi-
dues of the ABC importer. Because YpHmuT
contains conserved surface glutamates in similar
locations, it is expected to interact with similarly
conserved arginine patches of HmuUV. These
interactions are crucial for docking, as the
BtuCD-F complex could not be formed if the
glutamates of BtuF were mutated to alanines.8
Also, mutations of the analogous glutamates in the
S. aureus ferrichrome binding protein FhuD2
resulted in decreased uptake of this siderophore.28
Binding and transport stoichiometry
Until now, SBPs have been believed to feed single
substrate molecules into the translocation pathways
of their cognate ABC importers. Our finding that
YpHmuT binds two stacked heme molecules was
unexpected and surprising, in particular because the
close homologues SaIsdE, SdShuT, and PaPhuT all
bind single heme molecules.11,12 Our sequence
alignments (Fig. 3c and d) suggest that a subclass
of heme-binding SBPs may bind two hemes, whereas
others, including SdShuT and PaPhuT, bind only
one, depending on the presence or absence of a
histidine residue coordinating the Fe atom of the
228
second heme molecule. The Photobacterium damselae
subsp. piscicida HutB protein would, according to
our prediction, be able to bind two hemes (Fig. 3d).
HutB was purified and reported to bind heme in a 1:1
ratio.19 However, the spectroscopic binding data
presented in that report demonstrate that saturation
of heme binding occurs at 10 μM heme. Given that
5 μM HutB was used in the experiment, we conclude
that this could support the interpretation that there is
in fact a 2:1 heme-to-HutB binding ratio rather than
an equimolar binding.
What is the physiological relevance of our observa-
tion? Although we demonstrate that YpHmuT can
simultaneously bind two heme molecules in vitro, it
remains to be established whether these can be
translocated into the cytoplasm during a single
reaction cycle of the cognate ABC transporter YpH-
muUV. The binding constants for YpHmuT and the
two heme molecules are in the range described for
other SBPs.21 In particular, EcBtuF binds B12 with an
affinity of ∼ 15 nM,29 only a twofold higher affinity
than the second heme molecule in YpHmuT
(∼29 nM). Although the concentrations of nutrients,
such as heme, in the periplasm are difficult to estimate,
we assume, on the basis of the comparison with other
SBP's KD values, that diheme binding by YpHmuT
might well be a physiologically relevant phenomenon.
If the cognate ABC transporter YpHmuUV trans-
ported both hemes in one step, the stoichiometry of the
transport reaction would likely be one ATP to one
heme, because most ABC transporters are thought to
hydrolyze two ATP molecules during one reaction
cycle, as was demonstrated for the OpuA system.30
This would be energetically favorable over the
reactions of other ABC importers.
Heme is a relatively hydrophobic molecule that
oligomerizes to π–π dimers or larger π–π stacks in
aqueous solution, a process that is dependent on the
heme concentration, pH, temperature, and the
presence of salts.31 Heme uptake by Y. pestis is
facilitated by the gene products of the hmu and the
has loci,5,32,33 but it is unknown at present whether
diheme can be transported across any of the two OM
transporters HasR and HmuR. The related HasR
protein of S. marcescens probably imports monomeric
heme, as suggested by the HasR–HasA cocrystal
structure.9 We therefore assume that monomeric
heme crosses the OM and, depending on the heme
concentration, monomeric heme or diheme crosses
the inner membrane.
Conclusions and outlook
The two pathways of heme uptake across the Y.
pestis envelope merge at the level of the periplasmic
HmuT protein that feeds the substrate into the
cognate ABC importer HmuUV.34 YpHmuUV is a
type II ABC importer whose detailed transport
mechanism is currently poorly understood. The
Structure of bacterial heme binding protein HmuT
best-studied type II ABC importer, the vitamin B12
transporter BtuCD-F, has been demonstrated to
have a distinct mechanism from that of ABC
exporters or type I ABC importers.35,36 Among the
most significant differences are the apparent
absence of a binding pocket for the substrate in the
transmembrane domain, distinct transmembrane
folds and topologies, and a different coupling
mechanism in response to ATP binding and hydro-
lysis. The heme uptake system HmuUV-T is a
physiologically relevant member of type II ABC
importers that warrants further study, as iron
acquisition is essential for the growth of pathogenic
microbes. The structure of YpHmuT, described in
this report, combined with our observation that two
hemes are bound with physiologically relevant
affinities, are steps in the direction of a detailed
understanding of the heme uptake system.
Materials and Methods
Plasmid construction and protein expression
The gene hmuT of Y. pestis was amplified from pHMU75
without its predicted signal sequence (residues 2–25) and
ligated into a modified pET-19b (Novagen, Madison, WI)
vector to yield the plasmid phmuT+his. This fused a
C-terminal hexahistidine tag to the protein, which was used
for the structure determination of substrate-bound YpH-
muT. A nontagged version of YpHmuT was expressed from
the plasmid phmuT−his, which was generated by inserting
a stop codon into phmuT+his using the QuickChange Site-
directed mutagenesis kit (Stratagene, La Jolla, CA). The
phmuT−his construct was used for the crystallization of the
substrate-free form of HmuT.
Both constructs were expressed in Escherichia coli BL21
CodonPlus (DE3)-RIPL cells (Stratagene, La Jolla, CA) in
Terrific Broth medium containing 1% (w/v) glucose and
ampicillin (0.1 mg/ml) at 37 °C. The temperature of the
cultures was shifted to 25 °C at an optical density at 600 nm
(OD600) of 0.5, and the induction was done with 0.4 mM
IPTG at an OD600 of 1.6. The cultures were harvested after
4 h (phmuT+his) or 5 h 30 min (phmuT−his).
A selenomethionine (SeMet) derivative of YpHmuT was
expressed using the phmuT+his plasmid in E. coli BL21
CodonPlus (DE3)-RIPL cells. Expression was performed in
minimal medium [17 mM KH2PO4, 72 mM K2HPO4,
18 mM NH4Cl, 10 mM NaCl, 1 mM MgSO4, 0.5% (w/v)
glucose, vitamin B1 hydrochloride (0.5 mg/ml)]. The
temperature was shifted from 37 °C to 25 °C at an OD600 of
0.2 and 0.02% (w/v) L-lysine, L-threonine, L-phenylala-
nine, L-leucine, L-isoleucine, L-valine, and 0.01% (w/v)
L-SeMet were added. Expression was induced 60 min
later by addition of 0.4 mM IPTG. The culture was
harvested after 4 h. All cells were stored at − 80 °C.
Protein purification
All of the following purification steps for the native and
SeMet YpHmuT (phmuT+his) were done at room
Structure of bacterial heme binding protein HmuT
temperature unless stated otherwise. Cells containing the
native and the SeMet affinity-tagged HmuT were dis-
rupted with an M-110L microfluidizer (Microfluidics) at
15,000 psi (103 MPa) external pressure (chamber: H10Z,
100 μm) in 150 mM NaCl and 50 mM Tris–HCl (pH 7.5)
(additionally, 5 mM β-mercaptoethanol for SeMet HmuT).
The ratio of cells to cracking buffer was 1:10 (weight to
volume). The lysate was centrifuged (40,000g for 30 min at
4 °C) and the supernatant was loaded onto a Ni–NTA
superflow affinity column (QIAGEN). The column was
washed with 20 mM imidazole (pH 8.0) and the protein
was eluted with 250 mM imidazole (pH 8.0) in 150 mM
NaCl and 50 mM Tris–HCl (pH 7.5) (additionally, 5 mM β-
mercaptoethanol for SeMet HmuT). Purified proteins
were desalted into 150 mM NaCl, 20 mM Tris–HCl
(pH 7.5), and 0.5 mM EDTA (ethylenediaminetetraacetic
acid)–NaOH (pH 8.0) using a HiPrep 26/10 desalting
column (GE Healthcare, Piscataway, NJ).
Purification of nontagged YpHmuT was done at 4 °C.
Cells were disrupted as above but in 20 mM NaCl and
20 mM Tris–HCl (pH 8.0). After centrifugation, the
supernatant was diluted 10-fold in 20 mM Tris–HCl
(pH 8.0) and loaded onto a HiTrap Q HP column (GE
Healthcare). The column was washed with 2 mM NaCl
and the protein was eluted with 80 mM NaCl and 20 mM
Tris–HCl (pH 8.0).
Protein purity was analyzed by SDS-PAGE, and protein
concentrations were determined by measuring the UV
absorbance at 280 nm. The extinction coefficients were
calculated with the prot-param tool of the ExPASy
proteomics server.37 Protein concentrations were con-
firmed by performing amido black assays.38
Crystallization and data collection
For heme-bound YpHmuT, DMSO-dissolved hemin
(Fluka) was added to native, affinity-tagged HmuT or
SeMet affinity-tagged YpHmuT (both at 36 μM) to a
final concentration of 70 μM. For crystallization, apo and
holo YpHmuT were concentrated to 40–45 mg/ml with
an Amicon Ultra centrifugal filter (10,000 molecular
weight cutoff). Aggregates were removed by a 10 -min
ultracentrifugation step in an airfuge (Beckman Coulter),
and the stability of the concentrated protein was
analyzed by gel filtration (Superdex 200 10/300 GL
column, GE Healthcare).
Apo YpHmuT was crystallized at 10 °C by mixing
protein 1:1 with reservoir solution [29% (w/v) polyeth-
ylene glycol 5000 monomethyl ether, 100 mM magne-
sium acetate, 100 mM Mes (pH 6.5)]. Colorless crystals
appeared after 5 days. Heme-bound YpHmuT was also
crystallized at 10 °C by mixing protein 1:1 with reservoir
solution [13% (w/v) polyethylene glycol 4000, 200 mM
magnesium acetate, and 100 mM sodium cacodylate
(pH 6.5)]. Brown, hexagonal crystals appeared after
4 days.
Crystals were cryoprotected using 25% (v/v) glycerol
and frozen in liquid nitrogen. In the case of the apo
YpHmuT, the best diffraction data were obtained from a
crystal incubated in 500 mM sodium bromide, even
though the bromide was not used for phasing.
Diffraction data sets were collected from single crystals
at the beamline X06SA [Swiss Light Source (SLS), Villigen,
Switzerland] and were processed using XDS.39
229
Structure determination and refinement
Experimental phases for heme-bound YpHmuT were
obtained from a multiwavelength anomalous dispersion
collected from the SeMet derivative crystals. Selenium
positions were found using SHELX40 and refined using
SHARP, 41 and maps were calculated using CCP4
programs.42 The anomalous difference maps showed 24
out of 26 SeMet positions. Apo YpHmuT data were
phased by molecular replacement using Phaser.43 Several
rounds of model building were performed with the
software package O,44 and refinement was carried out in
CNS45 and PHENIX.46 For TLS refinement, the structures
were submitted to the TLSMD Web server.47
Both heme-bound and heme-free YpHmuT crystals
contained two molecules per asymmetric unit, designated
A and B. Heme-bound YpHmuT was modeled from
residues 26 to 279. Heme-free YpHmuT was modeled
from residues 26 to 278. Residues 166 to 171 in molecule A
of the heme-free state did not have interpretable electron
density map, therefore, the occupancy was set to zero.
Model quality was checked by PROCHECK.48 The
refined, heme-bound YpHmuT model had 93.3% of the
residues in the most favored regions, 6.0% in additionally
allowed regions, and 0.7% of the residues in the
generously allowed regions of the Ramachandran plot.
Heme-free YpHmuT had 94.8% of the residues in the most
favored regions, 4.8% in additionally allowed regions, and
0.5% in the generously allowed regions of the Ramachan-
dran plot.
Domain movements were investigated using DynDom,22
and illustrations were prepared using PyMOL.49 R.m.s.d
values were calculated using the least-squares calculation
in O.
Spectroscopic titration assays
For hemin titrations, purified, his-tagged YpHmuT
was desalted into 150 mM NaCl and 20 mM Tris–HCl
(pH 7.5) using a HiPrep 26/10 desalting column (GE
Healthcare) and diluted in the same buffer to a
concentration of 5 μM as determined by amido black
assay. A 0.5 mM monomeric hemin stock solution was
prepared as described earlier16 by initially dissolving
3.3 mg hemin chloride (Fluka) in 500 μL of 0.1 M NaOH
to which 500 μL of 1 M Tris (pH 8.0) was added. This
solution was diluted with 20 mM Tris (pH 7.5) and
150 mM NaCl to a total volume of 10 mL. Successive
aliquots of 0.5 mM hemin (1–20 μM, in 1 μM steps) were
added into a sample cuvette containing 5 μM YpHmuT
and a reference cuvette containing buffer alone.
Samples were equilibrated for 5 min after addition of
each heme aliquot, and UV/Vis spectra between 300 and
700 nm were recorded with a Varian Cary 50 spectro-
photometer at 20 °C.
For YpHmuT titrations, purified, nontagged YpHmuT
was desalted into titration buffer [150 mM NaCl, 20 mM
Tris–HCl, 0.1% (w/v) dodecyl maltoside (pH 8.0)] using
a HiPrep 26/10 desalting column and concentrated to
43 μM. Various samples were prepared, all containing
20 μM hemin (in the same buffer) and varying
concentrations (0–40 μM) of YpHmuT. Samples were
allowed to equilibrate before UV/Vis spectra were
recorded as above.
230
Isothermal titration calorimetry
A 0.5 mM monomeric hemin stock solution was
prepared as described above for the spectroscopic binding
assays. A 20 μM YpHmuT solution was prepared in the
same buffer as hemin. Both the protein and the heme
solutions were centrifuged and degassed for 5 min prior to
the measurements. ITC measurements were carried out at
30 °C by means of a MicroCalorimetry system (MicroCal)
by titrating hemin into the 20 μM YpHmuT solution. Heme
dilution enthalpies were determined in separate experi-
ments and subtracted. Data were analyzed with the Origin
software package (OriginLab), and the thermodynamic
parameters were determined by fitting the observed
curves of the integrated heat per mole of added titrant.
PDB accession numbers
Atomic coordinates and structure factor amplitudes
have been deposited in the PDB with the accession
numbers 3MD9 (apo YpHmuT) and 3NU1 (heme-bound
YpHmuT).
Supplementary materials related to this article can be
found online at doi:10.1016/j.jmb.2010.09.005
Acknowledgements
We thank R. D. Perry (University of Kentucky)
who kindly provided the plasmid pHMU7 contain-
ing the Y. pestis hmu system. We also thank the
beamline staff at the SLS for support with data
collection. This research was supported by the
NCCR Structural Biology Zurich, the Swiss National
Science Foundation SNF, and the FP7 program
EDICT of the European Union.
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