Structures of a Na+-coupled, substrate-bound MATE
Min Lua,1, Jindrich Symerskya, Martha Radchenkoa, Akiko Koideb, Yi Guoa, Rongxin Niea, and Shohei Koideb
aDepartment of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064; andbDepartment
of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637
Edited by Richard Henderson, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom, and approved December 19, 2012 (received for review
November 15, 2012)
Multidrug transporters belonging to the multidrug and toxic
compound extrusion (MATE) family expel dissimilar lipophilic and
cationic drugs across cell membranes by dissipating a preexisting
Na+or H+gradient. Despite its clinical relevance, the transport
mechanism of MATE proteins remains poorly understood, largely
owing to a lack of structural information on the substrate-bound
transporter. Here we report crystal structures of a Na+-coupled
MATE transporter NorM from Neisseria gonorrheae in complexes
with three distinct translocation substrates (ethidium, rhodamine
6G, andtetraphenylphosphonium), as wellas Cs+(a Na+congener),
all captured in extracellular-facing and drug-bound states. The
structures revealed a multidrug-binding cavity festooned with four
negatively charged amino acids and surprisingly limited hydropho-
bic moieties, in stark contrast to the general belief that aromatic
amino acids play a prominent role in multidrug recognition. Fur-
thermore, we discovered an uncommon cation–π interaction in the
Na+-binding site located outside the drug-binding cavity and vali-
dated the biological relevance of both the substrate- and cation-
binding sites by conducting drug resistance and transport assays.
Additionally, we uncovered potential rearrangement of at least
two transmembrane helices upon Na+-induced drug export. Based
on our structural and functional analyses, we suggest that Na+
triggers multidrug extrusion by inducing protein conformational
changes rather than by directly competing for the substrate-bind-
ing amino acids. This scenario is distinct from the canonical antiport
mechanism, in which both substrate and counterion compete for
a shared binding site in the transporter. Collectively, our findings
provide an important step toward a detailed and mechanistic un-
derstanding of multidrug transport.
cation coordination|substrate recognition|membrane protein|
ated by integral membrane proteins called “multidrug trans-
porters” is a major mechanism underlying multidrug resistance,
a serious and growing public health threat (1, 2). The ∼900
multidrug and toxic compound extrusion (MATE) transporters
are the most recently recognized members of multidrug efflux
pumps (3), which are unique among the known multidrug trans-
porters in that they can harness the energy stored in either Na+or
H+electrochemical gradient (4, 5). In particular, human MATE
transporters, hMATE1 and hMATE2, are H+-coupled anti-
porters (6, 7), whereas many bacterial MATE proteins, including
NorM from Neisseria gonorrheae (NorM-NG), NorM from Vibrio
cholerae (NorM-VC), and NorM from Vibrio parahaemolyticus
(NorM-VP), are Na+-dependent (8–10) (Fig. S1). MATE sub-
strates exhibit highly diversified chemical structures, although
they are typically polyaromatic and cationic. MATE transporters
are promising drug targets because they extrude antibiotics and
therapeutic drugs in pathogenic bacteria and in mammals, re-
The 3.65-Å-resolution X-ray structure of NorM-VC trapped in
a cation-bound, drug-free state revealed the transporter archi-
tecture in an outward-open conformation and implicated nine
he extrusion of antimicrobials and therapeutic drugs medi-
amino acids in Na+binding (9). However, the Na+coordination
chemistry remains unclear because only a semiconserved Y367 is
positioned close enough to make plausible coordination to this
cation in NorM-VC. Therefore, it is largely unknown how MATE
antiporters accomplish polyspecific multidrug recognition and
how they couple drug efflux to the influx of counterions. To ad-
dress such questions, we present here the structures of NorM-NG
bound to an engineered crystallization chaperone termed “mono-
body” (15), crystallized in the absence and presence of three
translocation substrates: ethidium, rhodamine 6G (R6G), and
Structure Determination. We aimed at elucidating the molecular
basis of multidrug recognition and transport by MATE trans-
porters. To this end, we crystallized a well-characterized bacterial
MATE transporter, NorM-NG (8), in the presence of its trans-
location substrates. The cocrystals diffracted X-rays beyond 3.8-Å
resolutions but suffered from severe twinning defects that pre-
cluded a successful structure solution. To overcome this difficulty,
we generated a monobody, a single domain-binding protein based
on the fibronectin type III domain, directed to NorM-NG using
the phage display technology (15). We then prepared crystals
of NorM-NG bound to the monobody, which were amenable to
crystallographic analysis. We obtained crystals both in the pres-
ence and absence of three translocation substrates. We sub-
sequently determined the four structures by combining molecular
replacement and multiple isomorphous replacement and anom-
alous scattering (MIRAS) phasing and refined the structures to
3.5–3.6 Å resolutions (Figs. S2 and S3; Tables S1–S6).
As anticipated, most of the crystal contacts are mediated by the
monobody and only a few by head-to-tail packing interactions
between neighboring NorM-NG molecules (Fig. S4). The asym-
metric unit contains one chalice-shaped complex of NorM-NG
and monobody, which interacts mainly through the carboxyl-ter-
minal tail of the transporter (Fig. 1 A and B). Unexpectedly, we
found an unidentified ligand, likely of cellular origin, located at
the drug-binding site in the “apo” structure. The four structures
are largely identical (rms deviation ∼0.5 Å for 550 Cα positions),
except for some minor differences near the substrate-binding site,
all portraying the transporter in an outward-facing and drug-
Author contributions: M.L., M.R., A.K., and S.K. designed research; M.L., J.S., M.R., A.K.,
Y.G., R.N., and S.K. performed research; M.L., J.S., M.R., A.K., and S.K. analyzed data; and
M.L. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.pdb.org (PDB ID codes 4HUK–4HUN).
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| February 5, 2013
| vol. 110
| no. 6
R6G Efflux Assay. We performed the fluorescence-based transport assays as
previously described (8, 24), with the following modifications. Briefly, cultures
of E. coli BL21 (DE3) ΔacrABΔmacABΔyojHI cells expressing NorM-NG variants
were grown at 30 °C to ∼1.0 A600nmunits. Cells were harvested, washed with
100mM Tris-HCl, pH 7.0, resuspended in the same buffer containing 4.5 μg/mL
R6G and 100 μM carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and in-
cubated at 37 °C for 30 min. To initiate the NorM-NG–mediated R6G efflux, 200
mM NaCl was added to the sample. R6G efflux was monitored by measuring
the fluorescence with a respective excitation and emission wavelength of 480
and 570 nm. Assays were performed in 96-well plates, and the fluorescence
was measured using a microplate reader. The R6G efflux activity of NorM-
NG variants was evaluated based on the reduction of R6G fluorescence,
which was calculated by subtracting fluorescence in the absence of the
artificial Na+gradient from that in the presence of the Na+gradient.
ACKNOWLEDGMENTS. We thank H. Yamanaka for BL21 mutant strains and
the beam-line staff at 23-ID and 22-ID of Argonne National Laboratory for
help during data collection. We also thank C. Correll, R. Henderson, P. Nissen,
G. Rudnick, M. Glucksman, R. Kaplan, and A. Gross for comments on the
manuscript. This work was supported by National Institutes of Health Grants
R01-GM094195 (to M.L.) and U54-GM087519, R01-GM072688, and R01-
GM090324 (to S.K.) and Rosalind Franklin University of Medicine and
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