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
Central Cavity. NorM-NG traverses the membrane bilayer 12 times
to yield the 12 membrane-spanning segments. Near the middle of
the membrane, similarly folded amino- and carboxyl-terminal
domains (TM1-6, TM7-12) diverge and point away from one
another toward the periplasm, giving rise to an outward-facing
conformation. The 12 TMs are connected by 11 intracellular and
extracellular loops, denoted L1-2 to L11-12. Among them, L3-4,
L9-10, and L6-7 are conspicuously long. The intracellular L6-7
demarcates the amino and carboxyl halves of the transporter,
whereas the extracellular L3-4 and L9-10 extend into a drug-
binding central cavity that is formed between the amino- and
carboxyl-terminal domains (Fig. 1C).
The bottom of the central cavity is situated about halfway
through the membrane bilayer and is defined by D41, S61, F265,
and I292, projecting inward from TM1, TM2, TM7, and TM8,
respectively (Fig. S5). As such, the cavity is shielded from the
cytoplasm by highly ordered protein structure, ∼20 Å thick. The
wall of the cavity is lined by T42, A57, L58, V269, Q284, V285,
I287, and S288 and capped by L3-4 and L9-10, which insert S129,
D130, D355, D356, and P357 to restrict the dissociation of bound
within the cavity are conserved or semiconserved. The interior of
the cavity exhibits a surplus of negative charges but rather limited
hydrophobic moieties, emphasizing its electrostatic attraction for
cations and implying reduced affinity for hydrophobic substrates
(Fig. 1D). One portal that may enable the exit of drugs and the
entry of extracellular Na+is flanked by L3-4 and L9-10, both of
which donate small amino acid side chains to avoid potential
Substrate-Binding Site. The drug-binding site was identified from
conspicuous nonprotein electron densities within the central cavity,
which were congruent with both the size and shape of the bound
substrates (Fig. S6). Furthermore, we calculated anomalous
difference Fourier maps using data collected on NorM-NG
crystals grown in the presence of tetraphenylarsonium (TPP),
a substrate analog (16). A strong anomalous peak indicated the
position of the arsenic atom (equivalent of the phosphorus atom
in TPP) and provided additional support for the identification of
the drug-binding site (Fig. S6).
Because the NorM-NG crystals were obtained in the absence of
monovalent cations including Na+, we propose that the four
structures all represent a Na+-free, drug-bound state of the
transporter. Although at current resolutions our X-ray data were
insufficient to confirm the absence of a bound cation (Na+) in our
structures, we drew our conclusions because the inclusion of Na+
as little as 1 mM precluded crystallization. The substrates bury
∼70% of their accessible surface area on binding the transporter,
which is consistent with values obtained from multidrug-binding
transcription factor BmrR (17). Also similar to BmrR (17, 18), the
structures of NorM-NG revealed similar docking locations for all
three substrates, which are located near the membrane-periplasm
interface (Fig. 2A). Such peripherally located substrate-binding
sites were observed in several different transporters, including the
ABC multidrug transporter P-glycoprotein (19–23).
The key electrostatic component of drug binding by NorM-NG
appears to be conferred by D41, D355, and D356. In particular,
the distances between the Asp carboxyl groups and the positively
charged phosphorus or nitrogen atoms of the bound substrates
range from 3.4 to 7.2 Å (Fig. 2). Because the conjugated aromatic
rings in the three substrates are all expected to carry a partial
positive charge, those electrostatic attractions may be further
enhanced. Consistent with favorable charge-charge interactions,
the distances between the Asp carboxyl groups and the closest
atoms in the substrates are within 3.7 Å. Also potentially con-
tributing to drug-charge complementation are the charge-dipole
interactions mediated by S61, Q284, and S288, whose hydroxyl
in substrates. Notably, the hydrophobic interactions between
NorM-NG monobody complex as viewed from the membrane plane. The
views in A and B are related by ∼180° rotation around the membrane nor-
mal. The amino (residues 5–230) and carboxyl (residues 231–459) halves of
NorM-NG are colored cyan and yellow, respectively. Monobody is shown as
a magenta ribbon and bound TPP as magenta sticks. (C) The arrangement of
transmembrane helices in NorM-NG as viewed from the periplasmic side. (D)
NorM-NG surface as viewed from the periplasmic side, which is colored
according to electrostatic potentials from −20 (red) to +20 kTe−1(blue).
Structure of NorM-NG-monobody complex. (A and B) Structure of
a ribbon, whereas the substrates are in stick representation. (B–D) Closeup of
the binding site for TPP, ethidium (ET), and R6G, respectively. Amino acids
within 4.5 Å (given the ∼0.6-Å coordinate errors at current resolutions) of
the substrate are illustrated as sticks. L3-4 was omitted in B and C for clarity.
Structure of the multidrug-binding site. (A) NorM-NG is drawn as
| www.pnas.org/cgi/doi/10.1073/pnas.1219901110 Lu et al.
NorM-NG and substrates are rather scarce, primarily involving
A57 and F265. In all cases, F265 makes an edge-to-face aromatic
stacking interaction with the substrate.
Functional Importance of the Substrate-Binding Site. To examine the
biological relevance of the drug-binding site, we generated a se-
ries of NorM-NG variants, each with a missense mutation tar-
geting a drug-binding amino acid (Table S7). Among the eight
single mutants that were tested, F265A and S288A were poorly
expressed and therefore excluded from further study. The re-
maining six mutants including F265L were expressed at levels
comparable to that of the WT protein (Fig. S7) and were exam-
ined further for their ability to confer cellular resistance to various
drugs. As shown in Table S7, expression of NorM-NG rendered
bacteria two- to fourfold more resistant toward drugs. By contrast,
the growth of bacteria expressing NorM-NG mutants D41A, F265L,
Q284A, D355A, and D356A at subinhibitory concentrations of
R6G was drastically reduced (Fig. S7). Indeed, those NorM-NG
mutants were unable to relieve the sensitivity of bacteria toward
ethidium, R6G, or TPP (Table S7). Notably, expression of S61A
conferred drug resistance to the same level as seen with the
Furthermore, we used a fluorescence-based, Na+-dependent
R6G effluxassay(8, 24) toevaluate the transport functionofthose
NorM-NG variants. As shown in Fig. S7, the retention of R6G
within the cells expressing NorM-NG gave rise to high fluores-
cence, which could be significantly reduced (>60%) by the extru-
sion of R6G in the presence of an inwardly directed Na+gradient.
As a comparison, there was little reduction (<5%) of R6G fluo-
rescence in cells bearing the empty expression vector, even in the
presence of a Na+gradient. Therefore, the observed fluorescence
reduction was largely due to the NorM-NG mediated R6G efflux.
Importantly, F265L, Q284A, D355A, and D356A all significantly
reduced the R6G efflux activity of NorM-NG, whereas D41A
completely abolished the transport activity (Fig. S7). By contrast,
S61A had little detrimental effect on the transport function, mir-
roring the drug resistance results.
Altogether, our biochemical data strongly suggested that the
drugresistance conferredbyNorM-NG wasdue to theNorM-NG–
mediated drug export, and D41, F265, Q284, D355, and D356
play critical roles in the transport function. Among them, D41 is
the only membrane-embedded charged residue and mutations of
its counterpart, NorM-VPD32, severely crippled transporter ac-
tivity (24). Additionally, hMATE1E300Aabolished transport
function, whereas hMATE1E278Dand hMATE1E300Ddecreased
drug-binding affinity (25). hMATE1E278and hMATE1E300cor-
implying a common substrate-binding site among those prokaryotic
and eukaryotic MATE orthologs.
Cation-Bound Structure of NorM-NG. Because the NorM-VC struc-
ture represents a cation-bound and drug-free state and NorM-NG
shares significant amino acid sequence homology (∼70% similar-
ity) with NorM-VC, there is an opportunity to uncover the con-
formational changes accompanying drug extrusion. Despite the
same protein fold, superimposition of the two structures gave an
rms deviation exceeding 5.2 Å for 450 common Cα positions.
Moreover, at least two conformational differences around the
drug-binding site arediscernible (Fig. 3A). First, whereas L3-4 and
L9-10 in NorM-NG extend into the central cavity and shield the
drug-binding site from the periplasm, their counterparts in
NorM-VC splay apart and move away from the central cavity.
Second, substantial rearrangement of the TMs occurs. Among
them, TM7 and TM8 exhibit the most significant spatial alter-
ations, followed by TM4 and TM11. In particular, TM7 and TM8
both tilt roughly 20° relative to the membrane normal, and as a
consequence, their periplasmic ends shift by ∼6 Å away from the
central cavity in NorM-VC. By contrast, the positioning of TM2,
TM5, TM9, TM10, and TM12 remains almost unchanged.
Because both NorM-NG and NorM-VC are Na+coupled, we
reason that the conformational changes associated with drug re-
lease are likely triggered by Na+binding. As such, we envision that
NorM-NG is capable of binding Na+even when it is in a drug-
bound state. To test this prediction, we soaked the apo NorM-NG
crystals in solutions containing Cs+(a more electron-dense Na+
analog) and solved the structure by molecular replacement. Likely
restrained by both the crystal contacts and binding of the un-
identified ligand, the resulting costructure showed no significant
conformational differences from that of Na+-free, drug-bound
NorM-NG (rms deviation ∼0.5 Å). Nevertheless, we identified
difference Fourier analysis (Fig. 3B). Therefore, in contrast to the
cation-bound, substrate-free structure of NorM-VC (9), the Cs+
-bound structure of NorM-NG represents a drug-bound, cation-
boundstate ofthetransporter,wheretheendogenousligand acted
as a substrate surrogate (Fig. S6). This finding is unexpected, be-
cause according to the canonical antiport mechanism in which
counterion and substrate compete for a shared binding-site in the
transporter (26), substrate and counterion cannot bind to the
Cation-Binding Site. In the Cs+-bound structure of NorM-NG, the
carboxyl group of a conserved E261 and the aromatic ring of
of NorM-NG (cyan and yellow, PDB ID code 4HUK) and NorM-VC (gray, PDB
ID code 3MKU) (9). TPP (stick model) is colored magenta; red arrows high-
light the rearrangement of TM7 and TM8 relative to TM10. (B) Cation-bound
structure of NorM-NG (purplish blue ribbon, PDB ID code 4HUL). Cs+(green
sphere) is overlaid with a difference isomorphous Fourier map (magenta
mesh) contoured at 6σ. Red arrows indicate proposed movement of TM7 and
TM8 toward TM10. TPP (magenta) taken from the TPP-bound structure (PDB
4HUK) is shown in stick representation to indicate the substrate-binding site.
(C) Hypothetical Na+(gray sphere) coordination arrangement that corre-
sponds to state 3 in Fig. 4. Relevant amino acids are depicted as stick models
and NorM-NG is colored gray.
Na+-induced protein conformational changes. (A) Structural overlay
Lu et al.PNAS
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a conserved Y294 are within 3.2 and 4.4 Å of the bound cation,
respectively (Fig. S8). As the radius of Cs+(1.69 Å) is larger than
that of Na+(0.95 Å), the distance between Cs+and its co-
ordination ligands should be larger than that of Na+by ∼0.7 Å. Our
structure thus suggested both E261 and Y294 as Na+-coordinating
ligands. Notably, no drug-binding amino acids appear to directly
coordinate the counterion. Significantly, Y294 makes an uncommon
cation–π interaction (27), for which the average distance between
29) and likely 4.3 Å for Cs+. Furthermore, the growth of bacteria
expressing NorM-NG mutants E261A and Y294L under a sub-
inhibitory concentration of R6G was significantly decreased
compared with that of the WT protein (Fig. S9). Indeed, both
mutants were unable to relieve the sensitivity of bacteria toward
ethidium, R6G, or TPP (Table S7). The inability of E261A and
Y294L to confer drug resistance was most likely due to the impaired
transport function, because both mutations severely suppressed the
R6G efflux activity of NorM-NG (Fig. S9). Notably, mutations of
the counterparts of E261, NorM-VPE251, and hMATE1E273also
abrogatedtransporter activity (6,24, 25),reinforcing the functional
relevance of the observed cation-binding site in NorM-NG.
Previous studies also suggested NorM-VCD371(equivalent of
NorM-NGD377) as a Na+-coordinating residue in the cation-
bound, substrate-free state, because NorM-VCD371Ncompletely
abolished the binding of Na+congeners (i.e., Cs+and Rb+) to the
drug-free transporter (9). Notably, NorM-NGD377is positioned
too far away from the central cavity (>14 Å) to bind substrate
directly. Mutagenesis studies of NorM-NGD377(Fig. S9; Table S7)
and its counterparts, NorM-VPD367and hMATE1E389(24, 25), on
the other hand, lent additional support to the essential role of
NorM-NGD377in Na+-coupled drug transport. Specifically, hMA-
TE1E389Dreduced transporter activity, and NorM-NGD377A,
NorM-VPD367A, NorM-VPD367K, and hMATE1E389Aabrogated
activity. However, in the structure of cation-bound NorM-NG, the
distance between the carboxyl group of D377 and Cs+exceeds
7.2 Å (Fig. 3B). Therefore, for D377 to participate in cation
coordination, a significant TM rearrangement must take place
Proposed Antiport Mechanism. As described above, when com-
paring the structures of cation-free, drug-bound NorM-NG and
cation-bound, drug-free NorM-VC, both TM7 and TM8 appear
to move significantly relative to TM10 on drug release (Fig. 3A).
Furthermore, superimposition of the cation-bound structures of
NorM-VC and NorM-NG placed the bound cation within the
drug-free transporter in close proximity to the carboxyl group of
D377 (TM10) in NorM-NG (Fig. S10). This finding bolstered the
contention that, on initial Na+loading, TM7 and TM8 move
toward TM10 to engage D377 in Na+coordination during drug
extrusion. This Na+-driven TM rearrangement appears to be
critical for the efflux function, as it will pull F265 (TM7), Q284,
and S288 (TM8) away from the central cavity to disrupt drug
binding (Fig. 3C). We posit that these Na+-induced protein con-
formational changes provide the molecular basis for Na+-coupled
drug release. This scenario may also explain why NorM-VPD367E
stimulated, whereas hMATE1E389Dsuppressed, transporter ac-
tivity (24, 25), as perturbation of side-chain length at the posi-
tion equivalent to NorM-NGD377should affect the scale of TM
rearrangement required for counterion coupling.
In our working model (Fig. 4), the outward-facing, drug-bound
NorM-NG (state 1) uses E261 and Y294 to initiate Na+loading
Na+coordination as TM7 and TM8 approach TM10. As a result,
F265, Q284, and S288 move away from the drug-binding site, trig-
gering the release of the bound substrate into the periplasm (state
3). The Na+-bound, drug-free transporter then assumes an inward-
open conformation to capture a new drug molecule (state 4). We
cytoplasm.The drug-bound,Na+-freetransporter (state 6)canthen
return to the extracellular-facing conformation for drug export. As
such, Na+and substrate alternately bind to two spatially distinct
sites during the transport cycle, rather than competing for a com-
mon subset of amino acids. Importantly, this noncanonical, indirect
competition-based antiport mechanism involves a fully loaded in-
termediate state in which substrate and counterion bind to the
transporter simultaneously (state 2), as evidenced by the drug-
bound, cation-bound NorM-NG structure (Fig. 3B).
Unusual Multidrug-Binding Site. The NorM-NG structures de-
scribed here revealed a multidrug-binding site located at the in-
terface between the amino and carboxyl halves of the protein.
circle) binds to a cation-free, drug-bound trans-
porter (state 1) and elicits the movement of TM7
and TM8 (red arrow) in the cation-bound, drug-
bound protein (state 2), causing the drug to disso-
ciate. The cation-bound, drug-free transporter
(state 3) then switches to the inward-facing con-
formation (state 4), before it binds another drug
molecule (magenta). Drug-binding triggers the
movement of TM7 and TM8 (red arrow), thereby
weakening the Na+binding (state 5). Na+releases
into the cytoplasm in an inward-facing, drug-bound
transporter (state 6), and the transporter returns to
(state 1) to complete the transport cycle. Our cat-
ion-free, drug-bound NorM-NG structures (PDB ID
codes 4HUK, 4HUM, and 4HUN) represent state 1,
whereas the cation-bound NorM-NG (PDB ID code
4HUL) and NorM-VC structures (PDB ID codes 3MKU
and 3MKT) emulate state 2 and state 3, re-
spectively. TM1 and TM2 are simplified as a cyan
stick, TM7 and TM8 as a thick yellow stick, and
TM10 as a thin yellow stick.
Proposed antiport mechanism. Na+(green
| www.pnas.org/cgi/doi/10.1073/pnas.1219901110 Lu et al.
In contrast to many other multidrug transporters including
P-glycoprotein (ABC family) and EmrE (SMR family), which use
numerous hydrophobic and especially aromatic amino acids to
bind substrate(s) within a relatively large, voluminous hydropho-
bic cavity (16, 20), NorM-NG uses a surprisingly small number of
hydrophobic residues for drug binding (Fig. 2). Additionally, at
least three acidic residues are used by NorM-NG to neutralize the
substrates, which may be essential for precluding negatively
charged or electroneutral compounds from binding and transport
by the protein, thereby conferring substrate specificity. The un-
expected paucity of aromatic and nonpolar residues, on the other
hand, may allow NorM-NG to avoid the deleterious consequences
of tight association with hydrophobic drugs when the transporter
is poised to release its bound substrate (30, 31). Moreover, there
are no significant alterations in the positions of amino acid side
chains on binding different substrates in NorM-NG, similar to
what had been observed in BmrR (17, 18). As in BmrR, the
presence of multiple acidic residues may enable versatile orien-
tation and charge complementation of structurally dissimilar
cationic drugs in NorM-NG without the need to revamp the drug-
binding site. Also, L3-4 and L9-10 cap the drug-binding site of
NorM-NG, which may provide flexibility to allow each substrate
to make optimal contacts with the transporter.
Uncommon Na+Coordination Arrangement. Membrane protein–
ligand interactions mediated by cation–π interactions usually in-
volve cationic amines, which had been observed in a number of
ligand-gated acetylcholine receptors and G protein–coupled
receptors (27, 32), as well as in the betaine and carnitine trans-
porters (33–35). In fact, cation–π interactions have been considered
as largely electrostatic in nature and well suited for a hydrophobic
environment, including the membrane bilayer (27). However, to
ourknowledge, the Na+–π interaction had never been observed in
any membrane protein before. Our data strongly suggested that
Y294 in NorM-NG makes a cation–π interaction with Na+, which
is also the preferred counterion for NorM-NG compared with K+
(8).This cationpreference maystemfromE261carboxylate being
a high-field-strength ligand that favors the smaller Na+(radius,
0.95 Å) over K+(radius 1.33 Å) (36), as well as the higher binding
affinity for the Na+–π relative to the K+–π interaction (37). Fur-
thermore, the relatively weak Na+–π interaction may also afford
NorM-NG certain kinetic advantages, i.e., faster rates of Na+-
binding and/or unbinding (38), in contrast to the common Na+
coordination arrangement that exclusively involves oxygen atoms
Unconventional Antiport Mechanism. Previous studies on secondary
transporters, including NhaA (41, 42), a Na+/Ca2+exchanger (43),
and the multidrug transporter EmrE (16, 44), supported the ca-
nonical antiport mechanism wherein counterion and substrate
compete for a shared binding site in the transporter. This direct
competition-based coupling mechanism implies that counterion
and substrate cannotbindtothe protein simultaneously. Emerging
experimental evidence, however, has suggested that this may not
be the only plausible antiport mechanism. Specifically, recent
studies on the H+-coupled multidrug transporter MdfA (MFS
family) indicated that H+and drug bind to distinct amino acids in
the protein(45).Furthermore,themultiple structures ofNorM-NG
presented here showed that Na+and substrate interact with
distinct amino acids in the transporter, and they can bind to the
protein simultaneously (Fig. 3B). Therefore, the coupling be-
tween drug and counterion in NorM-NG is indirect and mediated
by protein conformational changes. Notably, in both MdfA and
NorM-NG,the counterion-binding aminoacids areevolutionarily
more conserved than those that interact directly with substrates
(45)(Fig. S1),probably reflecting their broad substrate specificity.
a wide variety of substrates, they can also extrude drugs via distinct
antiport mechanisms, i.e., direct vs. indirect competition.
Materials and Methods
NorM-NG Expression and Purification. The gene encoding NorM from N. gon-
orrheae (NorM-NG) was cloned from genomic DNA into vector pET-15b. For
protein expression, Escherichia coli BL21 (DE3) cells were grown in Luria-Ber-
tani (LB) media to an attenuance of 0.6 at 600 nm and induced with 1 mM
isopropyl β-D-1-thiogalactopyranoside (IPTG) at 37 °C for 3 h. Cells were har-
vested by centrifugation and ruptured by multiple passages through a micro-
fluidizer. Cell membranes were collected by ultracentrifugation and extracted
with 1% (wt/vol) n-dodecyl-β-maltoside (DDM; Anatrace) in 20 mM Hepes, pH
7.5, 100 mM NaCl, 20% (vol/vol) glycerol, and 1 mM tris(2-carboxyethyl)
phosphine (TCEP). The soluble fraction was loaded onto Ni-NTA resin in the
same buffercontaining0.05% DDM. Proteinwas elutedusingthesame buffer
supplemented with 500 mM imidazole, and the protein sample was further
purified by gel filtration chromatography (Superdex 200).
Monobody Generation. Phage-display selection, phage ELISA, and monobody
preparation were conducted as described previously (46, 47). Briefly, a
monobody phage-display library in which three loop regions were di-
versified was sorted by using His-tagged NorM-NG bound to BTtrisNTA.
NorM-NG-binding monobodies were identified using phage ELISA. The gene
encoding the monobody was cloned into the pHFT2 expression vector. The
monobody with an N-terminal decahistidine-tag was then expressed in BL21
(DE3) cells and purified by using Ni-NTA affinity chromatography.
Protein Crystallization. NorM-NG and monobody were mixed at a 1:1 molar
ratioanddialyzedagainst 10%(vol/vol)glycerol,0.05%DDM, and1mMTCEP.
Removal of salt including NaCl was essential for obtaining crystals. Crystalli-
zation experiments were performed using the hanging-drop vapor diffusion
method at 22 °C. For cocrystallization with substrates, NorM-NG and mono-
body were incubated with various compounds at 0.4 mM on ice for 24 h. The
protein samples (2 mg/mL) were then mixed with equal volume of a crystal-
lization solution containing 20–30% PEG400, 10% glycerol, 0.05% DDM, and
1 mM TCEP. For derivatization, protein crystals were incubated with 6 mM
heavy metal compounds or 50 mM CsCl for 4 h at 22 °C.
Structure Determination. X-raydiffractiondatawereprocessedusingHKL2000
(48) and further analyzed using the CCP4 package (49) unless specified oth-
erwise. All structures were solved using a combination of molecular re-
placement and MIRAS phasing. Initially, a structural model of NorM-VC (PDB
ID code 3MKT) was placed into the unit cell for the apo crystal form using the
program PHASER (50). Heavy metal binding sites were identified by differ-
ence Fourier analysis, and MIRAS phases were calculated using the program
SHARP (51). Table S1 represents the optimal subset of derivative data for
MIRAS phasing. The resulting electron density maps were further improved
by solvent flattening, histogram matching, cross-crystal averaging, and phase
extension. Both theelectron density for somearomatic amino acid side chains
and heavy metal binding sites were used as markers to aid protein sequence
assignment (Table S6). Regions of the published structures of NorM-VC and
monobody were useful as a guide for model building, which was carried out
using the program O (52). Structure refinement was conducted using the
program REFMAC with experimental phases as restraints (53), except for the
Cs+-bound crystal form. All structure figures were prepared using the pro-
gram PyMol (www.pymol.org).
Drug Resistance Assay. Mutations were introduced into the norM-NG gene in
the pET-15b vector using the QuikChange method and were confirmed by
DNA sequencing. E. coli BL21 (DE3) ΔacrABΔmacABΔyojHI cells (54) were
transformed with pET-15b vector containing the inserted genes encoding the
NorM-NG variants. The expression of NorM-NG was not significantly affected
by mutations discussed in the text except for mutations F265A, S288A, and
Y294A, which completely abrogated NorM-NG expression, as judged by
Western blot using an antibody against the His-tag. Drug susceptibility
experiments were conducted based on established protocols (55). Briefly, the
exponential-phase bacterial culture from freshly transformed cells was di-
luted to ∼5 × 105CFUs/mL with LB broth containing IPTG (0.1 mM) and
ampicilin (100 μg/mL) at each drug concentration. The culture was incubated
at 30 °C with shaking, and bacterial growth was monitored after 10 h. We
defined the minimal inhibitory concentration as the lowest concentration
of antimicrobial compounds that prevents growth of E. coli under our
Lu et al.PNAS
| 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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| www.pnas.org/cgi/doi/10.1073/pnas.1219901110Lu et al.