Chapter 1

Structures and Transport Mechanisms of RND

Efflux Pumps

Satoshi Murakami

Abstract Multidrug efflux transporters of the resistance-nodulation-cell divi-

sion (RND) superfamily are one of the main causes of multidrug resistance
(MDR) in Gram-negative bacteria. These proton motive force-dependent trans-
porters generally occur as tripartite efflux complexes that span the cytoplasmic
and outer membranes, thus providing a mechanism to directly extrude antimi-
crobial substrates out of the cell. RND transporters can accommodate an incred-
ibly wide range of antimicrobial agents and play an important role in clinically
relevant antimicrobial resistance. The first crystal structure of a multidrug efflux
transporter was solved in 2002 for the AcrB RND transporter of Escherichia
coli. Subsequently over the last decade, several structures of RND transporters
have been successfully crystallized both in the presence and absence of their
antimicrobial substrates, providing a fundamental understanding of the struc-
tural basis for functional transport mechanisms. This chapter describes the cur-
rent knowledge of the structural and functional characteristics of RND efflux
pumps based on these crystal structures.

Keywords Multidrug resistance • Transporters • Structure • Transport mechanism

• RND efflux pumps • Escherichia coli • Pseudomonas aeruginosa • AcrB • MexB

S. Murakami
Division of Structure and Function of Biomolecules, Department of Life Science, Graduate
School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
e-mail: murakami@bio.titech.ac.jp

© Springer International Publishing Switzerland 2016 3

X.-Z. Li et al. (eds.), Efflux-Mediated Antimicrobial Resistance in Bacteria,
DOI 10.1007/978-3-319-39658-3_1
4 S. Murakami

1.1 Introduction

Emerging and reemerging infectious diseases are still one of the major global health
problems. Meanwhile, antimicrobial resistance of microorganisms and pathogens
has rapidly evolved, posing a challenge for chemotherapy of these diseases.
Nowadays, antimicrobial-resistant pathogens are widely seen in hospital- and
community-acquired infections, including life-threatening diseases. Many resistant
microbes possess simultaneous resistance to multiple structurally unrelated antimi-
crobial agents, a phenotype which is referred as multidrug resistance (MDR).
Several frequently reported Gram-negative pathogens, such as Acinetobacter bau-
mannii, Klebsiella pneumoniae, Neisseria gonorrhoeae, and Pseudomonas aerugi-
nosa, are prevalent with MDR phenotypes and greatly contribute to the major
threats from antimicrobial resistance, thus rendering many antimicrobial drugs inef-
fective [1].
A common structural feature of Gram-negative bacteria is their cell envelope
consisting of two cell membranes – the cytoplasmic (inner) membrane and outer
membrane [2]. It is well known that Gram-negative bacteria are much less suscep-
tible to many antimicrobial agents that are active against Gram-positive bacteria.
This phenomenon is attributable to both the outer membrane permeability barrier
[3–5] and the presence of drug efflux systems [6–8]. The latter can actively export
a wide range of antimicrobial agents (such as conventional antibiotics, antiseptics,
and many cytotoxic chemicals), thus reducing the intracellular concentration of
these agents. In bacteria, drug efflux transporters generally belong to one of five
transporter superfamilies: (i) ATP-binding cassette (ABC) superfamily; (ii) The
major facilitator superfamily (MFS) [9]; (iii) The drug/metabolite transporter
(DMT) superfamily (which contains the small multidrug resistance [SMR] family)
[10]; (iv) The multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter
superfamily (which includes the multidrug and toxic compound extrusion [MATE]
family) [11]; (v) The resistance-nodulation-cell division (RND) superfamily [12].
Recent studies have described novel drug transporter families including antimetabo-
lite transporters (the AbgT family) [13] and the proteobacterial antimicrobial com-
pound efflux transporters (the PACE family) [14].
Of the known multidrug transporters, those within the RND superfamily are
typically described as tripartite molecular complexes that span both cell membranes
[15]. RND transporters are further divided into seven families that include the
hydrophobic/amphiphilic efflux (HAE) family, the heavy metal efflux (HME) fam-
ily, and the SecDF protein-secretion accessory protein (SecDF) family [12] that are
to be discussed in this chapter. Transporters of the HAE subfamily, such as AcrB of
Escherichia coli, are featured by the broadest substrate profiles. They accommodate
drug molecules that not only have cytosolic targets (such as macrolides, quinolones,
and tetracyclines) but also target the outer leaflet of the cytoplasmic membrane (i.e.,
the periplasmic side) (and include agents such as β-lactams). RND pumps export
these agents out of the cell across the outer membrane [15]. Thus, they are thought
to be the major efflux transporters conferring drug resistance. In fact, RND pumps
1 Structures and Transport Mechanisms of RND Efflux Pumps 5

are recognized as one of the most effective systems for host defense since detrimen-
tal substances are actively extruded out of the cell. Genes encoding RND transport-
ers are found in all Gram-negative bacteria [12]. It is important to note that certain
Gram-positive bacteria (such as mycobacteria, Bacillus subtilis, and Staphylococcus
aureus) also possess RND pumps [16–19].
AcrB from E. coli and MexB of P. aeruginosa were the earliest reported RND
pumps. In E. coli, the acr locus influencing drug susceptibility was first discovered
by Nakamura et al. in the 1960s and 1970s [20, 21], although its role for drug efflux
remained unknown until in 1993 when Ma et al. [22] reported the molecular
characterization of the acrAE (i.e., acrAB) genes and their involvement in drug
efflux. For P. aeruginosa, Lei et al. [23] showed in 1991 the elevated ofloxacin
extrusion by in vitro-selected ofloxacin-resistant mutants. Multidrug efflux medi-
ated by the mexAB-oprM (initially named mexAB-oprK) operon of P. aeruginosa
was reported in 1993 by Poole et al. [24], which was substantiated by studies from
Li et al. [25–27] that demonstrated the predominant role of drug efflux pumps in the
intrinsic resistance (including resistance to β-lactams) of P. aeruginosa. However,
despite various additional molecular studies revealing the clinical importance of
efflux pumps in Gram-negative bacteria in the 1990s [28–30], structural informa-
tion for these transporters remained unknown until 2002, when the crystal structure
of AcrB was reported by Murakami et al. [31]. Subsequently, various structural
analyses, protein engineering, and molecular dynamics (MD) simulation studies of
RND pumps have been conducted based on these crystal structures. Indeed, RND
transporters are one of the most studied transporter superfamilies, with much in-
depth knowledge generated for the overall structural and functional basis of trans-
port mechanisms. In this chapter, crystal structures of RND transporters and their
functional relevance to transport mechanisms are discussed.

1.2 Crystal Structures of the RND Multidrug Efflux

Transporters

The first crystal structure of an RND transporter was described for the AcrB pump
of the E. coli AcrAB-TolC efflux system in 2002 [31]. Not only is this the first crys-
tal structure of a multidrug efflux transporter but is also the first structure of second-
ary active transporters driven by the proton motive force across the membrane.
Following this accomplishment, various groups have determined crystal structures
for several transporters of all five major families including ABC-type Sav1866 [32],
MFS-type EmrD [33], SMR-type EmrE [34], and MATE-type NorM [35] (Fig. 1.1).
As for the RND superfamily, in addition to crystallized AcrB (both with and without
drug substrates) [31, 36], multidrug efflux transporters MexB of P. aeruginosa [36,
37] and MtrD of N. gonorrhoeae [38] and two heavy metal ion efflux transporters,
CusA of E. coli [39] and ZneA from Cupriavidus metallidurans [40], were reported
in the last decade (Fig. 1.2). SecDF, with a pseudosymmetric, 12-helix
6 S. Murakami

Outer membrane

Periplasm

ABC MFS SMR MATE

H+ H+ H+ Na+

Inner membrane

RND Cytoplasm

ATP ADP

Fig. 1.1 The first reported crystal structures of five major drug efflux transporter superfamilies/
families. From left to right, Sav1866 from S. aureus (ABC), EmrD (MFS), AcrB (RND; together
with the periplasmic membrane fusion protein AcrA and the outer membrane channel protein
TolC), EmrE (SMR) from E. coli, and NorM of Vibrio cholerae (MATE) are shown

AcrB MexB MtrD CusA ZneA SecDF

Fig. 1.2 Crystal structures of six RND transporters. AcrB of E. coli (PDB code 1IWG), MexB of
P. aeruginosa (2V50), MrtD of N. gonorrhoeae (4MT1), CusA of E. coli (3KO7), ZneA of
Cupriavidus metallidurans (4K0J), and SecDF from Thermus thermophilus (3AQP) are shown in
the ribbon representation

transmembrane domain belonging to the RND superfamily (Fig. 1.2) [41], mediates
protein translocation across the cytoplasmic membrane when in complex with
SecYGE as a protein secretion system. A role of SecDF in antimicrobial resistance
was demonstrated in S. aureus [19]. As expected, the crystal structures of these
pumps are not the same for the pumps from different bacterial species with varied
substrate specificities. However, they do share very high structural similarity
between each other. In this section, fundamental structural features of RND trans-
porters are first introduced for AcrB, the prototypical representative of the RND
superfamily, and then described for several other RND transporters.
1 Structures and Transport Mechanisms of RND Efflux Pumps 7

1.2.1 AcrB from E. coli

The crystal structure of AcrB (Protein Data Bank [PDB] codes 1IWG and 2DHH)
was solved at 3.5 Å resolution (Fig. 1.3) [31]. In this analysis, AcrB monomers
comprise tight trimeric conformation with domain swapping by mutual insertion of
their long hairpin loop in the periplasmic domain (Figs. 1.3 and 1.4). This trimeric
conformation has threefold symmetry owing the threefold crystallographic sym-
metry of the crystal used in this crystallographic analysis. Thus, the structure of

TolC Docking
Domains
(~30Å)

Porter
(Pore)
Domains
(~40Å)

Transmambrane
Domains
(~50Å)

Fig. 1.3 Crystal structure of AcrB. (a) Side view, parallel to the membrane plane. Three pro-
tomers are individually colored (blue, green, and red). The N-terminal and C-terminal halves of the
protomers are depicted as dark and pale colors, respectively. The periplasmic region (TolC-docking
domains and pore domains) is at the top, and the transmembrane region is at the bottom. (b) Top
view. The protomers are individually colored as in A. (c) Structure within a slab (23 Å) of the
transmembrane domain parallel to the membrane plane near the periplasmic surface. The pro-
tomers are individually colored as in A and B. Threefold and pseudo-twofold rotation axes are
indicated. The label numbers indicate the transmembrane helix numbers (TMx in c)
8 S. Murakami

Fig. 1.3 (continued)

1 Structures and Transport Mechanisms of RND Efflux Pumps 9

Fig. 1.3 (continued)

10 S. Murakami

Fig. 1.4 Topology diagram of AcrB monomer. Secondary structure elements are indicated: TM,
transmembrane helices; Nα and Nβ, helices and strands, respectively, in the N-terminal half of the
headpiece; Cα and Cβ, helices and strands, respectively, in the C-terminal half of the headpiece; lα,
a helix attached to the cytoplasmic surface of the membrane. N- and C-terminal halves are depicted
in dark and pale blue, respectively. The porter domain is divided into four subdomains; PN1, PN2,
PC1, and PC2. The TolC-docking domain is divided into two subdomains, DN and DC. The hair-
pin structures for mutual insertion between TolC-docking domains of neighboring protomers are
depicted in green and pink

each protomer included in this trimer is averaged and identical by the crystallo-
graphic symmetry. In each protomer, there is a pseudo-twofold symmetry (Fig. 1.4).
It is not only found at structural level but also at the primary sequence level.
Moreover, it has been hypothesized that gene duplication and fusion events evolu-
tionally drove this pseudo-twofold property of RND transporters. A similar pseudo-
twofold symmetry can also be found in other transporter families, like the MFS,
SMR, and ABC families [32–34].
In 2006, two groups [42, 43], and in 2007, one group [44] reported new crystal
structures of the same AcrB from E. coli. These independent studies used different
crystal forms belonging to differing space groups. All crystals used by each group
did not have threefold crystallographic symmetry in their crystallographic analysis.
Thus, these groups solved essentially consistent asymmetric structures of AcrB. At
the same time, the Murakami group also solved the AcrB structure with bound sub-
strates (doxorubicin and minocycline) with a lower symmetry conformation and
showed that only one substrate molecule is bound asymmetrically to one of the
protomers of the AcrB trimer (Fig. 1.5). This protomer has a distinct conformation
suitable to accommodate substrates inside the molecule. All three groups concluded
1 Structures and Transport Mechanisms of RND Efflux Pumps 11

Fig. 1.5 Structure of the AcrB-minocycline complex in the asymmetric conformation. Three pro-
tomers are individually colored as same as those in Fig. 1.2. The minocycline molecule is shown
in CPK (Corey-Pauling-Koltun) representation, with the position of the C, N, and O atoms indi-
cated by yellow, blue, and red balls, respectively. (a) Side view, parallel to the membrane plane. (b)
Top view. (c) Cut view of the porter domain from the outside of the cell. Phenylalanine residues,
including those in the distal binding pocket, are shown in the ball-and-stick representation. The
difference Fourier map of bound minocycline is depicted by an orange-colored cage. Arrows indi-
cate the relative movements of the subdomains in comparison with those in the former state (The
figure is from Murakami et al. [42])
12 S. Murakami

Fig. 1.5 (continued)

that this asymmetric conformation is likely the physiological relevant form and pro-
posed either a functionally rotating mechanism or a peristaltic pump mechanism
(Fig. 1.6) [42, 43]. This idea is conceived from the analogy of the rotational cataly-
sis proposed and proved for F1F0-ATPase [45].
In asymmetric crystal structures of the AcrB trimer, there are three distinct con-
formations: “access” (“loose” as in another explanation by Seeger et al. [43]),
“binding” (“tight”), and “extrusion” (“open”) conformations [42] (Fig. 1.6). These
conformations are considered to be the three major functional states during the
substrate translocation cycle. Even though AcrB is a homo-trimeric complex, each
protomer included within this trimer exhibits asymmetrically different
conformations.
Each protomer is divided into three layers: the cytoplasmic domain (~10 Å), the
transmembrane domain (~40 Å), and the periplasmic domain (~70 Å) (which
protrudes into the periplasmic space) (Fig. 1.3). Within the cytoplasmic domain,
there is an α-helix almost parallel to the membrane plane, attaching inner leaflet of
the cytoplasmic membrane from the cytoplasmic side (Figs. 1.3 and 1.4). This helix
is regarded to play an important role as a stabilizer float of AcrB. Similar parallel
1 Structures and Transport Mechanisms of RND Efflux Pumps 13

Fig. 1.6 Schematic illustration of the functionally rotating mechanism with the peristaltic pump-
like motion in the substrate translocation pathway mediated by AcrB. The top view from the out-
side of the cell (upper). The view from the side parallel to the membrane plane (middle). Schematic
drawing of the substrate translocation pathway in the protomer colored in blue, and its peristaltic
movement during the substrate extrusion cycle (“left” → “center” → “right” corresponds
“access”→ “binding”→ “extrusion”). Substrate is transferred from the vestibule through the proxi-
mal and distal pockets to the exit toward the TolC outer membrane channel. Arrows indicate in the
bottom figures the relative peristaltic movement of the translocation pathway during the substrate
extrusion cycle. Dotted arrows refer to the substrate movement. The indicated “switch loop” sepa-
rates the proximal and distal binding pockets. Orange hexagon represents substrate molecule

helix can be observed not only in other transporters but also in many other mem-
brane proteins [46].
The membrane-embedded region consists of 12 α-helix bundles from each pro-
tomer. The bundles of each protomer are inserted circularly into the membrane
(Fig. 1.3), and these three bundles create a central spatial hole that is approximately
14 S. Murakami

Fig. 1.7 Switch-like conformational change by proton conduction via important titratable resi-
dues, D407, D408, and K940, in the TM4 and TM10. A cut view of the transmembrane region
viewed from the periplasm. Dotted lines show interaction between atoms (The figure is from
Eicher et al. [50] with slight modifications)

40 Å in diameter. This hole, surrounded by the helix bundles, is thought to be filled

with phospholipid molecules. The transmembrane helices TM4 and TM10 form a
central pair in this helix bundle (Fig. 1.3). In these helices, there are functionally
important charged residues, two acidic residues (Asp407 and Asp408), and one
basic residue (Lys970). These titratable residues produce charge pairs (Fig. 1.7).
The protonation and deprotonation events occurring in these residues trigger molec-
ular motion; this motion is transmitted to the periplasmic domain by remote confor-
mational changes for substrate translocation, which mainly occurs in the periplasmic
region [47–50]. Even though the greatest resolution of the AcrB crystal structure is
at 1.9 Å, protons are far beyond the observation by means of X-ray crystallography.
Thus, the protonation state of the aspartates including individual states has not been
elucidated so far, but by the MD calculation based on the crystal structure, we can
hypothesize a reasonable structure for these residues [49].
It is still unclear whether the substrate translocation pathway across the cytoplas-
mic membrane exists in the transmembrane region. Only in the case of CusA (see
the subsection on CusA below) was substrate (Cu+) binding observed in this domain
[39]. However, there is no other indication of substrate translocation through this
transmembrane domain for the other transporters, especially drug exporters. Hence,
the transmembrane domain is thought to be a power generator for these types of
transporters by exchanging proton conduction into structural changes in this domain.
Intriguingly, it was found that SecD, the transmembrane subunit of the SecDF
complex, shares structural similarity to the transmembrane region of AcrB [41].
This complex likely stimulates protein translocation through the Sec translocon by
proton motive force. The overall configuration of SecD and the transmembrane
region of RND transporters are similar, yielding a root-mean-square deviation of
around 2.7 Å for the Cα atoms of this region, in spite of their dissimilarity in their
primary sequences (~15 %) [41]. SecD also has equivalent charged residues in its
middle transmembrane region. Although SecD is very different in function, it shares
this structural module to capture the proton motive force and convert this energy
into movement of the protein molecule (Fig. 1.2).
1 Structures and Transport Mechanisms of RND Efflux Pumps 15

In comparison with the transmembrane regions, the periplasmic regions produce

a tighter interaction between the protomers and thus affect each other. These regions
can be divided in two layers as the lower porter domain and the upper TolC-docking
domain. Furthermore, the porter domain can be split into four subdomains (two in
N-terminus and two in C-terminus): PN1, PN2, PC1, and PC2 (Fig. 1.4). In the
three individual protomers, these four subdomains have different configurations.
For instance, the subdomain interactions are looser in the “binding” state to create
greater space between the subdomains; the resulting protomer forms a voluminous
pocket in the porter region to accommodate substrate binding. On the other hand,
the interactions between the subdomains are tighter in the “extrusion” state. In this
case, the pocket is shrunken without enough space to accommodate potential sub-
strates. These two different pocket states (i.e., the expanded and shrunken binding
pockets) are to control the binding affinity of substrates to the binding pocket.
Subdomains in the “access” state also have different conformations; the pocket con-
figuration is not as clearly open as the “binding” state.
The TolC-docking domain, which lies in the upper periplasmic region, includes
two subdomains dubbed DN and DC. Two subdomains from each of the three pro-
tomers (thus a total of six subdomains in this region) form the rim of a funnel-like
conformation. This structure can fit the connecting AcrA subunit in the tripartite
efflux complex. At the bottom of the funnel, three exits of the substrate transloca-
tion pathways from the porter domain of each protomer are connected together to
this funnel-like opening that is linked to TolC directly or indirectly. Another remark-
able feature of this region is its domain swapping characteristic by mutual insertion
of their long hairpin loop in the periplasmic domain. This long hairpin structure
from the DN subdomain inserts into the DC subdomain of the neighboring pro-
tomer, thus forming a four-stranded β-sheet. For the pseudo-twofold symmetry of
the molecule, the DC subdomain also has a hairpin, but is shorter. This hairpin is
inserted into the DN subdomain of the same protomer, yielding another four-
stranded β-sheet as in the case of the DN subdomain. Domain swapping within each
protomer and between neighboring protomers tightens the trimeric structure.
Although molecular movement in this region is rarely observed, however, structural
changes in this region may occur upon moving of whole tripartite complex. The
outer membrane protein, TolC, is also thought to have open/close mechanism in its
helix bundle that protrudes into the periplasm. Therefore, TolC-docking domain
may also change its conformation cooperatively with the membrane fusion protein
(periplasmic adaptor proteins) (i.e., AcrA) and TolC.

1.2.2 MexB from P. aeruginosa

The crystal structure of the MexB pump of the MexAB-OprM efflux system from
P. aeruginosa (PDB code 2V50) was solved at 3.0 Å resolution (Fig. 1.2) [37]. MexB
is a close homolog of AcrB, as their overall structures are basically similar, with a
comparably high primary sequence similarity (70 % identity and 92 % similarity).
16 S. Murakami

However, some of the secondary structural elements in the loop regions between the
transmembrane helices are different. The major difference is found around the ves-
tibule, the location of drug entrance in the “access” state. In AcrB, the vestibule is
widely open and thus accessible to the substrates at this opening. In comparison, the
MexB vestibule in its “access” state is rather closed [37]. Flexibility in the loop
structure around the vestibule is highlighted with MexB crystal structure.

1.2.3 MtrD from N. gonorrhoeae

The MtrD crystal structure of the MtrCDE efflux system from human pathogen
N. gonorrhoeae (PDB code 4MT1) was solved at 3.53 Å resolution (Fig. 1.2) [38].
In this structural analysis by Bolla et al. [38], crystals having threefold crystallo-
graphic symmetry were also used as same as the analysis for AcrB in 2002 [31], and
thus resulting structure also has threefold crystallographic symmetry. Interestingly,
only one protomer of the MtrD structure is included in the asymmetric unit. Similar
to the symmetric structure of AcrB [31], MtrD shares a similar “access” conforma-
tion shown in the asymmetric structure of AcrB. Bolla et al. [38] successfully solved
MtrD structure by using the “access” protomer in the asymmetric AcrB (PDB code
2DHH) as the search model to solve the phase problem of the crystallographic anal-
ysis by the molecular replacement method. Yet, in the case of MtrD, the superposi-
tion of these two structures shows a significant difference with a high root-mean-square
deviation around 7 Å, suggesting that the structures of AcrB and MtrD are quite
distinct. An interesting structural feature of MtrD has been observed in its trans-
membrane region. Its TM9 extends into the periplasm and contributes to the forma-
tion of the periplasmic domain. This elongation reaches around the PC1 and PC2
subdomains, the vestibule for the transporter incorporation. The elongated trans-
membrane helix is proposed to assist substrate incorporation from the outer leaflet of
the membrane to the substrate translocation pathway in the periplasmic domain [38].

1.2.4 CusA from E. coli

The crystal structure of CusA of the CusCBA efflux system from E. coli (PDB code
3KO7) was solved at 3.52 Å resolution (Fig. 1.2) [39]. CusA is not a multidrug
efflux transporter, but instead belongs to the HME subfamily of the RND transport-
ers. It can export copper (I) and silver (I) and confer resistance to these toxic heavy
metal ions [51]. CusA cooperates with the membrane fusion protein CusB and the
outer membrane protein CusC, producing a tripartite complex similar to the AcrAB-
TolC system. Interestingly, the complex crystal structure of CusBA was also solved
by the same group that solved the CusA structure. This structure is the only available
example for the crystallographic analysis of a complex structure of the components
including the tripartite transporter complexes [52]. Overall, the configuration of
1 Structures and Transport Mechanisms of RND Efflux Pumps 17

CusA is similar to that of other multidrug transporters such as AcrB and

MexB. Nevertheless, in CusA, the substrates are coordinated by three methionine
residues in the periplasmic and transmembrane regions [39]. In contrast to multidrug
transporters, the substrates of the HME subfamily are ions and not hydrophobic
compounds. Hence, the substrate binding sites of HME transporters are significantly
different from those of the multidrug efflux transporters. Substrate (metal) binding
sites of the HME family are composed of ion coordination residues (e.g., methionine
residues) and not the hydrophobic residues in the multidrug exporters. Secondary
structure elements can also differ from multidrug efflux transporters. Notably, the
substrate translocation pathway in the transmembrane region was also observed for
CusA, but was not obvious for multidrug exporters. However, the titratable residues
in the TM4 and TM10 and the proton translocation pathway with these residues
seem to have a similarity between HME transporters and multidrug exporters.
The CusBA complex structure was solved at 2.9 Å resolution [52]. This structure
is the only example of a complex crystal structure available for a tripartite RND
transporter. Although the correct stoichiometry of tripartite complex components
remains under debates, in the case of CusBA, two CusB molecules attach to the
periplasmic region of one CusA molecule, and a hexameric CusB seems to form a
continuous channel on the top of CusA. This channel also connects to trimeric
CusC, the outer membrane component of the CusCBA system. Long et al. [52] have
proposed two possible arrangements of CusB and CusC docking: (i) direct docking
of CusC on the top funnel of CusA with the hexameric CusB surrounding this com-
plex and (ii) indirect docking in which the CusB channel may connect to the CusC
channel.

1.2.5 ZneA from C. metallidurans

The crystal structure of ZneA from the metal-enduring bacterium, C. metallidurans

(PDB code 4K0J), was solved at 3.0 Å resolution (Fig. 1.2) [40]. ZneA also falls
into the HME family (similar to CusA). It exports zinc (II) ion and confers zinc
resistance. ZneA is also comprised as a trimer, and its overall conformation is basi-
cally similar to other RND transporters. However, the conformation and composi-
tion of the amino acid residues in the substrate binding site are fairly different from
those of CusA. In CusA, the sulfur atoms of methionine residues coordinate the
binding and exporting of monovalent metal ions like copper (I) and silver (I); on the
other hand, in the case of ZneA, carboxyl groups of aspartate and glutamate resi-
dues coordinate zinc (II) ions. The positions of the binding site remain within the
periplasmic substrate translocation pathway (similar to what is observed among
many other RND transporters), yet they are not identical to those of CusA. Crystals
free from threefold crystallographic symmetry were used for ZneA crystal structure
analysis. Structural differences between each protomer were observed in different
substrate translocation states, and a unique substrate transport mechanism on this
transporter was proposed [40]. No zinc binding in the transmembrane region of
18 S. Murakami

ZneA was observed. However, there are three titratable residues along the TM4
region that may provide a path for protons across the membrane.
Additionally, substrate sizes for the HAE and HME transporter families differ
greatly, and thus the sizes of the substrate binding pockets of these transporters are also
different. However, similar substrate translocation (through peristaltic or serial switch-
ing mechanisms) may share the same evolutionary origin in these RND families.

1.3 Drug Recognition Mechanisms

Crystal structures of the AcrB transporter in complex with substrates in the asym-
metric conformation were solved for doxorubicin, erythromycin, minocycline, and
rifampicin (Fig. 1.8) [42, 48, 53]. These substrates are observed in various positions
along the substrate translocation pathway from the entrance (or vestibule) to the exit
where there are two major substrate binding pockets – “proximal (access)” pocket
and “distal (deep)” binding pocket [48, 53]. These binding pockets are named by
their proximity to the cell membrane and the entrance. These two pockets are sepa-
rated by a characteristic “switch loop” (Figs. 1.4 and 1.6) [48]. Because of the gly-
cine residues, this loop has a flexible property and is proposed to play an important
role in drug efflux activity [48].
Binding of doxorubicin and minocycline was observed in the distal binding pocket
in the “binding” state [42, 48]. Within this pocket, there are a few of phenylalanine
residues and hydrophobic residues responsible for the substrate binding by hydro-
phobic interaction or π electron stacking interaction. Accumulation of hydrophobic
residues is commonly observed in the core region of the protein molecules, espe-
cially for globular soluble proteins or hydrophilic part of the membrane proteins
[54]. In the case of the HAE transporters, the accumulating hydrophobic residues are
relocated upon the substrate translocation cycle, which is powered by proton conduc-
tion across the membrane. Expansive shifting of the subdomains, accompanied with
aromatic and hydrophobic residues, produces a voluminous and hydrophobic bind-
ing pocket. Binding of doxorubicin, erythromycin, and rifampicin was observed in
the proximal binding pocket in the “access” state [48, 53]. This pocket is thought to
be the binding site for the substrates which have comparably larger molecular masses.
AcrB is known to export more than 30 structurally unrelated chemical com-
pounds. However, there are only a very limited number of drugs that have been
successfully co-crystallized with AcrB. As for other substrates, binding characteris-
tics are predicted by means of in silico docking simulation analysis, as co-
crystallization of these compounds have not yet succeeded [55, 56]. Studies by the
other methods, including protein engineering (including chimeric proteins) and
mutagenesis, have elucidated certain mechanisms of the substrate recognition and
identified key residues related to substrate recognition [57–62]. Naturally, these
important residues are located around the substrate translocation pathway from the
vestibule to the exit. In the case of β-lactam recognition, key residues can be found
around the proximal binding pocket, but their complex structures are not success-
fully determined [62].
1 Structures and Transport Mechanisms of RND Efflux Pumps 19

a b

c d

Fig. 1.8 Substrate binding pockets with multidrug binding and their difference Fourier maps.
Distal binding pocket with minocycline binding (a) and doxorubicin binding (b) in the “binding”
state of the AcrB trimer. Proximal binding pocket with doxorubicin dimer (c) and erythromycin
binding (d) in the “access” state of the AcrB trimer (Figures a–c are from Eicher et al. [48], and d
is from Nakashima et al. [53] with slight modifications)

1.4 Proton Translocation Pathway and Energy Coupling

Mechanism

Even before the crystal structures of RND transporters were solved, it was recog-
nized that three charged residues in the TM4 and TM10 regions are highly con-
served and aided the prediction of their functional importance. Site-directed
mutagenesis of these residues severely impairs the functionality of the drug efflux
systems [60, 63–69]. These residues are located triangularly and form ion pair in the
middle of the transmembrane helix bundle. In high-resolution structural analysis,
differences of interaction between these charged residues are clearly observed upon
20 S. Murakami

protonation/deprotonation of these residues using a switch-like mechanism

(Fig. 1.7) [48, 50].
Despite the advances in structural and biochemical studies of the RND transport-
ers, there remain inevitable obstacles that hinder our understanding of the proton
translocation pathway for RND transporters. Firstly, protons are virtually invisible
in protein X-ray crystallographic analysis. Secondly, it is impossible to control the
protonation states of each individual protomer within these crystals. Soaking of
crystals into acidic or basic buffer solution during the experimental process influ-
ences all the titratable residues in an uncontrollable manner. Thirdly, for a tripartite
transporter, it is somewhat useless to reconstitute of a solo RND transporter into
proteoliposomes for conventional biochemical analysis. All three components
including the membrane fusion protein and outer membrane protein components are
likely required to assess the full function of an RND transporter system. It is easy to
imagine that reconstitution of all three components, which includes two different
membrane proteins, can be difficult to achieve by common biochemical techniques.
Accordingly, only a few studies were successful in quantitatively analyzing RND
transporters through authentic biochemical methods [69, 70].
For this reason, MD simulation attracts much interest as a complementary
method in structural biology [56, 71–75]. This methodology incorporates
hypothetical models with protonation or deprotonation states of individual residues
for MD calculation. These models are then assessed for their accuracy following the
MD calculation. MD simulation has helped propose models ranging from proton
utility and generation of molecular movement in the transporters to transmission of
the movement to the porter domain via a remote conformational change [49, 72,
76–78].

1.5 Substrate Translocation Pathways

Among the RND transporters with known crystal structures, drug exporters like
AcrB and heavy metal ion exporters like CusA have different substrate transloca-
tion pathways. For drug exporters, the substrate translocation pathway has only
been found in the periplasmic domains (Fig. 1.6). Thus, substrates may be taken
up from the periplasm or from just above the outer leaflet of the cytoplasmic
membrane. In other words, a transmembrane translocation pathway may not
exist, even though many classes of substrates exert their action in the cytosol. In
these cases, the substrates might be transferred from the cytosol to the periplasm
(or at least the outer leaflet of the cytoplasmic membrane) by diffusion. The result
of the substrates taken by RND pumps via these pathways would shift the equi-
librium toward efflux of the substrates. Consequently, there would be no positive
substrate translocation pathway across the membrane for drug efflux transporters.
In contrast, for CusA, binding substrates are observed in the transmembrane
region. Thus, there may exist a substrate translocation pathway not only in the
porter domain like drug exporters but also in their transmembrane region.
1 Structures and Transport Mechanisms of RND Efflux Pumps 21

The first substrate translocation pathway (i.e., through the central pore helices at
the center of the trimer) was mistakenly proposed by a simplistic interpretation of
the structure solved in 2002 [31]. The first physiological pathway was proposed by
Lomovskaya and Totrov [79] through a model calculation and took into consider-
ation experimental observations made by Aires and Nikaido [80]. At the same time,
the asymmetric structure of AcrB with bound substrate (doxorubicin and minocy-
cline) was solved, and the previous model was corrected according to the crystal
structural analysis [42].
The substrate translocation pathway in the periplasmic part of AcrB starts from
the vestibule or entrance opening to the proximal part of periplasm and/or just above
the outer leaflet of the cytoplasmic membrane. These locations are some entrances
that were proposed by structural features and/or by MD calculation [53, 71, 73].
These pathways from several entrances merge into the proximal binding pocket for
substrates and continue together. A distinct loop structure divides the middle of this
pathway. Following the loop, in the latter part of this pathway, lies the distal pocket
(another substrate binding). This pocket has an exit formed by a gap between two
protomers, the N-terminal porter subdomains PN1 and PN2. Each exit connects to
the funnel-like opening in the TolC-docking domain. Simply put, substrates are
translocated between the subdomains in the periplasmic domain from the vestibule
to this exit. All along this pathway, substrates are recognized and bound elsewhere
in this long pathway. This vast extent of the pathway provides many sites that can
accommodate a wide variety of chemical compounds as substrates.

1.6 Peristaltic Pump Mechanism in Multidrug Efflux

In the asymmetric crystal structures, asymmetric configuration of the subdomains

including in the porter domain of each protomer is interpreted as consequent of their
cooperative movement. In the porter domain, molecular movement of the subdo-
mains is converted to the sequential movement of the substrate translocation path-
way. Initially, this movement is triggered by the proton conduction through the
transmembrane region and then transmitted by the remote conformational change.
The molecular movement in the porter domain includes opening and closing of the
vestibule, proximal and distal substrate binding pockets, and the exit gate (Fig. 1.6).
The opening of the entrance and the proximal half of the translocation pathway
allow the substrates to gain access through the pathway in the “access” state
(Fig. 1.6, left). Some parts of this pathway (i.e., the proximal binding pocket) are
expanded. In the subsequent “binding” state, the opening of the latter half of the
pathway makes the distal binding pocket accessible to the substrates through the
loop region that separates the two pockets (Fig. 1.6, middle). In this case, PN2 and
PC1 move away from the trimeric center to expand the distal binding pocket, result-
ing in a gap between the PN1 and PN2 porter subdomains to serve as the exit of the
pathway (Fig. 1.5c). Following this event, an α-helix belonging to the PN1 subdo-
main of the neighboring protomer closes this exit by inserting the α-helix.
22 S. Murakami

Consequently, the expanded distal binding pocket closes the exit, thus keeping the
substrates inside the pocket. The helix bundle which is comprised of the α-helix
from each protomer is the so-called pore helix, which plays a crucial role for drug
export [42]. In this case, the inserted helix is greatly inclined in comparison with the
other equivalent helices belonging to the other protomers. In the final “extrusion”
state, the PN2 and PC1 subdomains move back toward the center of the trimer
(Fig. 1.5c). PC2 moves closer to PC1 to shrink the expanded distal binding pocket
in the “binding” state and constrict the pocket and forces the bound substrates out of
the pocket (Fig. 1.6, right). During the “extrusion” state, the vestibule closes its
conformation by changing the secondary structure around that region. The proximal
half of the translocation pathway also gets shrunken. The helix is inclined toward
the expanded pocket in the “binding” protomer and contributes to shutting the exit
gate (Fig. 1.6, right). However, at the same time, the inclination widely opens the
exit gate for the “extrusion” state itself. As a result, the protomer in the “extrusion”
state has a closed entrance and opens the exit with a shrunken distal pocket.
Accordingly, it forms a suitable conformation for substrate extrusion from the distal
pocket to the exit. Next, as PC2 approaches PC1, it cycles back to the “access” state.
Drugs are exported through this three-step functionally rotating mechanism in
which substrates undergo an ordered binding change. This cyclical process is analo-
gous to the rotational catalysis of ATP synthesis by the F1F0-ATPase [45]. The cur-
rent understanding of this transport cycle is mainly derived from our molecular
comprehension of pump crystal structures [42–44]. Although intermediate steps
might exist among the “access,” “binding,” and “extrusion” states, they remain to be
elucidated.

1.7 Structure of the Tripartite Efflux Pump Complex

Crystal structures of tripartite RND transporters have only been solved through their
components separately, though the one exception, CusBA crystal structure, has
been successfully solved at 3.2 Å resolution [52]. In the latter CusBA study, six
membrane fusion proteins were demonstrated to be bound to the trimeric RND
transporter. It is also thought to connect one trimer of the outer membrane protein
to this complex, thus yielding a tripartite efflux complex comprised of three trans-
porter, six membrane fusion protein, and three outer membrane protein molecules.
Difficulties in the sample preparation for the whole tripartite complex hinder the
identification of a complete tripartite efflux structure. As a result, there have been no
solved crystal structures to date. In 2014, the Luisi group [81] successfully prepared
a stable tripartite efflux complex by employing a protein fusion technique. This
group was able to connect the transporter, the membrane fusion protein, and the
outer membrane protein with peptide linkers. A potent tripartite transporter com-
plex was obtained in vivo and was then used for structural determination in sub-
atomic resolution under cryo-electron microscopy. The observed structure was
noted to be essentially the same as the computer-generated model. However, more
1 Structures and Transport Mechanisms of RND Efflux Pumps 23

detailed information was obtained from this study. In particular, the distance
between the RND transporter (i.e., AcrB) and the outer membrane protein (i.e.,
TolC) was experimentally determined from these methods, revealing that there is
less potential for direct interactions between the RND transporter and the outer
membrane protein component (Fig. 1.1) [82]. A recent study using the reconstitu-
tion of native AcrAB-TolC and MexAB-OprM systems in a lipid nanodisc system
also showed no physical interaction between the cytoplasmic and outer membrane
efflux components [83]. To note, an earlier report had potentiated a direct interac-
tion between AcrB and TolC by means of a cross-linking study [84]. The interaction
between RND proteins and outer membrane protein components remains to be fur-
ther investigated; however, the distance between the outer and cytoplasmic mem-
branes is expected to be flexible. Thus, both direct and indirect interactions may be
possible for the tripartite RND efflux complex [52].

1.8 Concluding Remarks

RND efflux transporters are one of the most studied among the major transporter
families. Based on identified crystal structures (especially asymmetric conforma-
tions with bound substrates), plausible functional mechanisms are proposed and
proved by many molecular biological analyses [15, 47, 70, 85–87] and MD simula-
tions [55, 56, 72, 77]. High-resolution X-ray crystallographic analysis is a reliable
technique to elucidate protein structure, as diffraction data is a direct output of
structural averaging between the billions of molecules included in the crystals. At
the same time, however, it is still important to note that the dynamic aspects of these
molecules within crystals may also be averaged; resulting crystal structures are
merely static snapshots of the protein molecules. Additionally, the factor of tem-
perature for the peptide chains within protein molecules may contain information
regarding structural flexibility, but dynamic aspects of the molecules are mostly
lost. In order to observe live images of protein molecules, other methodologies like
MD simulation can produce supplemental information for their dynamic aspect.
MD calculation for macromolecules is an established methodology that has already
been applied in structural biology [88, 89]. To note, precise MD simulation first
requires a definite high-resolution crystal structure, and therefore, crystallographic
analysis and MD calculation are mutually complementary. Moreover, to continue
MD calculation in an appropriate period of time in each biological system, espe-
cially for large molecules like RND transporters and their tripartite complexes,
supercomputer-assisted techniques with reliable high-resolution starting model and
reasonable force field for the MD calculation will be critical for elucidating the
structural and functional basis of the drug efflux systems. Overall, additional struc-
tures (especially those with great complexity), more quantitative biochemical analy-
ses (especially involving kinetics), and more precise long-term MD simulations are
required for an in-depth understanding of RND drug efflux complexes to facilitate
our efforts in tackling the efflux-mediated multidrug resistance problem.
24 S. Murakami

Acknowledgments Studies in the Murakami laboratory are supported by Grant-in-Aid for

Targeted Proteins Research Program and Platform for Drug Discovery, Informatics, and
Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology
of Japan, the funding program for the Next Generation World-Leading Researchers (NEXT
Program) from the Japan Society for the Promotion of Science, ERATO “Lipid Active Structure
Project” from the Japan Science and Technology Agency, and the Advanced Research for
Medical Products Mining Program of the National Institute of Biomedical Innovation, Japan. We
are also grateful for the funding by the International Human Frontier Science Program
Organization.

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