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Stoichiometry and mechanistic implications of the MacAB-TolC tripartite efflux pump
Accepted Manuscript Stoichiometry and mechanistic implications of the MacAB-TolC tripartite efflux pump Inseong Jo, Seokho Hong, Minho Lee, Saemee Song, Jin-Sik Kim, Alok Mitra, Jaekyung Hyun, Kangseok Lee, Nam-Chul Ha PII:
S0006-291X(17)32080-6
DOI:
10.1016/j.bbrc.2017.10.102
Reference:
YBBRC 38719
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 12 October 2017 Accepted Date: 19 October 2017
Please cite this article as: I. Jo, S. Hong, M. Lee, S. Song, J.-S. Kim, A. Mitra, J. Hyun, K. Lee, N.-C. Ha, Stoichiometry and mechanistic implications of the MacAB-TolC tripartite efflux pump, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.10.102. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Stoichiometry and mechanistic implications of the MacAB-TolC tripartite efflux pump Inseong Jo1†, Seokho Hong1†, Minho Lee2, Saemee Song1, Jin-Sik Kim1, Alok Mitra4,
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Jaekyung Hyun3, Kangseok Lee2*, and Nam-Chul Ha1* Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, and
Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826,
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Republic of Korea
Department of Life Science, Chung-Ang University, Seoul 06974, Republic of Korea
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Electron Microscopy Research Center, Korea Basic Science Institute, Chungcheongbukdo
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2
28119, Republic of Korea 4
Department of Biological Sciences, the University of Auckland, Auckland 1010, New
†
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Zealand
These authors equally contributed to this work.
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* To whom correspondence should be addressed: Nam-Chul Ha (e-mail: [email protected], Tel.: +82-2-880-4853, Fax: +82-2-873-5095) and Kangseok Lee (email: [email protected],
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Tel.: +82-2-820-5241, Fax: +82-2-825-5206)
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Abstract The MacAB-TolC tripartite efflux pump is involved in resistance to macrolide antibiotics and secretion of protein toxins in many Gram-negative bacteria. The pump spans the entire cell
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envelope and operates by expelling substances to extracellular space. X-ray crystal and electron microscopic structures have revealed the funnel-like MacA hexamer in the periplasmic space and the cylindrical TolC trimer. Nonetheless, the inner membrane
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transporter MacB still remains ambiguous in terms of its oligomeric state in the functional complex. In this study, we purified a stable binary complex using a fusion protein of MacA
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and MacB of Escherichia coli, and then supplemented MacA to meet the correct stoichiometry between the two proteins. The result demonstrated that MacB is a homodimer in the complex, which is consistent with results from the recent complex structure using cryoelectron microscopy single particle analysis. Structural comparison with the previously
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reported MacB periplasmic domain structure suggests a molecular mechanism for regulation of the activity of MacB via an interaction between the MacB periplasmic domain and MacA.
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Keywords
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Our results provide a better understanding of the tripartite pumps at the molecular level.
Escherichia coli; Actinobacillus actinomycetemcomitans; tripartite efflux pump; MacABTolC; electron microscopy
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Introduction Many Gram-negative bacteria express the MacAB-TolC tripartite efflux pump, which consists of the inner membrane transporter MacB, the membrane fusion protein MacA, and
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the outer membrane channel protein TolC [1]. This pump is involved in efflux of macrolide antibiotics and in secretion or transport of the heat-stable enterotoxin II, outer membrane glycolipids, protoporphyrin, and lipopeptides [1-5]. The active processes of efflux and
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transport are carried out by MacB, which contains an ATP-binding cassette (ABC) that uses ATP hydrolysis as a driving force [6].
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MacA is anchored in the outer leaflet of the inner membrane via the N-terminal noncleavable signal peptide [7-9]. MacA is also called the periplasmic adaptor protein since it connects the inner membrane transporter to the outer membrane channel protein in the periplasm.
Crystal
structures
of
MacA
from
E.
coli
and
Actinobacillus
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actinomycetemcomitans (Aa) revealed a linear arrangement of the membrane proximal (MP) domain, beta-barrel domain, lipoyl domain, and α-hairpin domain [7, 10]. The crystal structure of Aa MacA further revealed a funnel-like hexameric assembly [7]. The funnel stem
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portion of the MacA hexamer forms an α-barrel structure with six cogwheels at the end of the α-barrel. This characteristic cogwheel structure was implicated as a binding and channel
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opening motif for the similar α-barrel of the TolC trimer, by intermeshing the two α-barrels. The flared cone structure, or funnel mouth, in the MacA hexamer was proposed to accommodate the periplasmic domain of MacB [10]. A membrane topology study suggested that MacB contains an N-terminal nucleotide
binding domain (NBD), transmembrane domain (TM) 1, a large periplasmic domain, TM 2, cytoplasmic region, TM 3, a small periplasmic region, TM 4, and a C-terminal cytoplasmic tail [11]. The crystal structure of the large periplasmic domain of Aa MacB revealed a 3
ACCEPTED MANUSCRIPT relatively large domain with two compartments [12]. The domain arrangement and the number of TM regions of MacB are different from typical B-family ABC transporters, which operate as a homodimer with 6 α-helices per monomer, a small periplasmic domain, and a C-
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terminal NBD [13, 14]. Therefore, the oligomeric state of MacB in the functional complex was unclear.
Due to the low affinity between the components in the complex, many researchers
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have stabilized the complex by fusing the components or introducing an artificial disulfide bond between the proteins for structural studies [8, 10, 15-18]. The MacAB-TolC complex
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structure was recently determined by cryo-electronmicroscopy (cryo-EM) using a MacBMacA fusion protein [8]. The MacB homodimer in the complex showed a structure consistent with previous results from the topology study. Interestingly, although the protein sample has a stoichiometry of 6:6:3 for the complex of MacA:MacB:TolC, the cryo-EM structure had a
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stoichiometry of 6:2:3. Only two of six MacB molecules were visible in the cryo-EM structure. Since the other four MacB molecules were not fixed in the complex, they diffused and disappeared during the particle processing.
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In this study, we removed the redundant MacB molecules in the complex by supplementing with MacA, and we suggest a plausible mechanism for how the activity of the
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efflux pump is activated in the functional complex.
Materials and Methods Construction of the MacB-MacA fusion protein and the intact MacA protein Full-length MacB and MacA were each amplified from E. coli K12 genomic DNA without the stop codon by PCR. The linker between MacB and MacA was designed to code for the following amino acids: GGGGSGGGGSGGGGS. MacB, the linker, and MacA were then 4
ACCEPTED MANUSCRIPT ligated by PCR-driven overlap extension. The resulting DNA fragment was inserted into the multiple cloning site of pET21c using the Nde1 and Xho1 restriction enzyme sites. The PreScission protease cleavage site (LEVLFQGP) and Protein G tag (residues 302-427) from
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Streptococcus dysgalactiae were added to the C-terminus of pET21c-MacB-MacA using the Cold Fusion ligation method, resulting in the pET21c-MacB-MacA-protein G tag plasmid. To express intact MacA from E. coli, the full-length MacA gene including the stop codon was
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inserted into pET29b using the Nde1 and Xho1 restriction enzyme sites (BD Sciences, USA),
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resulting in the pET29b-EcMacA plasmid.
Measurement of MIC
The measurement of MIC was carried out as previously described [7] except that cultures were grown in LB containing 100 µg/ml kanamycin and 100 µg/ml ampicillin and the
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expression of MacA and MacB proteins was induced by adding 0.5 mM isopropyl β-D-1thiogalactopyranoside (IPTG) to the cultures.
MacA protein
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Co-expression and co-purification of the MacB-MacA fusion protein and the intact
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The E.coli strain C43 (DE3) was transformed with plasmids encoding MacB-MacA and MacA, and cultured in 10 L TB medium supplemented with 100 µg/ml ampicillin and 50 µg/ml kanamycin until the culture reached an OD600 of ~4 at 37°C. Co-expression of the proteins was induced by treatment with 0.5 mM IPTG for 8 hr at 16°C. The cells were harvested at 5000 x g and resuspended with 200 ml lysis buffer, containing 20 mM HEPES (pH 7.5), 10% (v/v) glycerol and 150 mM NaCl. Suspensions were disrupted by passing twice through a continuous-type French Press (Constant Systems Limited) at 30 kpsi pressure. 5
ACCEPTED MANUSCRIPT The cell debris was removed by centrifugation at 10,000 x g. The membrane fraction was obtained by ultracentrifugation at 250,000 x g for 1 hr and stored at -70°C until further purification. The membrane fraction was solubilized in 20 mM HEPES (pH 7.5) buffer
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containing 150 mM NaCl, 10% (v/v) glycerol and 2% (w/v) n-dodecyl β-D-maltoside (DDM) using a homogenizer. After removing the non-solubilized fraction by centrifugation, the supernatant containing membrane proteins was loaded onto an open column packed with
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bovine IgG-coupled resin [15]. To eliminate non-target proteins, the resin was washed with 300 ml of washing buffer containing 20 mM HEPES (pH 7.5), 300 mM NaCl, 10% (v/v)
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glycerol and 0.04% (w/v) DDM. The protein complex containing MacB-MacA and MacA was eluted by cleaving the Protein G tag with PreScission protease. To replace the detergents, amphipol A8-34 was added to the sample in a 1:5 (w/w) ratio of protein/amphipol and then incubated for 30 min. The remaining DDM was absorbed by incubating with Bio-Beads SM-
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2 (Bio-Rad) and the Bio-Beads were then removed by gentle centrifugation. The complex was further purified by size-exclusion chromatography using a Superdex 200 10/300 GL column (GE Healthcare) pre-equilibrated with a buffer containing 20 mM HEPES (pH 7.5),
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150 mM NaCl, and 5% (v/v) glycerol.
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Purification of the ternary complex The construction and purification of the TolC-hybrid protein were described in a previous study [10]. Prior to size-exclusion chromatography, the purified complex with MacB-MacA and MacA was incubated with purified TolC-hybrid protein for 30 min. For purification and analysis, size exclusion chromatography was performed using a Superdex 200 10/300 GL column (GE Healthcare) pre-equilibrated with 20 mM HEPES (pH 7.5), 150 mM NaCl, and 5% (v/v) glycerol. 6
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Transmission electron microscopy The ternary complex was loaded onto an EM grid with a layer of amorphous carbon film that
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was rendered hydrophilic by glow discharge. The grid was stained with 1% (w/v) uranyl acetate solution followed by blotting of excess solution and air-drying. Images of negatively stained particles were collected using a Talos L120C TEM (Thermo Fisher Scientific Inc.,
Results and Discussion
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USA) operating at 120kV acceleration voltage. [19].
A strategy to make a stable complex of MacB and MacA.
To investigate the structure of the MacAB-TolC complex, we generated a MacB-MacA fusion
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protein, which is similar to the approach used to determine the cryo-EM structure [8]. Fulllength MacA was fused to the C-terminal end of full-length MacB, whose stop codon was deleted. Protein G tag was attached to the C-terminus of the MacA protein for affinity
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purification [15] (Fig. 1). However, we found redundant blobs near the MacB portion in electron micrographs of negatively stained complex, similar to recently published cryo-EM
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images [8] (data not shown). To remove these blobs, which represent extra MacB molecules in the complex, we supplemented intact MacA protein by co-expression with a different plasmid in E. coli under an acrAB-deletion background (Fig. 1). In line with previous results, an increased resistance to macrolide antibiotics was shown only when MacA and MacB were overexpressed in the absence of the functional AcrAB-TolC pump, which is the major efflux pump for diverse antibiotics [7, 11]. As seen in Table 1, increased resistance to erythromycin was conferred when MacA was added to the MacB-MacA fusion protein. This result indicates 7
ACCEPTED MANUSCRIPT that a more functional complex could be generated by supplementing additional MacA protein to the MacB-MacA fusion protein.
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Stoichiometry of 6:2:3 for MacA:MacB:TolC in the complex The MacB-MacA fusion protein and intact MacA were overexpressed in E. coli, and were readily purified in the presence of the detergent dodecyl-maltoside. A binary complex
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containing the MacB-MacA fusion protein and intact MacA protein was co-eluted on a size exclusion chromatographic column. Since the E.coli MacA protein exists as a monomeric
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protein in solution in the absence of a chemical cross linker [7], this co-elution suggests that intact MacA protein bound to the MacB-MacA fusion protein in order to form a full protein complex. We speculate that the N-terminal transmembrane region of MacA destabilizes the MacA protein in solution and that MacA is stabilized by interacting with the integral
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membrane protein MacB in the complex. The intensity of the MacB-MacA fusion protein band on SDS-PAGE is ~20% stronger than that of the intact MacA protein (Fig. 2A). The molar ratio between these two proteins suggests that the number of MacA proteins is about
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two times as many as the number of MacB-MacA fusion proteins (Fig. 2A; right). Assuming that MacA is a hexamer in the complex, the number of free MacA monomers in the complex
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corresponds to 4, along with 2 MacB-MacA fusion protein protomers. To form the ternary complex, we incubated this binary protein complex with a TolC-
hybrid protein containing the MacA binding motifs at the TolC periplasmic tip regions [10]. This TolC-hybrid protein was used as a TolC surrogate in this study, similar to previous studies [7, 10, 15, 16, 20, 21]. The protein mixture was applied to the size exclusion chromatography to purify and analyze the complex. The three proteins were co-eluted on a size exclusion chromatographic column (Fig. 2B). Further SDS-PAGE analysis of the 8
ACCEPTED MANUSCRIPT fractions suggested that the molar ratio of the MacB-MacA fusion protein : MacA protein : TolC-hybrid protein is 2.0:2.6:4.1 or approximately 2:3:4. Based on this result, the source of the MacA hexamer can be derived: 2 from the MacB-MacA fusion protein and 4 from the
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intact MacA protein. Likewise, MacB is a dimer in the complex since MacB is derived from the MacB-MacA fusion protein. Taken together, we conclude that the stoichiometry for MacA:MacB:TolC is 6:2:3, which is consistent with the previous EM structure [8].
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To examine the structure of the ternary complex, we obtained electron micrographs of negatively stained MacAB-TolC. Each particle lacked the redundant MacB density below
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the MacA portion in the EM images, consistent with the previous complex structure [8] (Fig. 2C). As a whole, the previous cryo-EM structure [8] represents the complex of the MacABTolC pump well, even though a protein sample with a different stoichiometry was applied.
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Periplasmic domain-gated MacB activation mechanism
We revisited the crystal structure of the Aa MacB periplasmic domain, which was previously reported by our research group (PDB code: 3FTJ) [12]. Although the Aa MacB and E. coli
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MacB have limited sequence homology (sequence identity: 35%) in the periplasmic region (residues 331-337, 322-328 in Aa MacB and E. coli MacB, respectively), the two structures
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are very similar in their protomers (RMSD = 1.708 Å between 179 Cα atoms; Fig. 3A). The E. coli MacB dimer in the complex makes a hole in the center of the two periplasmic domains (PDB code: 5NIL) [8] (Fig. 3B). We found that a dimeric assembly was formed by the two adjacent molecules in the crystal of the Aa MacB periplasmic domain [12]. The MacB dimer in the crystal forms a similar structure to the E. coli MacB despite the lack of a hole between the two protomers (Fig. 3C). Dimeric assembly of the Aa MacB periplasmic domain has not been noted before because the purified protein was eluted as a monomer on size exclusion 9
ACCEPTED MANUSCRIPT chromatography [12], and the residues at the dimeric interface are not well-conserved. By comparing these two structures of MacB periplasmic domains, we propose a channel-opening mechanism for MacB, depending on its interaction with MacA in the
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complex. In the complex structure, the outer rim parts of the MacB periplasmic domain make contacts with the central hollow region formed by the MP and the beta-barrel domains of the MacA hexamer [8]. In the Aa MacB periplasmic domain structure, only minor interactions
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were found between the two MacB periplasmic domains. These observations suggest that the two periplasmic domains of the MacB dimer are separated in the context of the central
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hollow of the MacA hexamer, leading to creation of the central hole of MacB. The central hole of MacB may be important in activation of this efflux pump because it is the only gateway to the MacA channel in the MacAB-TolC pump. The weak propensity to form a dimer in the periplasmic domain appears rather reasonable, since the weak affinity between
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the proteins could be suitable for the dynamic association and dissociation of the proteins. Furthermore, the lack of sequence conservation at the dimeric interface of the MacB periplasmic domain could be accounted for by the interaction being mediated by the
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backbone, rather than by side chains.
In a previous study, the ATPase activity of MacB was increased by MacA [6]. Given
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the lack of a full-length MacB structure in the absence of MacA, we generated a full-length model of MacB (Fig. 4A). Since the periplasmic domain and the cytoplasmic NBD domain are connected by rigid transmembrane α-helices, the relative locations of the two NBD domains in the cytosol should be affected by the close contact between the periplasmic domains, resulting in a ‘locked’ conformation. This locked conformation could be changed by interaction with the MacA hexamer, which is in turn facilitated by the interaction with the TolC trimer (Fig. 4B). Thus, the activation of the efflux pump is coupled with the synergistic 10
ACCEPTED MANUSCRIPT formation of the ternary complex in bacteria. Gram-positive bacteria have homologous efflux pumps, such as the sporulationdelaying efflux pump YknWXYZ [22]. Recent structural studies revealed that the inner
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membrane ABC transporter YknZ has a similar extracellular domain to the periplasmic domain of MacB [23], and the MFP YknX displays a funnel-like hexameric arrangement as observed in MacA [24]. Therefore, our findings suggest that a similar mechanism could occur
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in the efflux pumps of Gram-positive bacteria.
Acknowledgements
This work was supported by grants from the National Research Foundation of Korea (NRF2017H1A2A1042661-Global Ph.D. Fellowship Program to SH, 2017R1A2B2003992 to
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NCH and 2017R1A2B2011008 to KL). We made use of the EM facility at the Korea Basic
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Science Institute (Ochang, Republic of Korea).
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References [1] N. Kobayashi, K. Nishino, A. Yamaguchi, Novel macrolide-specific ABC-type efflux transporter in Escherichia coli, J Bacteriol, 183 (2001) 5639-5644.
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[2] H. Yamanaka, H. Kobayashi, E. Takahashi, K. Okamoto, MacAB is involved in the secretion of Escherichia coli heat-stable enterotoxin II, J Bacteriol, 190 (2008) 7693-7698. [3] I. Vallet-Gely, A. Novikov, L. Augusto, P. Liehl, G. Bolbach, M. Pechy-Tarr, P. Cosson, C.
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Wandersman, Protoporphyrin (PPIX) efflux by the MacAB-TolC pump in Escherichia coli, Microbiologyopen, 3 (2014) 849-859.
[6] H.T. Lin, V.N. Bavro, N.P. Barrera, H.M. Frankish, S. Velamakanni, H.W. van Veen, C.V.
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Robinson, M.I. Borges-Walmsley, A.R. Walmsley, MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion
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protein MacA, J Biol Chem, 284 (2009) 1145-1154. [7] S. Yum, Y. Xu, S. Piao, S.H. Sim, H.M. Kim, W.S. Jo, K.J. Kim, H.S. Kweon, M.H. Jeong, H. Jeon, K. Lee, N.C. Ha, Crystal structure of the periplasmic component of a tripartite macrolide-specific efflux pump, J Mol Biol, 387 (2009) 1286-1297. [8] A.W.P. Fitzpatrick, S. Llabres, A. Neuberger, J.N. Blaza, X.C. Bai, U. Okada, S. Murakami, H.W. van Veen, U. Zachariae, S.H.W. Scheres, B.F. Luisi, D. Du, Structure of the MacAB-TolC ABC-type tripartite multidrug efflux pump, Nat Microbiol, 2 (2017) 17070. 12
ACCEPTED MANUSCRIPT [9] S. Song, J.S. Kim, K. Lee, N.C. Ha, Molecular architecture of the bacterial tripartite multidrug efflux pump focusing on the adaptor bridging model, J Microbiol, 53 (2015) 355364.
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[10] Y. Xu, S. Song, A. Moeller, N. Kim, S. Piao, S.H. Sim, M. Kang, W. Yu, H.S. Cho, I. Chang, K. Lee, N.C. Ha, Functional implications of an intermeshing cogwheel-like interaction between TolC and MacA in the action of macrolide-specific efflux pump MacAB-
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TolC, J Biol Chem, 286 (2011) 13541-13549.
[11] N. Kobayashi, K. Nishino, T. Hirata, A. Yamaguchi, Membrane topology of ABC-type
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macrolide antibiotic exporter MacB in Escherichia coli, FEBS Lett, 546 (2003) 241-246. [12] Y. Xu, S.H. Sim, K.H. Nam, X.L. Jin, H.M. Kim, K.Y. Hwang, K. Lee, N.C. Ha, Crystal structure of the periplasmic region of MacB, a noncanonic ABC transporter, Biochemistry, 48 (2009) 5218-5225.
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[13] S. Wilkens, Structure and mechanism of ABC transporters, F1000Prime Rep, 7 (2015) 14.
[14] K.P. Locher, Mechanistic diversity in ATP-binding cassette (ABC) transporters, Nat
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Struct Mol Biol, 23 (2016) 487-493.
[15] J.S. Kim, H. Jeong, S. Song, H.Y. Kim, K. Lee, J. Hyun, N.C. Ha, Structure of the
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tripartite multidrug efflux pump AcrAB-TolC suggests an alternative assembly mode, Mol Cells, 38 (2015) 180-186.
[16] H. Jeong, J.S. Kim, S. Song, H. Shigematsu, T. Yokoyama, J. Hyun, N.C. Ha, Pseudoatomic Structure of the Tripartite Multidrug Efflux Pump AcrAB-TolC Reveals the Intermeshing Cogwheel-like Interaction between AcrA and TolC, Structure, 24 (2016) 272276. [17] D. Du, Z. Wang, N.R. James, J.E. Voss, E. Klimont, T. Ohene-Agyei, H. Venter, W. Chiu, 13
ACCEPTED MANUSCRIPT B.F. Luisi, Structure of the AcrAB-TolC multidrug efflux pump, Nature, 509 (2014) 512-515. [18] Z. Wang, G. Fan, C.F. Hryc, J.N. Blaza, Serysheva, II, M.F. Schmid, W. Chiu, B.F. Luisi, D. Du, An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump, Elife,
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6 (2017). [19] J.-K. Hyun, H.S. Jung, 3D Electron Microscopy, Biodesign, 1 (2013) 13-19.
[20] Y. Xu, M. Lee, A. Moeller, S. Song, B.Y. Yoon, H.M. Kim, S.Y. Jun, K. Lee, N.C. Ha,
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Funnel-like hexameric assembly of the periplasmic adapter protein in the tripartite multidrug efflux pump in gram-negative bacteria, J Biol Chem, 286 (2011) 17910-17920.
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[21] J.S. Kim, S. Song, M. Lee, S. Lee, K. Lee, N.C. Ha, Crystal Structure of a Soluble Fragment of the Membrane Fusion Protein HlyD in a Type I Secretion System of GramNegative Bacteria, Structure, 24 (2016) 477-485.
[22] Y. Yamada, E.B. Tikhonova, H.I. Zgurskaya, YknWXYZ is an unusual four-component
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transporter with a role in protection against sporulation-delaying-protein-induced killing of Bacillus subtilis, J Bacteriol, 194 (2012) 4386-4394. [23] Y. Xu, J. Guo, L. Wang, R. Jiang, X. Jin, J. Liu, S. Fan, C.S. Quan, N.C. Ha, The Crystal
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Structure of the YknZ Extracellular Domain of ABC Transporter YknWXYZ from Bacillus amyloliquefaciens, PLoS One, 11 (2016) e0155846.
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[24] Y. Xu, I. Jo, L. Wang, J. Chen, S. Fan, Y. Dong, C.S. Quan, N.C. Ha, Hexameric assembly of membrane fusion protein YknX of the sporulation delaying efflux pump from Bacillus amyloliquefaciens, Biochemical and Biophysical Research Communications, (2017).
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Figure Legends Figure 1. Construction of the ternary complex of MacA, MacB, and TolC using a fusion protein and co-expression.
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(A) Construct for the MacB-MacA fusion protein. Full-length MacB and MacA proteins from E. coli (Ec) were inserted into the pET21c-Protein G vector containing a Cterminal Protein G tag for purification (Novagen, USA) [15].
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(B) Construct for the intact Ec MacA protein. The full-length MacA protein was inserted into the pET29b vector (Novagen, USA). This construct was co-expressed with the
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expression vector depicted in Fig. 1(A).
(C) Construct for the TolC-hybrid protein, which was previously reported [10]. Two Aa MacA proteins without the signal sequence were fused, and Ec TolC R1 (residues 136-159) and R2 (residues 354-377) replaced the corresponding regions in MacA.
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The TolC R1 and R2 are solely responsible for binding to MacA [10]. One molecule of TolC-hybrid protein is equivalent to one molecule of TolC.
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Figure 2. Purified protein complexes
(A) The elution profile of the MacB-MacA fusion protein with intact MacA protein on a
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size exclusion chromatographic column (left panel). Each fraction was analyzed by SDS-PAGE (middle panel). The molar ratio was calculated based on the protein band
intensity using lane 3 and the molecular weight of each protein (right panel). Lane 3 was selected to minimize the effect of other proteins, and the relative molar mass of MacB-MacA was set to 2.
(B) The elution profile of the MacB-MacA fusion protein, the intact MacA protein, and the TolC-hybrid protein on size exclusion chromatography (left panel). Each fraction 15
ACCEPTED MANUSCRIPT (middle panel) and the molar ratio (right panel) was analyzed as described in (A). The molar ratio was calculated based on lane 1. (C) A representative electron micrograph of negatively stained MacAB-TolC ternary
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complex (left panel). Insets show selected particles (red color in the EM image) that show the overall arrangement of MacA, MacB, and TolC with the name of each
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protein labeled (right panels). Scale bars represent 200 nm.
Figure 3. Structural comparison of the MacB periplasmic domain alone to the protein
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complex with MacA.
(A) Structural superposition of protomers of the Aa MacB periplasmic domain (PDB code: 3FTJ; orange) and the E. coli MacB periplasmic domain in the protein complex (PDB code: 5NIL; blue).
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(B) The E. coli MacB periplasmic domain in complex with the MacA hexamer. (side view and top view). The circle indicates the outermost diameter of the inner rim of the MacA hexamer.
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(C) The Aa MacB periplasmic domain alone (side view and top view). The circle for the
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inner rim of the MacA hexamer in (B) is also drawn for comparison.
Figure 4. The MacA-dependent MacB activation mechanism. Left, a molecular model of the full-length MacB dimer in the locked conformation. The periplasmic domain (PD) of MacB is connected to the cytoplasmic NBD via the transmembrane domain (TM). The two PDs tightly interact, resulting in blockage of the central substrate path in PD and TM and hydrolysis of ATP in NBDs. Right, an assembly model for MacA, MacB, and TolC. The PDs of MacB directly 16
ACCEPTED MANUSCRIPT interact with the MacA hexamer, whose oligomerization is facilitated by its interaction with the TolC trimer. For MacB PD to interact with MacA, the two MacB PDs should dissociate from each other, leading to a change to the unlocked conformation of MacB,
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which allows ATP hydrolysis in NBDs and the efflux of substrates through the central
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channel.
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ACCEPTED MANUSCRIPT Table 1. Effects of overexpression of wild-type MacA and/or MacB-MacA hybrid proteins on the resistance of E. coli strain BW25113 ∆acrAB to erythromycin. BW25113 ∆acrAB
a
pET29b pET21c
pMacA pET21c
pET29b pMacB-MacA
pMacA pMacB-MacA
2.5
7.5
10.0
17.5
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MIC (µg of erythromycin/ml)a
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Background Plasmid
In the BW25113 ∆acrAB background with the indicated plasmid. 0, 2.5, 5.0, 7.5, 10.0, 12.5,
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15.0, 17.5 and 20 µg doses of erythromycin/ml were used to measure MICs.
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Highlights The exact stoichiometry of the MacAB-TolC pump was confirmed.
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Structural comparison provides a mechanism for MacA-dependent MacB activation.
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Improving understanding of the regulatory mechanisms of other efflux pumps.
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S0006-291X(17)32080-6
DOI:
10.1016/j.bbrc.2017.10.102
Reference:
YBBRC 38719
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 12 October 2017 Accepted Date: 19 October 2017
Please cite this article as: I. Jo, S. Hong, M. Lee, S. Song, J.-S. Kim, A. Mitra, J. Hyun, K. Lee, N.-C. Ha, Stoichiometry and mechanistic implications of the MacAB-TolC tripartite efflux pump, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.10.102. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Stoichiometry and mechanistic implications of the MacAB-TolC tripartite efflux pump Inseong Jo1†, Seokho Hong1†, Minho Lee2, Saemee Song1, Jin-Sik Kim1, Alok Mitra4,
1
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Jaekyung Hyun3, Kangseok Lee2*, and Nam-Chul Ha1* Department of Agricultural Biotechnology, Center for Food Safety and Toxicology, and
Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826,
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Republic of Korea
Department of Life Science, Chung-Ang University, Seoul 06974, Republic of Korea
3
Electron Microscopy Research Center, Korea Basic Science Institute, Chungcheongbukdo
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28119, Republic of Korea 4
Department of Biological Sciences, the University of Auckland, Auckland 1010, New
†
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Zealand
These authors equally contributed to this work.
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* To whom correspondence should be addressed: Nam-Chul Ha (e-mail: [email protected], Tel.: +82-2-880-4853, Fax: +82-2-873-5095) and Kangseok Lee (email: [email protected],
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Tel.: +82-2-820-5241, Fax: +82-2-825-5206)
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Abstract The MacAB-TolC tripartite efflux pump is involved in resistance to macrolide antibiotics and secretion of protein toxins in many Gram-negative bacteria. The pump spans the entire cell
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envelope and operates by expelling substances to extracellular space. X-ray crystal and electron microscopic structures have revealed the funnel-like MacA hexamer in the periplasmic space and the cylindrical TolC trimer. Nonetheless, the inner membrane
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transporter MacB still remains ambiguous in terms of its oligomeric state in the functional complex. In this study, we purified a stable binary complex using a fusion protein of MacA
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and MacB of Escherichia coli, and then supplemented MacA to meet the correct stoichiometry between the two proteins. The result demonstrated that MacB is a homodimer in the complex, which is consistent with results from the recent complex structure using cryoelectron microscopy single particle analysis. Structural comparison with the previously
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reported MacB periplasmic domain structure suggests a molecular mechanism for regulation of the activity of MacB via an interaction between the MacB periplasmic domain and MacA.
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Keywords
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Our results provide a better understanding of the tripartite pumps at the molecular level.
Escherichia coli; Actinobacillus actinomycetemcomitans; tripartite efflux pump; MacABTolC; electron microscopy
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Introduction Many Gram-negative bacteria express the MacAB-TolC tripartite efflux pump, which consists of the inner membrane transporter MacB, the membrane fusion protein MacA, and
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the outer membrane channel protein TolC [1]. This pump is involved in efflux of macrolide antibiotics and in secretion or transport of the heat-stable enterotoxin II, outer membrane glycolipids, protoporphyrin, and lipopeptides [1-5]. The active processes of efflux and
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transport are carried out by MacB, which contains an ATP-binding cassette (ABC) that uses ATP hydrolysis as a driving force [6].
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MacA is anchored in the outer leaflet of the inner membrane via the N-terminal noncleavable signal peptide [7-9]. MacA is also called the periplasmic adaptor protein since it connects the inner membrane transporter to the outer membrane channel protein in the periplasm.
Crystal
structures
of
MacA
from
E.
coli
and
Actinobacillus
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actinomycetemcomitans (Aa) revealed a linear arrangement of the membrane proximal (MP) domain, beta-barrel domain, lipoyl domain, and α-hairpin domain [7, 10]. The crystal structure of Aa MacA further revealed a funnel-like hexameric assembly [7]. The funnel stem
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portion of the MacA hexamer forms an α-barrel structure with six cogwheels at the end of the α-barrel. This characteristic cogwheel structure was implicated as a binding and channel
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opening motif for the similar α-barrel of the TolC trimer, by intermeshing the two α-barrels. The flared cone structure, or funnel mouth, in the MacA hexamer was proposed to accommodate the periplasmic domain of MacB [10]. A membrane topology study suggested that MacB contains an N-terminal nucleotide
binding domain (NBD), transmembrane domain (TM) 1, a large periplasmic domain, TM 2, cytoplasmic region, TM 3, a small periplasmic region, TM 4, and a C-terminal cytoplasmic tail [11]. The crystal structure of the large periplasmic domain of Aa MacB revealed a 3
ACCEPTED MANUSCRIPT relatively large domain with two compartments [12]. The domain arrangement and the number of TM regions of MacB are different from typical B-family ABC transporters, which operate as a homodimer with 6 α-helices per monomer, a small periplasmic domain, and a C-
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terminal NBD [13, 14]. Therefore, the oligomeric state of MacB in the functional complex was unclear.
Due to the low affinity between the components in the complex, many researchers
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have stabilized the complex by fusing the components or introducing an artificial disulfide bond between the proteins for structural studies [8, 10, 15-18]. The MacAB-TolC complex
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structure was recently determined by cryo-electronmicroscopy (cryo-EM) using a MacBMacA fusion protein [8]. The MacB homodimer in the complex showed a structure consistent with previous results from the topology study. Interestingly, although the protein sample has a stoichiometry of 6:6:3 for the complex of MacA:MacB:TolC, the cryo-EM structure had a
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stoichiometry of 6:2:3. Only two of six MacB molecules were visible in the cryo-EM structure. Since the other four MacB molecules were not fixed in the complex, they diffused and disappeared during the particle processing.
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In this study, we removed the redundant MacB molecules in the complex by supplementing with MacA, and we suggest a plausible mechanism for how the activity of the
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efflux pump is activated in the functional complex.
Materials and Methods Construction of the MacB-MacA fusion protein and the intact MacA protein Full-length MacB and MacA were each amplified from E. coli K12 genomic DNA without the stop codon by PCR. The linker between MacB and MacA was designed to code for the following amino acids: GGGGSGGGGSGGGGS. MacB, the linker, and MacA were then 4
ACCEPTED MANUSCRIPT ligated by PCR-driven overlap extension. The resulting DNA fragment was inserted into the multiple cloning site of pET21c using the Nde1 and Xho1 restriction enzyme sites. The PreScission protease cleavage site (LEVLFQGP) and Protein G tag (residues 302-427) from
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Streptococcus dysgalactiae were added to the C-terminus of pET21c-MacB-MacA using the Cold Fusion ligation method, resulting in the pET21c-MacB-MacA-protein G tag plasmid. To express intact MacA from E. coli, the full-length MacA gene including the stop codon was
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inserted into pET29b using the Nde1 and Xho1 restriction enzyme sites (BD Sciences, USA),
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resulting in the pET29b-EcMacA plasmid.
Measurement of MIC
The measurement of MIC was carried out as previously described [7] except that cultures were grown in LB containing 100 µg/ml kanamycin and 100 µg/ml ampicillin and the
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expression of MacA and MacB proteins was induced by adding 0.5 mM isopropyl β-D-1thiogalactopyranoside (IPTG) to the cultures.
MacA protein
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Co-expression and co-purification of the MacB-MacA fusion protein and the intact
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The E.coli strain C43 (DE3) was transformed with plasmids encoding MacB-MacA and MacA, and cultured in 10 L TB medium supplemented with 100 µg/ml ampicillin and 50 µg/ml kanamycin until the culture reached an OD600 of ~4 at 37°C. Co-expression of the proteins was induced by treatment with 0.5 mM IPTG for 8 hr at 16°C. The cells were harvested at 5000 x g and resuspended with 200 ml lysis buffer, containing 20 mM HEPES (pH 7.5), 10% (v/v) glycerol and 150 mM NaCl. Suspensions were disrupted by passing twice through a continuous-type French Press (Constant Systems Limited) at 30 kpsi pressure. 5
ACCEPTED MANUSCRIPT The cell debris was removed by centrifugation at 10,000 x g. The membrane fraction was obtained by ultracentrifugation at 250,000 x g for 1 hr and stored at -70°C until further purification. The membrane fraction was solubilized in 20 mM HEPES (pH 7.5) buffer
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containing 150 mM NaCl, 10% (v/v) glycerol and 2% (w/v) n-dodecyl β-D-maltoside (DDM) using a homogenizer. After removing the non-solubilized fraction by centrifugation, the supernatant containing membrane proteins was loaded onto an open column packed with
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bovine IgG-coupled resin [15]. To eliminate non-target proteins, the resin was washed with 300 ml of washing buffer containing 20 mM HEPES (pH 7.5), 300 mM NaCl, 10% (v/v)
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glycerol and 0.04% (w/v) DDM. The protein complex containing MacB-MacA and MacA was eluted by cleaving the Protein G tag with PreScission protease. To replace the detergents, amphipol A8-34 was added to the sample in a 1:5 (w/w) ratio of protein/amphipol and then incubated for 30 min. The remaining DDM was absorbed by incubating with Bio-Beads SM-
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2 (Bio-Rad) and the Bio-Beads were then removed by gentle centrifugation. The complex was further purified by size-exclusion chromatography using a Superdex 200 10/300 GL column (GE Healthcare) pre-equilibrated with a buffer containing 20 mM HEPES (pH 7.5),
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150 mM NaCl, and 5% (v/v) glycerol.
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Purification of the ternary complex The construction and purification of the TolC-hybrid protein were described in a previous study [10]. Prior to size-exclusion chromatography, the purified complex with MacB-MacA and MacA was incubated with purified TolC-hybrid protein for 30 min. For purification and analysis, size exclusion chromatography was performed using a Superdex 200 10/300 GL column (GE Healthcare) pre-equilibrated with 20 mM HEPES (pH 7.5), 150 mM NaCl, and 5% (v/v) glycerol. 6
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Transmission electron microscopy The ternary complex was loaded onto an EM grid with a layer of amorphous carbon film that
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was rendered hydrophilic by glow discharge. The grid was stained with 1% (w/v) uranyl acetate solution followed by blotting of excess solution and air-drying. Images of negatively stained particles were collected using a Talos L120C TEM (Thermo Fisher Scientific Inc.,
Results and Discussion
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USA) operating at 120kV acceleration voltage. [19].
A strategy to make a stable complex of MacB and MacA.
To investigate the structure of the MacAB-TolC complex, we generated a MacB-MacA fusion
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protein, which is similar to the approach used to determine the cryo-EM structure [8]. Fulllength MacA was fused to the C-terminal end of full-length MacB, whose stop codon was deleted. Protein G tag was attached to the C-terminus of the MacA protein for affinity
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purification [15] (Fig. 1). However, we found redundant blobs near the MacB portion in electron micrographs of negatively stained complex, similar to recently published cryo-EM
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images [8] (data not shown). To remove these blobs, which represent extra MacB molecules in the complex, we supplemented intact MacA protein by co-expression with a different plasmid in E. coli under an acrAB-deletion background (Fig. 1). In line with previous results, an increased resistance to macrolide antibiotics was shown only when MacA and MacB were overexpressed in the absence of the functional AcrAB-TolC pump, which is the major efflux pump for diverse antibiotics [7, 11]. As seen in Table 1, increased resistance to erythromycin was conferred when MacA was added to the MacB-MacA fusion protein. This result indicates 7
ACCEPTED MANUSCRIPT that a more functional complex could be generated by supplementing additional MacA protein to the MacB-MacA fusion protein.
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Stoichiometry of 6:2:3 for MacA:MacB:TolC in the complex The MacB-MacA fusion protein and intact MacA were overexpressed in E. coli, and were readily purified in the presence of the detergent dodecyl-maltoside. A binary complex
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containing the MacB-MacA fusion protein and intact MacA protein was co-eluted on a size exclusion chromatographic column. Since the E.coli MacA protein exists as a monomeric
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protein in solution in the absence of a chemical cross linker [7], this co-elution suggests that intact MacA protein bound to the MacB-MacA fusion protein in order to form a full protein complex. We speculate that the N-terminal transmembrane region of MacA destabilizes the MacA protein in solution and that MacA is stabilized by interacting with the integral
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membrane protein MacB in the complex. The intensity of the MacB-MacA fusion protein band on SDS-PAGE is ~20% stronger than that of the intact MacA protein (Fig. 2A). The molar ratio between these two proteins suggests that the number of MacA proteins is about
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two times as many as the number of MacB-MacA fusion proteins (Fig. 2A; right). Assuming that MacA is a hexamer in the complex, the number of free MacA monomers in the complex
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corresponds to 4, along with 2 MacB-MacA fusion protein protomers. To form the ternary complex, we incubated this binary protein complex with a TolC-
hybrid protein containing the MacA binding motifs at the TolC periplasmic tip regions [10]. This TolC-hybrid protein was used as a TolC surrogate in this study, similar to previous studies [7, 10, 15, 16, 20, 21]. The protein mixture was applied to the size exclusion chromatography to purify and analyze the complex. The three proteins were co-eluted on a size exclusion chromatographic column (Fig. 2B). Further SDS-PAGE analysis of the 8
ACCEPTED MANUSCRIPT fractions suggested that the molar ratio of the MacB-MacA fusion protein : MacA protein : TolC-hybrid protein is 2.0:2.6:4.1 or approximately 2:3:4. Based on this result, the source of the MacA hexamer can be derived: 2 from the MacB-MacA fusion protein and 4 from the
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intact MacA protein. Likewise, MacB is a dimer in the complex since MacB is derived from the MacB-MacA fusion protein. Taken together, we conclude that the stoichiometry for MacA:MacB:TolC is 6:2:3, which is consistent with the previous EM structure [8].
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To examine the structure of the ternary complex, we obtained electron micrographs of negatively stained MacAB-TolC. Each particle lacked the redundant MacB density below
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the MacA portion in the EM images, consistent with the previous complex structure [8] (Fig. 2C). As a whole, the previous cryo-EM structure [8] represents the complex of the MacABTolC pump well, even though a protein sample with a different stoichiometry was applied.
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Periplasmic domain-gated MacB activation mechanism
We revisited the crystal structure of the Aa MacB periplasmic domain, which was previously reported by our research group (PDB code: 3FTJ) [12]. Although the Aa MacB and E. coli
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MacB have limited sequence homology (sequence identity: 35%) in the periplasmic region (residues 331-337, 322-328 in Aa MacB and E. coli MacB, respectively), the two structures
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are very similar in their protomers (RMSD = 1.708 Å between 179 Cα atoms; Fig. 3A). The E. coli MacB dimer in the complex makes a hole in the center of the two periplasmic domains (PDB code: 5NIL) [8] (Fig. 3B). We found that a dimeric assembly was formed by the two adjacent molecules in the crystal of the Aa MacB periplasmic domain [12]. The MacB dimer in the crystal forms a similar structure to the E. coli MacB despite the lack of a hole between the two protomers (Fig. 3C). Dimeric assembly of the Aa MacB periplasmic domain has not been noted before because the purified protein was eluted as a monomer on size exclusion 9
ACCEPTED MANUSCRIPT chromatography [12], and the residues at the dimeric interface are not well-conserved. By comparing these two structures of MacB periplasmic domains, we propose a channel-opening mechanism for MacB, depending on its interaction with MacA in the
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complex. In the complex structure, the outer rim parts of the MacB periplasmic domain make contacts with the central hollow region formed by the MP and the beta-barrel domains of the MacA hexamer [8]. In the Aa MacB periplasmic domain structure, only minor interactions
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were found between the two MacB periplasmic domains. These observations suggest that the two periplasmic domains of the MacB dimer are separated in the context of the central
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hollow of the MacA hexamer, leading to creation of the central hole of MacB. The central hole of MacB may be important in activation of this efflux pump because it is the only gateway to the MacA channel in the MacAB-TolC pump. The weak propensity to form a dimer in the periplasmic domain appears rather reasonable, since the weak affinity between
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the proteins could be suitable for the dynamic association and dissociation of the proteins. Furthermore, the lack of sequence conservation at the dimeric interface of the MacB periplasmic domain could be accounted for by the interaction being mediated by the
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backbone, rather than by side chains.
In a previous study, the ATPase activity of MacB was increased by MacA [6]. Given
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the lack of a full-length MacB structure in the absence of MacA, we generated a full-length model of MacB (Fig. 4A). Since the periplasmic domain and the cytoplasmic NBD domain are connected by rigid transmembrane α-helices, the relative locations of the two NBD domains in the cytosol should be affected by the close contact between the periplasmic domains, resulting in a ‘locked’ conformation. This locked conformation could be changed by interaction with the MacA hexamer, which is in turn facilitated by the interaction with the TolC trimer (Fig. 4B). Thus, the activation of the efflux pump is coupled with the synergistic 10
ACCEPTED MANUSCRIPT formation of the ternary complex in bacteria. Gram-positive bacteria have homologous efflux pumps, such as the sporulationdelaying efflux pump YknWXYZ [22]. Recent structural studies revealed that the inner
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membrane ABC transporter YknZ has a similar extracellular domain to the periplasmic domain of MacB [23], and the MFP YknX displays a funnel-like hexameric arrangement as observed in MacA [24]. Therefore, our findings suggest that a similar mechanism could occur
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in the efflux pumps of Gram-positive bacteria.
Acknowledgements
This work was supported by grants from the National Research Foundation of Korea (NRF2017H1A2A1042661-Global Ph.D. Fellowship Program to SH, 2017R1A2B2003992 to
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NCH and 2017R1A2B2011008 to KL). We made use of the EM facility at the Korea Basic
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Science Institute (Ochang, Republic of Korea).
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References [1] N. Kobayashi, K. Nishino, A. Yamaguchi, Novel macrolide-specific ABC-type efflux transporter in Escherichia coli, J Bacteriol, 183 (2001) 5639-5644.
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[2] H. Yamanaka, H. Kobayashi, E. Takahashi, K. Okamoto, MacAB is involved in the secretion of Escherichia coli heat-stable enterotoxin II, J Bacteriol, 190 (2008) 7693-7698. [3] I. Vallet-Gely, A. Novikov, L. Augusto, P. Liehl, G. Bolbach, M. Pechy-Tarr, P. Cosson, C.
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Wandersman, Protoporphyrin (PPIX) efflux by the MacAB-TolC pump in Escherichia coli, Microbiologyopen, 3 (2014) 849-859.
[6] H.T. Lin, V.N. Bavro, N.P. Barrera, H.M. Frankish, S. Velamakanni, H.W. van Veen, C.V.
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Robinson, M.I. Borges-Walmsley, A.R. Walmsley, MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion
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protein MacA, J Biol Chem, 284 (2009) 1145-1154. [7] S. Yum, Y. Xu, S. Piao, S.H. Sim, H.M. Kim, W.S. Jo, K.J. Kim, H.S. Kweon, M.H. Jeong, H. Jeon, K. Lee, N.C. Ha, Crystal structure of the periplasmic component of a tripartite macrolide-specific efflux pump, J Mol Biol, 387 (2009) 1286-1297. [8] A.W.P. Fitzpatrick, S. Llabres, A. Neuberger, J.N. Blaza, X.C. Bai, U. Okada, S. Murakami, H.W. van Veen, U. Zachariae, S.H.W. Scheres, B.F. Luisi, D. Du, Structure of the MacAB-TolC ABC-type tripartite multidrug efflux pump, Nat Microbiol, 2 (2017) 17070. 12
ACCEPTED MANUSCRIPT [9] S. Song, J.S. Kim, K. Lee, N.C. Ha, Molecular architecture of the bacterial tripartite multidrug efflux pump focusing on the adaptor bridging model, J Microbiol, 53 (2015) 355364.
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[10] Y. Xu, S. Song, A. Moeller, N. Kim, S. Piao, S.H. Sim, M. Kang, W. Yu, H.S. Cho, I. Chang, K. Lee, N.C. Ha, Functional implications of an intermeshing cogwheel-like interaction between TolC and MacA in the action of macrolide-specific efflux pump MacAB-
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TolC, J Biol Chem, 286 (2011) 13541-13549.
[11] N. Kobayashi, K. Nishino, T. Hirata, A. Yamaguchi, Membrane topology of ABC-type
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macrolide antibiotic exporter MacB in Escherichia coli, FEBS Lett, 546 (2003) 241-246. [12] Y. Xu, S.H. Sim, K.H. Nam, X.L. Jin, H.M. Kim, K.Y. Hwang, K. Lee, N.C. Ha, Crystal structure of the periplasmic region of MacB, a noncanonic ABC transporter, Biochemistry, 48 (2009) 5218-5225.
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[13] S. Wilkens, Structure and mechanism of ABC transporters, F1000Prime Rep, 7 (2015) 14.
[14] K.P. Locher, Mechanistic diversity in ATP-binding cassette (ABC) transporters, Nat
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Struct Mol Biol, 23 (2016) 487-493.
[15] J.S. Kim, H. Jeong, S. Song, H.Y. Kim, K. Lee, J. Hyun, N.C. Ha, Structure of the
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tripartite multidrug efflux pump AcrAB-TolC suggests an alternative assembly mode, Mol Cells, 38 (2015) 180-186.
[16] H. Jeong, J.S. Kim, S. Song, H. Shigematsu, T. Yokoyama, J. Hyun, N.C. Ha, Pseudoatomic Structure of the Tripartite Multidrug Efflux Pump AcrAB-TolC Reveals the Intermeshing Cogwheel-like Interaction between AcrA and TolC, Structure, 24 (2016) 272276. [17] D. Du, Z. Wang, N.R. James, J.E. Voss, E. Klimont, T. Ohene-Agyei, H. Venter, W. Chiu, 13
ACCEPTED MANUSCRIPT B.F. Luisi, Structure of the AcrAB-TolC multidrug efflux pump, Nature, 509 (2014) 512-515. [18] Z. Wang, G. Fan, C.F. Hryc, J.N. Blaza, Serysheva, II, M.F. Schmid, W. Chiu, B.F. Luisi, D. Du, An allosteric transport mechanism for the AcrAB-TolC multidrug efflux pump, Elife,
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6 (2017). [19] J.-K. Hyun, H.S. Jung, 3D Electron Microscopy, Biodesign, 1 (2013) 13-19.
[20] Y. Xu, M. Lee, A. Moeller, S. Song, B.Y. Yoon, H.M. Kim, S.Y. Jun, K. Lee, N.C. Ha,
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Funnel-like hexameric assembly of the periplasmic adapter protein in the tripartite multidrug efflux pump in gram-negative bacteria, J Biol Chem, 286 (2011) 17910-17920.
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[21] J.S. Kim, S. Song, M. Lee, S. Lee, K. Lee, N.C. Ha, Crystal Structure of a Soluble Fragment of the Membrane Fusion Protein HlyD in a Type I Secretion System of GramNegative Bacteria, Structure, 24 (2016) 477-485.
[22] Y. Yamada, E.B. Tikhonova, H.I. Zgurskaya, YknWXYZ is an unusual four-component
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transporter with a role in protection against sporulation-delaying-protein-induced killing of Bacillus subtilis, J Bacteriol, 194 (2012) 4386-4394. [23] Y. Xu, J. Guo, L. Wang, R. Jiang, X. Jin, J. Liu, S. Fan, C.S. Quan, N.C. Ha, The Crystal
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Structure of the YknZ Extracellular Domain of ABC Transporter YknWXYZ from Bacillus amyloliquefaciens, PLoS One, 11 (2016) e0155846.
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[24] Y. Xu, I. Jo, L. Wang, J. Chen, S. Fan, Y. Dong, C.S. Quan, N.C. Ha, Hexameric assembly of membrane fusion protein YknX of the sporulation delaying efflux pump from Bacillus amyloliquefaciens, Biochemical and Biophysical Research Communications, (2017).
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Figure Legends Figure 1. Construction of the ternary complex of MacA, MacB, and TolC using a fusion protein and co-expression.
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(A) Construct for the MacB-MacA fusion protein. Full-length MacB and MacA proteins from E. coli (Ec) were inserted into the pET21c-Protein G vector containing a Cterminal Protein G tag for purification (Novagen, USA) [15].
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(B) Construct for the intact Ec MacA protein. The full-length MacA protein was inserted into the pET29b vector (Novagen, USA). This construct was co-expressed with the
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expression vector depicted in Fig. 1(A).
(C) Construct for the TolC-hybrid protein, which was previously reported [10]. Two Aa MacA proteins without the signal sequence were fused, and Ec TolC R1 (residues 136-159) and R2 (residues 354-377) replaced the corresponding regions in MacA.
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The TolC R1 and R2 are solely responsible for binding to MacA [10]. One molecule of TolC-hybrid protein is equivalent to one molecule of TolC.
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Figure 2. Purified protein complexes
(A) The elution profile of the MacB-MacA fusion protein with intact MacA protein on a
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size exclusion chromatographic column (left panel). Each fraction was analyzed by SDS-PAGE (middle panel). The molar ratio was calculated based on the protein band
intensity using lane 3 and the molecular weight of each protein (right panel). Lane 3 was selected to minimize the effect of other proteins, and the relative molar mass of MacB-MacA was set to 2.
(B) The elution profile of the MacB-MacA fusion protein, the intact MacA protein, and the TolC-hybrid protein on size exclusion chromatography (left panel). Each fraction 15
ACCEPTED MANUSCRIPT (middle panel) and the molar ratio (right panel) was analyzed as described in (A). The molar ratio was calculated based on lane 1. (C) A representative electron micrograph of negatively stained MacAB-TolC ternary
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complex (left panel). Insets show selected particles (red color in the EM image) that show the overall arrangement of MacA, MacB, and TolC with the name of each
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protein labeled (right panels). Scale bars represent 200 nm.
Figure 3. Structural comparison of the MacB periplasmic domain alone to the protein
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complex with MacA.
(A) Structural superposition of protomers of the Aa MacB periplasmic domain (PDB code: 3FTJ; orange) and the E. coli MacB periplasmic domain in the protein complex (PDB code: 5NIL; blue).
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(B) The E. coli MacB periplasmic domain in complex with the MacA hexamer. (side view and top view). The circle indicates the outermost diameter of the inner rim of the MacA hexamer.
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(C) The Aa MacB periplasmic domain alone (side view and top view). The circle for the
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inner rim of the MacA hexamer in (B) is also drawn for comparison.
Figure 4. The MacA-dependent MacB activation mechanism. Left, a molecular model of the full-length MacB dimer in the locked conformation. The periplasmic domain (PD) of MacB is connected to the cytoplasmic NBD via the transmembrane domain (TM). The two PDs tightly interact, resulting in blockage of the central substrate path in PD and TM and hydrolysis of ATP in NBDs. Right, an assembly model for MacA, MacB, and TolC. The PDs of MacB directly 16
ACCEPTED MANUSCRIPT interact with the MacA hexamer, whose oligomerization is facilitated by its interaction with the TolC trimer. For MacB PD to interact with MacA, the two MacB PDs should dissociate from each other, leading to a change to the unlocked conformation of MacB,
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which allows ATP hydrolysis in NBDs and the efflux of substrates through the central
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channel.
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ACCEPTED MANUSCRIPT Table 1. Effects of overexpression of wild-type MacA and/or MacB-MacA hybrid proteins on the resistance of E. coli strain BW25113 ∆acrAB to erythromycin. BW25113 ∆acrAB
a
pET29b pET21c
pMacA pET21c
pET29b pMacB-MacA
pMacA pMacB-MacA
2.5
7.5
10.0
17.5
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MIC (µg of erythromycin/ml)a
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Background Plasmid
In the BW25113 ∆acrAB background with the indicated plasmid. 0, 2.5, 5.0, 7.5, 10.0, 12.5,
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15.0, 17.5 and 20 µg doses of erythromycin/ml were used to measure MICs.
1
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Highlights The exact stoichiometry of the MacAB-TolC pump was confirmed.
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Structural comparison provides a mechanism for MacA-dependent MacB activation.
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Improving understanding of the regulatory mechanisms of other efflux pumps.
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