Energy-coupling mechanism of the multidrug resistance transporter AcrB: Evidence for membrane potential-driving hypothesis through mutagenic analysis.
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ABSTRACT:
SUBMITTER: Liu M
PROVIDER: S-EPMC5546932 | biostudies-literature | 2017 Aug
REPOSITORIES: biostudies-literature
Publications
Energy-coupling mechanism of the multidrug resistance transporter AcrB: Evidence for membrane potential-driving hypothesis through mutagenic analysis.
Protein & cell 20170801 8
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Project description:Gram-negative bacteria maintain an intrinsic resistance mechanism against entry of noxious compounds by utilizing highly efficient efflux pumps. The E. coli AcrAB-TolC drug efflux pump contains the inner membrane H+/drug antiporter AcrB comprising three functionally interdependent protomers, cycling consecutively through the loose (L), tight (T) and open (O) state during cooperative catalysis. Here, we present 13 X-ray structures of AcrB in intermediate states of the transport cycle. Structure-based mutational analysis combined with drug susceptibility assays indicate that drugs are guided through dedicated transport channels toward the drug binding pockets. A co-structure obtained in the combined presence of erythromycin, linezolid, oxacillin and fusidic acid shows binding of fusidic acid deeply inside the T protomer transmembrane domain. Thiol cross-link substrate protection assays indicate that this transmembrane domain-binding site can also accommodate oxacillin or novobiocin but not erythromycin or linezolid. AcrB-mediated drug transport is suggested to be allosterically modulated in presence of multiple drugs.
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Project description:AcrB is the inner membrane transporter of the tripartite multidrug efflux pump AcrAB-TolC in E. coli, which poses a major obstacle to the treatment of bacterial infections. X-ray structures have identified two types of substrate-binding pockets in the porter domains of AcrB trimer: the proximal binding pocket (PBP) and the distal binding pocket (DBP), and suggest a functional rotating mechanism in which each protomer cycles consecutively through three distinct conformational states (access, binding and extrusion). However, the details of substrate binding and translocation between the binding pockets remain elusive. In this work, we performed atomic simulations to obtain the free energy profile of the translocation of an antibiotic drug doxorubicin (DOX) inside AcrB. Our simulation indicates that DOX binds at the PBP and DBP with comparable affinities in the binding state protomer, and overcomes a 3 kcal/mol energy barrier to transit between them. Obvious conformational changes including closing of the PC1/PC2 cleft and shrinking of the DBP were observed upon DOX binding in the PBP, resulting in an intermediate state between the access and binding states. Taken together, the simulation results reveal a detailed stepwise substrate binding and translocation process in the framework of functional rotating mechanism.
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Project description:Energy coupling factor (ECF) transporters are responsible for the uptake of micronutrients in bacteria and archaea. They consist of an integral membrane unit, the S-component, and a tripartite ECF module. It has been proposed that the S-component mediates the substrate transport by toppling over in the membrane when docking onto an ECF module. Here, we present multi-scale molecular dynamics simulations and in vitro experiments to study the molecular toppling mechanism of the S-component of a folate-specific ECF transporter. Simulations reveal a strong bending of the membrane around the ECF module that provides a driving force for toppling of the S-component. The stability of the toppled state depends on the presence of non-bilayer forming lipids, as confirmed by folate transport activity measurements. Together, our data provide evidence for a lipid-dependent toppling-based mechanism for the folate-specific ECF transporter, a mechanism that might apply to other ECF transporters.
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Project description:This paper presented the dataset of correction parameters used in the determination of the energy transfer efficiencies from the spectrum-based fluorescence resonance energy transfer (FRET) measurement in a trimeric membrane protein AcrB. The cyan fluorescent protein (CFP) and yellow fluorescent protein (YPet) were used as the donor and acceptor, respectively. Two AcrB fusion proteins were constructed, AcrB-CFP and AcrB-YPet. The proteins were co-expressed in Escherichia coli cells, and energy transfer efficiency were determined in live cells. To obtain reliable energy transfer data, a complete set of correction parameters need to be first determined to accommodate for factors such as background fluorescence and spectra overlap. This paper described the methodology and determination of the correction factors, which are useful data and reference points for researchers working on fluorescence measurement of membrane protein complexes in live bacteria cells. Further interpretation and discussion of these data can be found in "Comparison of in vitro and in vivo oligomeric states of a wild type and mutant trimeric inner membrane multidrug transporter" (Wang et al., in press).
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Project description:Prokaryotic AcrB-like proteins belong to a family of transporters of the RND superfamily, and as main contributing factor to multidrug resistance pose a tremendous threat to future human health. A unique feature of AcrB transporters is the presence of two separate domains responsible for carrying substrate and generating energy. Significant progress has been made in elucidating the three-dimensional structures of the homo-trimer complexes of AcrB-like transporters, and a three-step functional rotation was identified for this class of transporters. However, the detailed mechanisms for the transduction of the substrate binding signal, as well as the energy coupling processes between the functionally distinct domains remain to be established. Here, we propose a model for the interdomain communication in AcrB that explains how the substrate binding signal from the substrate-carrier domain triggers protonation in the transmembrane domain. Our model further provides a plausible mechanism that explains how protonation induces conformational changes in the substrate-carrier domain. We summarize the thermodynamic principles that govern the functional cycle of the AcrB trimer complex.
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Project description:AcrAB(Z)-TolC is the main drug efflux transporter complex in Escherichia coli. The extrusion of various toxic compounds depends on several drug binding sites within the trimeric AcrB transporter. Membrane-localized carboxylated substrates, such as fusidic acid and hydrophobic β-lactams, access the pump via a groove between the transmembrane helices TM1 and TM2. In this article, the transport route from the initial TM1/TM2 groove binding site toward the deep binding pocket located in the periplasmic part has been addressed via molecular modeling studies followed by functional and structural characterization of several AcrB variants. We propose that membrane-embedded drugs bind initially to the TM1/TM2 groove, are oriented by the AcrB PN2 subdomain, and are subsequently transported via a PN2/PC1 interface pathway directly toward the deep binding pocket. Our work emphasizes the exploitation of multiple transport pathways by AcrB tuned to substrate physicochemical properties related to the polyspecificity of the pump.
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Project description:The multidrug resistance (MDR) system effectively expels antibiotics out of bacteria causing serious issues during bacterial infection. In addition to drug, indole, a common metabolic waste of bacteria, is expelled by MDR system of gram-negative bacteria for their survival. Experimental results suggest that AcrB, one of the key components of MDR system, undergoes large scale conformation changes during the pumping due to proton-motive process. However, due to extremely short time scale, it is difficult to observe (experimentally) those changes in the AcrB, which might facilitate the pumping process. Molecular simulations can shed light to understand the conformational changes for transport of indole in AcrB. Examination of conformational changes using all-atom simulation is, however, impractical. Here, we develop a hybrid coarse-grained force field to study the conformational changes of AcrB in presence of indole in the porter domain of monomer II. Using the coarse-grained force field, we investigated the conformational changes of AcrB for a number of model systems considering the effect of protonation in aspartic acid (Asp) residues Asp407 and Asp408 in the transmembrane domain of monomer II. Our results show that in the presence of indole, protonation of Asp408 or Asp407 residue causes conformational changes from binding state to extrusion state in monomer II, while remaining two monomers (I and III) approach access state in AcrB protein. We also observed that all three AcrB monomers prefer to go back to access state in the absence of indole. Steered molecular dynamics simulations were performed to demonstrate the feasibility of indole transport mechanism for protonated systems. Identification of indole transport pathway through AcrB can be very helpful in understanding the drug efflux mechanism used by the MDR bacteria.
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Project description:The resistance-nodulation-cell division (RND)-type xenobiotic efflux system plays a major role in the multidrug resistance of gram-negative bacteria. The only constitutively expressed RND system of Escherichia coli consists of the inner membrane transporter AcrB, the membrane fusion protein AcrA, and the outer membrane channel TolC. The latter two components are shared with another RND-type transporter AcrD, whose expression is induced by environmental stimuli. Here, we demonstrate how RND-type ternary complexes, which span two membranes and the cell wall, form in vivo. Total internal reflection fluorescence (TIRF) microscopy revealed that most fluorescent foci formed by AcrB fused to green fluorescent protein (GFP) were stationary in the presence of TolC but showed lateral displacements when tolC was deleted. The fraction of stationary AcrB-GFP foci decreased with increasing levels of AcrD. We propose that the AcrB-containing complex becomes unstable upon the induction of AcrD, which presumably replaces AcrB, a process we call "transporter exchange." This instability is suppressed by AcrB-specific substrates, suggesting that the ternary complex is stabilised when it is in action. These results suggest that the assembly of the RND-type efflux system is dynamically regulated in response to external stimuli, shedding new light on the adaptive antibiotic resistance of bacteria.
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Project description:The multidrug transporter AcrB transports a broad range of drugs out of the cell by means of the proton-motive force. The asymmetric crystal structure of trimeric AcrB suggests a functionally rotating mechanism for drug transport. Despite various supportive forms of evidence from biochemical and simulation studies for this mechanism, the link between the functional rotation and proton translocation across the membrane remains elusive. Here, calculating the minimum free energy pathway of the functional rotation for the complete AcrB trimer, we describe the structural and energetic basis behind the coupling between the functional rotation and the proton translocation at atomic resolution. Free energy calculations show that protonation of Asp408 in the transmembrane portion of the drug-bound protomer drives the functional rotation. The conformational pathway identifies vertical shear motions among several transmembrane helices, which regulate alternate access of water in the transmembrane as well as peristaltic motions that pump drugs in the periplasm.
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Project description:The multidrug resistance (MDR) system actively pumps antibiotics out of cells causing serious health problems. During the pumping, AcrB (one of the key components of MDR) undergoes a series of large-scale and proton-motive conformational changes. Capturing the conformational changes through all-atom simulations is challenging. Here, we implement a hybrid coarse-grained force field to investigate the conformational changes of AcrB in the porter domain under different protonation states of Asp407/Asp408 in the trans-membrane domain. Our results show that protonation of Asp408 in monomer III (extrusion) stabilizes the asymmetric structure of AcrB; deprotonation of Asp408 induces clear opening of the entrance and closing of the exit leading to the transition from extrusion to access state. The structural changes in the porter domain of AcrB are strongly coupled with the proton translocation stoichiometry in the trans-membrane domain. Moreover, our simulations support the postulation that AcrB should adopt the symmetric resting state in a substrate-free situation.
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