As active part of the major efflux system in E. coli bacteria, AcrB is responsible for the uptake and pumping of toxic substrates from the periplasm toward the extracellular space. In combination with the channel protein TolC and membrane fusion protein AcrA, this efflux pump is able to help the bacterium to survive different kinds of noxious compounds. With the present study we intend to enhance the understanding of the interactions between the domains and monomers, for example, the transduction of mechanical energy from the transmembrane domain into the porter domain, correlated motions of different subdomains within monomers, and cooperative effects between monomers. To this end, targeted molecular dynamics simulations have been employed either steering the whole protein complex or specific parts thereof. By forcing only parts of the complex towards specific conformational states, the risk for transient artificial conformations during the simulations is reduced. Distinct cooperative effects between the monomers in AcrB have been observed. Possible allosteric couplings have been identified providing microscopic insights that might be exploited to design more efficient inhibitors of efflux systems.

Corynebacterium jeikeium, a resident of human skin, is often associated with multidrug resistant nosocomial infections in immunodepressed patients. C. jeikeium K411 belongs to mycolic acid-containing actinomycetes, the mycolata and contains a channel-forming protein as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent treated cell walls and in extracts of whole cells using organic solvents. A gene coding for a 40 amino acid long polypeptide possibly responsible for the pore-forming activity was identified in the known genome of C. jeikeium by its similar chromosomal localization to known porH and porA genes of other Corynebacterium strains. The gene jk0268 was expressed in a porin deficient Corynebacterium glutamicum strain. For purification temporarily histidine-tailed or with a GST-tag at the N-terminus, the homogeneous protein caused channel-forming activity with an average conductance of 1.25 nS in 1M KCl identical to the channels formed by the detergent extracts. Zero-current membrane potential measurements of the voltage dependent channel implied selectivity for anions. This preference is according to single-channel analysis caused by some excess of cationic charges located in the channel lumen formed by oligomeric alpha-helical wheels. The channel has a suggested diameter of 1.4 nm as judged from the permeability of different sized hydrated anions using the Renkin correction factor. Surprisingly, the genome of C. jeikeium contained only one gene coding for a cell wall channel of the PorA/PorH type found in other Corynebacterium species. The possible evolutionary relationship between the heterooligomeric channels formed by certain Corynebacterium strains and the homooligomeric pore of C. jeikeium is discussed.

The permeation of water soluble molecules across cell membranes is controlled by channel forming proteins and particularly the channel surface determines the selectivity. An adequate method to study properties of these channels is electrophysiology and in particular analyzing the ion current fluctuation in the presence of permeating solutes provides information on possible interactions with the channel surface. The temperature-dependent transport of the ionic liquid 1-butyl-3-methyl-imidazolium chloride (BMIM-Cl) in aqueous solution is studied theoretically and experimentally. Using molecular dynamics simulations and ion-conductance measurements, the transport is examined in bulk as well as through a biological nanopore, OmpF and its mutant D113A. This investigation is motivated by the observation that aqueous solutions of BMIM-Cl drastically reduce the translocation speed of DNA or antibiotics through nanopores in electrophysiological measurements. This makes BMIM-Cl an interesting alternative salt to improve the time resolution. In line with previous investigations of simple salts, the size of the ions and their orientation adds another important degree of freedom to the ion transport, thereby slowing the transport through nanopores. An excellent agreement between theory and conductance measurements is obtained for wild type OmpF and a reasonable agreement for the mutant. Moreover, all-atom simulations allow an atomistic analysis revealing molecular details of the rate-limiting ion interactions with the channel. [1] Mahendran KR et al, J. Phys: Condens. Matter 22 (2010) 454131. [2] Niraj Modi, Pratik Raj Singh et al, J. Phys. Chem. Lett. 2 (2011) 2331-36.

The temperature-dependent transport of the ionic liquid 1-butyl-3-methylimidazolium chloride (BMIM-Cl) in aqueous solution is studied theoretically and experimentally. Using molecular dynamics simulations and ion-conductance measurements, the transport is examined in bulk as well as through a biological nanopore, that is, OmpF and its mutant D113A. This investigation is motivated by the observation that aqueous solutions of BMIM-Cl drastically reduce the translocation speed of DNA or antibiotics through nanopores in electrophysiological measurements. This makes BMIM-Cl an interesting alternative salt to improve the time resolution. In line with previous investigations of simple salts, the size of the ions and their orientation adds another important degree of freedom to the ion transport, thereby slowing the transport through nanopores. An excellent agreement between theory and conductance measurements is obtained for wild type OmpF and a reasonable agreement for the mutant. Moreover, all-atom simulations allow an atomistic analysis revealing molecular details of the rate-limiting ion interactions with the channel.

The tripartite efflux pump AcrAB-TolC is responsible for the intrinsic and acquired multidrug resistance in Escherichia coli. Its active part, the homotrimeric transporter AcrB, is in charge of the selective binding of substrates and energy transduction. The mutation F610A has been shown to significantly reduce the minimum inhibitory concentration of doxorubicin and many other substrates, although F610 does not appear to interact strongly with them. Biochemical study of transport kinetics in AcrB is not yet possible, except for some β-lactams, and other techniques should supply this important information. Therefore, in this work, we assess the impact of the F610A mutation on the functionality of AcrB by means of computational techniques, using doxorubicin as substrate. We found that the compound slides deeply inside the binding pocket after mutation, increasing the strength of the interaction. During subsequent conformational alterations of the transporter, doxorubicin was either not extruded from the binding site or displaced along a direction other than the one associated with extrusion. Our study indicates how subtle interactions determine the functionality of multidrug transporters, since decreased transport might not be simplistically correlated to decreased substrate binding affinity.

The major efflux system in Escherichia coli is the tripartite complex AcrAB-TolC. Its homotrimeric transporter AcrB is polyspecific and extrudes antibiotics out of the bacterium. This extrusion is performed via a functional rotation, in which each monomer assumes a particular conformation. In the present study, targeted molecular dynamics simulations have been employed to obtain a molecular level understanding of the transport process. A particular focus is put on the role of water molecules in this extrusion process. It is shown that the water flows from the binding pocket toward the exit gate in the extrusion step and helps the substrate to move along this path. These results are underpinned by a detailed analysis of the electrostatic interaction energy. Furthermore, the role of water for the polyspecificity of the transporter is discussed.

Bacteria, such as E. coli, use multidrug efflux pumps to export toxic substrates through their cell membranes, including antibiotics. The RND transporter of the AcrAB-TolC efflux pump is able to export structurally and chemically different substrates via a functional rotation. The three major states of this rotation cycle were found in several asymmetric crystal structures. After initially analyzing the basic mechanisms of opening of the TolC channel [1] and of substrate extrusion by AcrB [2] separately, we have continued the analysis of the latter one.

The tripartite complex AcrAB-TolC is the major efflux system in Escherichia coli. It extrudes a wide spectrum of noxious compounds out of the bacterium, including many antibiotics. Its active part, the homotrimeric transporter AcrB, is responsible for the selective binding of substrates and energy transduction. Based on available crystal structures and biochemical data, the transport of substrates by AcrB has been proposed to take place via a functional rotation, in which each monomer assumes a particular conformation. However, there is no molecular-level description of the conformational changes associated with the rotation and their connection to drug extrusion. To obtain insights thereon, we have performed extensive targeted molecular dynamics simulations mimicking the functional rotation of AcrB containing doxorubicin, one of the two substrates that were co-crystallized so far. The simulations, including almost half a million atoms, have been used to test several hypotheses concerning the structure-dynamics-function relationship of this transporter. Our results indicate that, upon induction of conformational changes, the substrate detaches from the binding pocket and approaches the gate to the central funnel. Furthermore, we provide strong evidence for the proposed peristaltic transport involving a zipper-like closure of the binding pocket, responsible for the displacement of the drug. A concerted opening of the channel between the binding pocket and the gate further favors the displacement of the drug. This microscopically well-funded information allows one to identify the role of specific amino acids during the transitions and to shed light on the functioning of AcrB.

Bacteria, such as Escherichia coli, use multidrug efflux pumps to export toxic substrates through their cell membranes. Upon formation of an efflux pump, the aperture of its outer membrane protein TolC opens and thereby enables the extrusion of substrate molecules. The specialty of TolC is its ability to dock to different transporters, making it a highly versatile export protein. Within this study, the transition between two conformations of TolC that are both available as crystal structures was investigated using all-atom molecular dynamics simulations. To create a partially open conformation from a closed one, the stability of the periplasmic aperture was weakened by a double point mutation at the constricting ring, which removes some salt bridges and hydrogen bonds. These mutants, which showed partial opening in previous experiments, did not spontaneously open during a 20-ns equilibration at physiological values of the KCl solution. Detailed analysis of the constricting ring revealed that the cations of the solvent were able to constitute ionic bonds in place of the removed salt bridges, which inhibited the opening of the aperture in simulations. To remove the ions from these binding positions within the available simulation time, an extra force was applied onto the ions. To keep the effect of this additional force rather flexible, it was applied in form of an artificial external electric field perpendicular to the membrane. Depending on the field direction and the ion concentration, these simulations led to a partial opening. In experiments, this energy barrier for the ions can be overcome by thermal fluctuations on a longer timescale.

Bacteria use multidrug efflux pumps to extrude toxic substrates through their cell membranes. The RND transporters of the AcrAB-TolC (E.Coli) and MexAB-OprM (P.Aeruginosa) systems are able to export structurally and chemically different substrates, being responsible of multidrug resistance. While the energy conversion takes place in the transmembrane domain of AcrB and MexB, the energy is transducted towards the periplasmic part and used there to initiate what is believed to be a three-cyclic peristaltic pumping. Using different computational methods like adaptive bias force (ABF) and targeted molecular dynamics (TMD), we have investigated the mechanism of substrate uptake and pumping. With ABF we have studied the passage of antibiotics from the periplasm and protein-lipid interface into the inner pore of the pump, while TMD has been used to assess the effect of conformational changes on the extrusion of drugs (located into one of the proposed binding pockets). Finally, analysis of water distribution in the transmembrane region represents an important step to identify features of the energy transduction process. Comparison between the active pumps AcrB and MexB (which show different resistance patterns despite their homology) provide insights into the microscopic details of their functioning.

Bacteria, such as E. coli, use multidrug efflux pumps to export toxic substrates through their cell membranes. Upon formation of the AcrAB-TolC efflux pump, the aperture of its outer membrane protein TolC opens and thereby enables the extrusion of substrate molecules. Within this study, TolC was investigated using all-atom molecular dynamics simulations in a system of approx. 200 000 atoms. The stability of the periplasmic aperture was examined by applying double point mutations at the constricting ring which remove some salt bridges and hydrogen bonds. These mutants, which showed partial opening in experiments, did not spontaneously open during a 20 ns equilibration at physiological values of the KCl solution. Detailed analysis of the constricting ring revealed that the cations of the solvent were able to constitute ionic bonds in place of the removed salt bridges, which inhibited the opening of the aperture. To remove the ions from their binding sites, an external force in the form of an electric field was applied. Depending on the direction of the field, the ions were removed from their binding sites and the mutated TolC partially opened in the simulations.