Arthur Neuberger

Crystal structure of tripartite-type ABC transporter MacB from Acinetobacter baumannii.

The MacA–MacB–TolC tripartite complex is a transmembrane machine that spans both plasma membrane and outer membrane and actively extrudes substrates, including macrolide antibiotics, virulence factors, peptides and cell envelope precursors. These transport activities are driven by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. Here, we present the crystal structure of MacB at 3.4-Å resolution. MacB forms a dimer in which each protomer contains a nucleotide-binding domain and four transmembrane helices that protrude in the periplasm into a binding domain for interaction with the membrane fusion protein MacA. MacB represents an ABC transporter in pathogenic microorganisms with unique structural features.The tripartite multidrug efflux pump MacA-MacB-TolC in Gram-negative bacterial pathogens is driven by the ATPase MacB, which belongs to the ATP-binding cassette (ABC) superfamily. Here the authors present the 3.4 Å resolution crystal structure of MacB, and compare it with other known ABC transporter structures.

Relocation of active site carboxylates in major facilitator superfamily multidrug transporter LmrP reveals plasticity in proton interactions

The expression of polyspecific membrane transporters is one important mechanism by which cells can obtain resistance to structurally different antibiotics and cytotoxic agents. These transporters reduce intracellular drug concentrations to subtoxic levels by mediating drug efflux across the cell envelope. The major facilitator superfamily multidrug transporter LmrP from Lactococcus lactis catalyses drug efflux in a membrane potential and chemical proton gradient-dependent fashion. To enable the interaction with protons and cationic substrates, LmrP contains catalytic carboxyl residues on the surface of a large interior chamber that is formed by transmembrane helices. These residues co-localise together with polar and aromatic residues, and are predicted to be present in two clusters. To investigate the functional role of the catalytic carboxylates, we generated mutant proteins catalysing membrane potential-independent dye efflux by removing one of the carboxyl residues in Cluster 1. We then relocated this carboxyl residue to six positions on the surface of the interior chamber, and tested for restoration of wildtype energetics. The reinsertion at positions towards Cluster 2 reinstated the membrane potential dependence of dye efflux. Our data uncover a remarkable plasticity in proton interactions in LmrP, which is a consequence of the flexibility in the location of key residues that are responsible for proton/multidrug antiport.

Hoechst 33342 Is a Hidden "Janus" amongst Substrates for the Multidrug Efflux Pump LmrP.

Multidrug transporters mediate the active extrusion of antibiotics and toxic ions from the cell. This reaction is thought to be based on a switch of the transporter between two conformational states, one in which the interior substrate binding cavity is available for substrate binding at the inside of the cell, and another in which the cavity is exposed to the outside of the cell to enable substrate release. Consistent with this model, cysteine cross-linking studies with the Major Facilitator Superfamily drug/proton antiporter LmrP from Lactococcus lactis demonstrated binding of transported benzalkonium to LmrP in its inward-facing state. The fluorescent dye Hoechst 33342 is a substrate for many multidrug transporters and is extruded by efflux pumps in microbial and mammalian cells. Surprisingly, and in contrast to other multidrug transporters, LmrP was found to actively accumulate, rather than extrude, Hoechst 33342 in lactococcal cells. Consistent with this observation, LmrP expression was associated with cellular sensitivity, rather than resistance to Hoechst 33342. Thus, we discovered a hidden “Janus” amongst LmrP substrates that is translocated in reverse direction across the membrane by binding to outward-facing LmrP followed by release from inward-facing LmrP. These findings are in agreement with distance measurements by electron paramagnetic resonance in which Hoechst 33342 binding was found to stabilize LmrP in its outward-facing conformation. Our data have important implications for the use of multidrug exporters in selective targeting of “Hoechst 33342-like” drugs to cells and tissues in which these transporters are expressed.

Multidrug efflux pumps: structure, function and regulation

Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this Review, we describe recent advances that have increased our understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria. Clinical and laboratory data indicate that efflux pumps function not only in the drug extrusion process but also in virulence and the adaptive responses that contribute to antimicrobial resistance during infection. The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities.One factor contributing to the emergence of antimicrobial resistance is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. This Review describes recent insights into the structure, function and regulation of efflux pumps.

Structure and mechanism of bacterial tripartite efflux pumps

Efflux pumps are membrane proteins which contribute to multi-drug resistance. In Gram-negative bacteria, some of these pumps form complex tripartite assemblies in association with an outer membrane channel and a periplasmic membrane fusion protein. These tripartite machineries span both membranes and the periplasmic space, and they extrude from the bacterium chemically diverse toxic substrates. In this chapter, we summarise current understanding of the structural architecture, functionality, and regulation of tripartite multi-drug efflux assemblies.

Author Correction: Multidrug efflux pumps: structure, function and regulation

In the version of this Review originally published, the author contributions of co-author Arthur Neuberger were incorrectly listed. The author contributions should have appeared as ‘D.D., X.W.-K., A.N., H.W.v.V., K.M.P., L.J.V.P. and B.F.L. researched data for the article, made substantial contributions to discussions of the content, wrote the article, and reviewed and edited the manuscript before submission’. This has now been corrected in all versions of the Review. The authors apologize to readers for this error.

IntelliEppi: Intelligent reaction monitoring and holistic data management system for the molecular biology lab

Daily alterations of routines and protocols create high, yet so far unmet demands for intelligent reaction monitoring, quality control and data management in molecular biology laboratories. To meet such needs, the “internet of things” is implemented here. We propose an approach which combines direct tracking of lab tubes, reactions and racks with a comprehensive data management system. Reagent tubes in this system are tagged with 2D data matrices or imprinted RFID-chips using a unique identification number. For each tube, individual content and all relevant information based on conducted experimental procedures are stored in an experimental data management system. This information is managed automatically but allow scientists to engage and interfere via user-friendly graphical interface. Tagged tubes are used in connection with a detectable RFID-tagged rack. We show that reaction protocols, HTS storage and complex reactions are easily planned and controlled.

Research data supporting “Crystal structure of tripartite-type ABC transporter MacB from Acinetobacter baumannii”

The MacA–MacB–TolC tripartite complex is a transmembrane machine that spans both plasma membrane and outer membrane and actively extrudes substrates, including macrolide antibiotics, virulence factors, peptides and cell envelope precursors. These transport activities are driven by the ATPase MacB, a member of the ATP-Binding Cassette (ABC) superfamily. Here, we present the crystal structure of MacB at 3.4 A resolution. MacB forms a dimer in which each protomer contains a nucleotide binding domain and four transmembrane helices that protrude in the periplasm into a binding domain for interaction with the membrane fusion protein MacA. MacB represents an ABC transporter in pathogenic microorganisms with unique structural features. The data sets included in this repository concern results of cell growth experiments in Fig. 1b and Table 2, and the raw immuno blot data for Supplementary Figure 14.

Lemons, or Squeezed for Resources? Information Symmetry and Asymmetric Resources in Biotechnology

Thousands of biotech companies are developing promising products, but have insufficient resources to complete the clinical testing process, while large, well-funded companies have increasingly focused on the need to access external innovation. As a result, licensing deals are an essential and growing part of this industry. Yet, casting a shadow over the licensing market is the classic Lemons Problem: Does asymmetrical information put licensees at a severe disadvantage, leading to a market dominated by inferior opportunities, with the best products retained for internal development? Our analysis of clinical stage products developed over three decades shows that there is no Lemons Problem. We discuss the results of this first apples-to-apples analysis of the biomedical licensing market, and suggest reasons why the Lemons Problem does not exist where it might be most expected—in a high technology, knowledge-based industry.

Research data supporting “Relocation of active site carboxylates in major facilitator superfamily multidrug transporter LmrP reveals plasticity in proton interactions”

The expression of polyspecific membrane transporters is one important mechanism by which cells can obtain resistance to structurally different antibiotics and cytotoxic agents. These transporters reduce intracellular drug concentrations to subtoxic levels by mediating drug efflux across the cell envelope. The major facilitator superfamily multidrug transporter LmrP from Lactococcus lactis catalyses drug efflux in a membrane potential and chemical proton gradient-dependent fashion. To enable the interaction with protons and cationic substrates, LmrP contains catalytic carboxyl residues on the surface of a large interior chamber that is formed by transmembrane helices. These residues co-localise together with polar and aromatic residues, and are predicted to be present in two clusters. To investigate the functional role of the catalytic carboxylates, we generated mutant proteins catalysing membrane potential-independent dye efflux by removing one of the carboxyl residues in Cluster 1. We then relocated this carboxyl residue to six positions on the surface of the interior chamber, and tested for restoration of wildtype energetics. The reinsertion at positions towards Cluster 2 reinstated the membrane potential dependence of dye efflux. Our data uncover a remarkable plasticity in proton interactions in LmrP, which is a consequence of the flexibility in the location of key residues that are responsible for proton/multidrug antiport. This repository contains the data sets that were used to prepare the figures.