P-glycoprotein (P-gp) detoxifies cells by exporting hundreds of chemically unrelated toxins but has been implicated in multidrug resistance (MDR) in the treatment of cancers. Substrate promiscuity is a hallmark of P-gp activity, thus a structural description of poly-specific drug-binding is important for the rational design of anticancer drugs and MDR inhibitors. The x-ray structure of apo P-gp at 3.8 angstroms reveals an internal cavity of ∼6000 angstroms cubed with a 30 angstrom separation of the two nucleotide-binding domains. Two additional P-gp structures with cyclic peptide inhibitors demonstrate distinct drug-binding sites in the internal cavity capable of stereoselectivity that is based on hydrophobic and aromatic interactions. Apo and drug-bound P-gp structures have portals open to the cytoplasm and the inner leaflet of the lipid bilayer for drug entry. The inward-facing conformation represents an initial stage of the transport cycle that is competent for drug binding.

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Structure of P-glycoprotein reveals a molecular basis for poly-specific drug binding.

Summary: High-affinity iron acquisition is mediated by siderophore-dependent pathways in the majority of pathogenic and nonpathogenic bacteria and fungi. Considerable progress has been made in characterizing and understanding mechanisms of siderophore synthesis, secretion, iron scavenging, and siderophore-delivered iron uptake and its release. The regulation of siderophore pathways reveals multilayer networks at the transcriptional and posttranscriptional levels. Due to the key role of many siderophores during virulence, coevolution led to sophisticated strategies of siderophore neutralization by mammals and (re)utilization by bacterial pathogens. Surprisingly, hosts also developed essential siderophore-based iron delivery and cell conversion pathways, which are of interest for diagnostic and therapeutic studies. In the last decades, natural and synthetic compounds have gained attention as potential therapeutics for iron-dependent treatment of infections and further diseases. Promising results for pathogen inhibition were obtained with various siderophore-antibiotic conjugates acting as “Trojan horse” toxins and siderophore pathway inhibitors. In this article, general aspects of siderophore-mediated iron acquisition, recent findings regarding iron-related pathogen-host interactions, and current strategies for iron-dependent pathogen control will be reviewed. Further concepts including the inhibition of novel siderophore pathway targets are discussed.

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Siderophore-Based Iron Acquisition and Pathogen Control

Multidrug transporters of the ABC family facilitate the export of diverse cytotoxic drugs across cell membranes. This is clinically relevant, as tumour cells may become resistant to agents used in chemotherapy. To understand the molecular basis of this process, we have determined the 3.0 A crystal structure of a bacterial ABC transporter (Sav1866) from Staphylococcus aureus. The homodimeric protein consists of 12 transmembrane helices in an arrangement that is consistent with cross-linking studies and electron microscopic imaging of the human multidrug resistance protein MDR1, but critically different from that reported for the bacterial lipid flippase MsbA. The observed, outward-facing conformation reflects the ATP-bound state, with the two nucleotide-binding domains in close contact and the two transmembrane domains forming a central cavity—presumably the drug translocation pathway—that is shielded from the inner leaflet of the lipid bilayer and from the cytoplasm, but exposed to the outer leaflet and the extracellular space. Multidrug efflux transporters cause serious problems in cancer chemotherapy and in the treatment of bacterial infections. A puzzling aspect of their biology is how a single transporter can recognize and transport such a wide variety of structurally dissimilar compounds. The publication of the crystal structures of two quite different multidrug efflux transporters will help to solve the mystery. In the first study, the structure of AcrB — a multidrug efflux transporter from E. coli — was determined. Its three constituent subunits were captured at different steps in the transport cycle: prior to substrate binding, substrate-bound, and post-extrusion. The voluminous multidrug binding pocket handles multiple substrates via multi-site binding. The second study determined the structure of an ATP-driven multidrug transporter from S. aureus. The clinical relevance of this 'ABC' family of transporters derives from the fact that they catalyse the extrusion of various cytotoxic compounds used in cancer therapy. The structure, with the transporter in the outward-facing conformation, is a useful model of human homologues and may initiate the rational design of drugs aimed at interfering with the extrusion of agents used in chemotherapy.

Structure of a bacterial multidrug ABC transporter

Summary: ATP-binding cassette (ABC) systems are universally distributed among living organisms and function in many different aspects of bacterial physiology. ABC transporters are best known for their role in the import of essential nutrients and the export of toxic molecules, but they can also mediate the transport of many other physiological substrates. In a classical transport reaction, two highly conserved ATP-binding domains or subunits couple the binding/hydrolysis of ATP to the translocation of particular substrates across the membrane, through interactions with membrane-spanning domains of the transporter. Variations on this basic theme involve soluble ABC ATP-binding proteins that couple ATP hydrolysis to nontransport processes, such as DNA repair and gene expression regulation. Insights into the structure, function, and mechanism of action of bacterial ABC proteins are reported, based on phylogenetic comparisons as well as classic biochemical and genetic approaches. The availability of an increasing number of high-resolution structures has provided a valuable framework for interpretation of recent studies, and realistic models have been proposed to explain how these fascinating molecular machines use complex dynamic processes to fulfill their numerous biological functions. These advances are also important for elucidating the mechanism of action of eukaryotic ABC proteins, because functional defects in many of them are responsible for severe human inherited diseases.

Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems

The lipid A moiety of lipopolysaccharide forms the outer monolayer of the outer membrane of most gram-negative bacteria. Escherichia coli lipid A is synthesized on the cytoplasmic surface of the inner membrane by a conserved pathway of nine constitutive enzymes. Following attachment of the core oligosaccharide, nascent core-lipid A is flipped to the outer surface of the inner membrane by the ABC transporter MsbA, where the O-antigen polymer is attached. Diverse covalent modifications of the lipid A moiety may occur during its transit from the outer surface of the inner membrane to the outer membrane. Lipid A modification enzymes are reporters for lipopolysaccharide trafficking within the bacterial envelope. Modification systems are variable and often regulated by environmental conditions. Although not required for growth, the modification enzymes modulate virulence of some gram-negative pathogens. Heterologous expression of lipid A modification enzymes may enable the development of new vaccines.

Lipid a modification systems in gram-negative bacteria

ATP-binding cassette (ABC) transporters are integral membrane proteins that translocate a wide variety of substrates across cellular membranes and are conserved from bacteria to humans. Here we compare four x-ray structures of the bacterial ABC lipid flippase, MsbA, trapped in different conformations, two nucleotide-bound structures and two in the absence of nucleotide. Comparison of the nucleotide-free conformations of MsbA reveals a flexible hinge formed by extracellular loops 2 and 3. This hinge allows the nucleotide-binding domains to disassociate while the ATP-binding half sites remain facing each other. The binding of the nucleotide causes a packing rearrangement of the transmembrane helices and changes the accessibility of the transporter from cytoplasmic (inward) facing to extracellular (outward) facing. The inward and outward openings are mediated by two different sets of transmembrane helix interactions. Altogether, the conformational changes between these structures suggest that large ranges of motion may be required for substrate transport.

https://www.pnas.org/content/pnas/104/48/19005.full.pdf

Flexibility in the ABC transporter MsbA: Alternating access with a twist

Select members of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter family couple ATP binding and hydrolysis to substrate efflux and confer multidrug resistance. We have determined the x-ray structure of MsbA in complex with magnesium, adenosine diphosphate, and inorganic vanadate (Mg·ADP·Vi) and the rough-chemotype lipopolysaccharide, Ra LPS. The structure supports a model involving a rigid-body torque of the two transmembrane domains during ATP hydrolysis and suggests a mechanism by which the nucleotide-binding domain communicates with the transmembrane domain. We propose a lipid “flip-flop” mechanism in which the sugar groups are sequestered in the chamber while the hydrophobic tails are dragged through the lipid bilayer.

https://science.sciencemag.org/content/sci/308/5724/1028.full.pdf

Structure of the ABC transporter MsbA in complex with ADP.vanadate and lipopolysaccharide.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.febslet.2005.11.033

The structures of MsbA: Insight into ABC transporter-mediated multidrug efflux

The ABC transporter MsbA is an integral membrane protein involved in the transport of lipid A and lipopolysaccharides to the outer leaflet of the inner membrane in bacteria. Here, the critical role of the natural substrate lipopolysaccharide in the crystallization and diffraction quality of MsbA crystals is reported. Initial crystals grown in complex with ATP–vanadate alone diffracted to ∼9 A. Screening of the natural substrate lipopolysaccharides led to the crystallization of MsbA in complex with ADP–vanadate and Ra lipopolysaccharide. The increased order within the crystal lattice allowed structure determination to 4.2 A.

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Lipopolysaccharide stabilizes the crystal packing of the ABC transporter MsbA

The invention relates to the three-dimensional structure of a crystal of a transporter protein complexed with a ligand. The three-dimensional structure of the transporter-ligand complex is disclosed. The transporter-ligand crystal structure, wherein the ligand is an inhibitor molecule, is useful for providing structural information that may be integrated into drug screening and drug design processes. Thus, the invention also relates to methods for utilizing the crystal structure of the transporter-ligand complex for identifying, designing, selecting, or testing inhibitors of the transporter protein, such inhibitors being useful as therapeutics for the treatment or modulation of i) diseases; ii) disease symptoms; or iii) the effect of other physiological events mediated by the transporter.

Christopher L. Reyes

Papers

Flexibility in the ABC transporter MsbA: Alternating access with a twist

Structure of the ABC transporter MsbA in complex with ADP.vanadate and lipopolysaccharide.

The structures of MsbA: Insight into ABC transporter-mediated multidrug efflux

Lipopolysaccharide stabilizes the crystal packing of the ABC transporter MsbA

Crystal of a transporter-ligand complex and methods of use