A Gram-negative-selective antibiotic that spares the gut microbiome.

Infections caused by Gram-negative pathogens are increasingly prevalent and are typically treated with broad-spectrum antibiotics, resulting in disruption of the gut microbiome and susceptibility to secondary infections1-3. There is a critical need for antibiotics that are selective both for Gram-negative bacteria over Gram-positive bacteria, as well as for pathogenic bacteria over commensal bacteria. Here we report the design and discovery of lolamicin, a Gram-negative-specific antibiotic targeting the lipoprotein transport system. Lolamicin has activity against a panel of more than 130 multidrug-resistant clinical isolates, shows efficacy in multiple mouse models of acute pneumonia and septicaemia infection, and spares the gut microbiome in mice, preventing secondary infection with Clostridioides difficile. The selective killing of pathogenic Gram-negative bacteria by lolamicin is a consequence of low sequence homology for the target in pathogenic bacteria versus commensals; this doubly selective strategy can be a blueprint for the development of other microbiome-sparing antibiotics.

Differential dynamics and direct interaction of bound ligands with lipids in multidrug transporter ABCG2.

ABCG2 is an ATP-binding cassette (ABC) transporter that extrudes a wide range of xenobiotics and drugs from the cell and contributes to multidrug resistance in cancer cells. Following our recent structural characterization of topotecan-bound ABCG2, here, we present cryo-EM structures of ABCG2 under turnover conditions in complex with a special modulator and slow substrate, tariquidar, in nanodiscs. The structures reveal that similar to topotecan, tariquidar induces two distinct ABCG2 conformations under turnover conditions (turnover-1 and turnover-2). µs-scale molecular dynamics simulations of drug-bound and apo ABCG2 in native-like lipid bilayers, in both topotecan- and tariquidar-bound states, characterize the ligand size as a major determinant of its binding stability. The simulations highlight direct lipid-drug interactions for the smaller topotecan, which exhibits a highly dynamic binding mode. In contrast, the larger tariquidar occupies most of the available volume in the binding pocket, thus leaving little space for lipids to enter the cavity and interact with it. Similarly, when simulating ABCG2 in the apo inward-open state, we also observe spontaneous penetration of phospholipids into the binding cavity. The captured phospholipid diffusion pathway into ABCG2 offers a putative general path to recruit any hydrophobic/amphiphilic substrates directly from the membrane. Our simulations also reveal that ABCG2 rejects cholesterol as a substrate, which is omnipresent in plasma membranes that contain ABCG2. At the same time, cholesterol is found to prohibit the penetration of phospholipids into ABCG2. These molecular findings have direct functional ramifications on ABCG2's function as a transporter.

Serotonin transporter-ibogaine complexes illuminate mechanisms of inhibition and transport.

The serotonin transporter (SERT) regulates neurotransmitter homeostasis through the sodium- and chloride-dependent recycling of serotonin into presynaptic neurons1-3. Major depression and anxiety disorders are treated using selective serotonin reuptake inhibitors-small molecules that competitively block substrate binding and thereby prolong neurotransmitter action2,4. The dopamine and noradrenaline transporters, together with SERT, are members of the neurotransmitter sodium symporter (NSS) family. The transport activities of NSSs can be inhibited or modulated by cocaine and amphetamines2,3, and genetic variants of NSSs are associated with several neuropsychiatric disorders including attention deficit hyperactivity disorder, autism and bipolar disorder2,5. Studies of bacterial NSS homologues-including LeuT-have shown how their transmembrane helices (TMs) undergo conformational changes during the transport cycle, exposing a central binding site to either side of the membrane1,6-12. However, the conformational changes associated with transport in NSSs remain unknown. To elucidate structure-based mechanisms for transport in SERT we investigated its complexes with ibogaine, a hallucinogenic natural product with psychoactive and anti-addictive properties13,14. Notably, ibogaine is a non-competitive inhibitor of transport but displays competitive binding towards selective serotonin reuptake inhibitors15,16. Here we report cryo-electron microscopy structures of SERT-ibogaine complexes captured in outward-open, occluded and inward-open conformations. Ibogaine binds to the central binding site, and closure of the extracellular gate largely involves movements of TMs 1b and 6a. Opening of the intracellular gate involves a hinge-like movement of TM1a and the partial unwinding of TM5, which together create a permeation pathway that enables substrate and ion diffusion to the cytoplasm. These structures define the structural rearrangements that occur from the outward-open to inward-open conformations, and provide insight into the mechanism of neurotransmitter transport and ibogaine inhibition.

Tumor-acquired somatic mutation affects conformation to abolish ABCG2-mediated drug resistance.

ABCG2 is an important ATP-binding cassette transporter impacting the absorption and distribution of over 200 chemical toxins and drugs. ABCG2 also reduces the cellular accumulation of diverse chemotherapeutic agents. Acquired somatic mutations in the phylogenetically conserved amino acids of ABCG2 might provide unique insights into its molecular mechanisms of transport. Here, we identify a tumor-derived somatic mutation (Q393K) that occurs in a highly conserved amino acid across mammalian species. This ABCG2 mutant seems incapable of providing ABCG2-mediated drug resistance. This was perplexing because it is localized properly and retained interaction with substrates and nucleotides. Using a conformationally sensitive antibody, we show that this mutant appears "locked" in a non-functional conformation. Structural modeling and molecular dynamics simulations based on ABCG2 cryo-EM structures suggested that the Q393K interacts with the E446 to create a strong salt bridge. The salt bridge is proposed to stabilize the inward-facing conformation, resulting in an impaired transporter that lacks the flexibility to readily change conformation, thereby disrupting the necessary communication between substrate binding and transport.

Role of internal loop dynamics in antibiotic permeability of outer membrane porins.

Gram-negative bacteria pose a serious public health concern due to resistance to many antibiotics, caused by the low permeability of their outer membrane (OM). Effective antibiotics use porins in the OM to reach the interior of the cell; thus, understanding permeation properties of OM porins is instrumental to rationally develop broad-spectrum antibiotics. A functionally important feature of OM porins is undergoing open-closed transitions that modulate their transport properties. To characterize the molecular basis of these transitions, we performed an extensive set of molecular dynamics (MD) simulations of Escherichia coli OM porin OmpF. Markov-state analysis revealed that large-scale motion of an internal loop, L3, underlies the transition between energetically stable open and closed states. The conformation of L3 is controlled by H bonds between highly conserved acidic residues on the loop and basic residues on the OmpF ß-barrel. Mutation of key residues important for the loop's conformation shifts the equilibrium between open and closed states and regulates translocation of permeants (ions and antibiotics), as observed in the simulations and validated by our whole-cell accumulation assay. Notably, one mutant system G119D, which we find to favor the closed state, has been reported in clinically resistant bacterial strains. Overall, our accumulated ∼200 µs of simulation data (the wild type and mutants) along with experimental assays suggest the involvement of internal loop dynamics in permeability of OM porins and antibiotic resistance in Gram-negative bacteria.

Computational Dissection of Membrane Transport at a Microscopic Level.

Membrane transporters are key gatekeeper proteins at cellular membranes that closely control the traffic of materials. Their function relies on structural rearrangements of varying degrees that facilitate substrate translocation across the membrane. Characterizing these functionally important molecular events at a microscopic level is key to our understanding of membrane transport, yet challenging to achieve experimentally. Recent advances in simulation technology and computing power have rendered molecular dynamics (MD) simulation a powerful biophysical tool to investigate a wide range of dynamical events spanning multiple spatial and temporal scales. Here, we review recent studies of diverse membrane transporters using computational methods, with an emphasis on highlighting the technical challenges, key lessons learned, and new opportunities to illuminate transporter structure and function.

The cellular membrane as a mediator for small molecule interaction with membrane proteins.

The cellular membrane constitutes the first element that encounters a wide variety of molecular species to which a cell might be exposed. Hosting a large number of structurally and functionally diverse proteins associated with this key metabolic compartment, the membrane not only directly controls the traffic of various molecules in and out of the cell, it also participates in such diverse and important processes as signal transduction and chemical processing of incoming molecular species. In this article, we present a number of cases where details of interaction of small molecular species such as drugs with the membrane, which are often experimentally inaccessible, have been studied using advanced molecular simulation techniques. We have selected systems in which partitioning of the small molecule with the membrane constitutes a key step for its final biological function, often binding to and interacting with a protein associated with the membrane. These examples demonstrate that membrane partitioning is not only important for the overall distribution of drugs and other small molecules into different compartments of the body, it may also play a key role in determining the efficiency and the mode of interaction of the drug with its target protein. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

Transient formation of water-conducting states in membrane transporters.

Membrane transporters rely on highly coordinated structural transitions between major conformational states for their function, to prevent simultaneous access of the substrate binding site to both sides of the membrane--a mode of operation known as the alternating access model. Although this mechanism successfully accounts for the efficient exchange of the primary substrate across the membrane, accruing evidence on significant water transport and even uncoupled ion transport mediated by transporters has challenged the concept of perfect mechanical coupling and coordination of the gating mechanism in transporters, which might be expected from the alternating access model. Here, we present a large set of extended equilibrium molecular dynamics simulations performed on several classes of membrane transporters in different conformational states, to test the presence of the phenomenon in diverse transporter classes and to investigate the underlying molecular mechanism of water transport through membrane transporters. The simulations reveal spontaneous formation of transient water-conducting (channel-like) states allowing passive water diffusion through the lumen of the transporters. These channel-like states are permeable to water but occluded to substrate, thereby not hindering the uphill transport of the primary substrate, i.e., the alternating access model remains applicable to the substrate. The rise of such water-conducting states during the large-scale structural transitions of the transporter protein is indicative of imperfections in the coordinated closing and opening motions of the cytoplasmic and extracellular gates. We propose that the observed water-conducting states likely represent a universal phenomenon in membrane transporters, which is consistent with their reliance on large-scale motion for function.

Conformational dynamics at the inner gate of KcsA during activation.

The potassium channel KcsA offers a unique opportunity to explicitly study the dynamics of the moving parts of ion channels, yet our understanding of the extent and dynamic behavior of the physiologically relevant structural changes at the inner gate in KcsA remains incomplete. Here, we use electron paramagnetic resonance, nuclear magnetic resonance, and molecular dynamics simulations to characterize the extent of pH-dependent conformational changes of the inner gate in lipid bilayers or detergent micelles. Our results show that under physiological conditions the inner gate experiences a maximal diagonal opening of ∼24 Šwith the largest degree of dynamics near the pKa of activation (pH ∼3.9). These results extend the observation that the C-terminus is necessary to limit the extent of opening and imply that the inner gate regulates the extent of conformational change at the zone of allosteric coupling and at the selectivity filter.

On the origin of large flexibility of P-glycoprotein in the inward-facing state.

P-glycoprotein (Pgp) is one of the most biomedically relevant transporters in the ATP binding cassette (ABC) superfamily due to its involvement in developing multidrug resistance in cancer cells. Employing molecular dynamics simulations and double electron-electron resonance spectroscopy, we have investigated the structural dynamics of membrane-bound Pgp in the inward-facing state and found that Pgp adopts an unexpectedly wide range of conformations, highlighted by the degree of separation between the two nucleotide-binding domains (NBDs). The distance between the two NBDs in the equilibrium simulations covers a range of at least 20 Å, including, both, more open and more closed NBD configurations than the crystal structure. The double electron-electron resonance measurements on spin-labeled Pgp mutants also show wide distributions covering both longer and shorter distances than those observed in the crystal structure. Based on structural and sequence analyses, we propose that the transmembrane domains of Pgp might be more flexible than other structurally known ABC exporters. The structural flexibility of Pgp demonstrated here is not only in close agreement with, but also helps rationalize, the reported high NBD fluctuations in several ABC exporters and possibly represents a fundamental difference in the transport mechanism between ABC exporters and ABC importers. In addition, during the simulations we have captured partial entrance of a lipid molecule from the bilayer into the lumen of Pgp, reaching the putative drug binding site. The location of the protruding lipid suggests a putative pathway for direct drug recruitment from the membrane.

Visualizing functional motions of membrane transporters with molecular dynamics simulations.

Computational modeling and molecular simulation techniques have become an integral part of modern molecular research. Various areas of molecular sciences continue to benefit from, indeed rely on, the unparalleled spatial and temporal resolutions offered by these technologies, to provide a more complete picture of the molecular problems at hand. Because of the continuous development of more efficient algorithms harvesting ever-expanding computational resources, and the emergence of more advanced and novel theories and methodologies, the scope of computational studies has expanded significantly over the past decade, now including much larger molecular systems and far more complex molecular phenomena. Among the various computer modeling techniques, the application of molecular dynamics (MD) simulation and related techniques has particularly drawn attention in biomolecular research, because of the ability of the method to describe the dynamical nature of the molecular systems and thereby to provide a more realistic representation, which is often needed for understanding fundamental molecular properties. The method has proven to be remarkably successful in capturing molecular events and structural transitions highly relevant to the function and/or physicochemical properties of biomolecular systems. Herein, after a brief introduction to the method of MD, we use a number of membrane transport proteins studied in our laboratory as examples to showcase the scope and applicability of the method and its power in characterizing molecular motions of various magnitudes and time scales that are involved in the function of this important class of membrane proteins.

Conformational coupling of the nucleotide-binding and the transmembrane domains in ABC transporters.

Basic architecture of ABC transporters includes two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). Although the transport process takes place in the TMDs, which provide the substrate translocation pathway across the cell membrane and control its accessibility between the two sides of the membrane, the energy required for the process is provided by conformational changes induced in the NBDs by binding and hydrolysis of ATP. Nucleotide-dependent conformational changes in the NBDs, therefore, need to be coupled to structural changes in the TMDs. Using molecular dynamics simulations, we have investigated the structural elements involved in the conformational coupling between the NBDs and the TMDs in the Escherichia coli maltose transporter, an ABC importer for which an intact structure is available both in inward-facing and outward-facing conformations. The prevailing model of coupling is primarily based on a single structural motif, known as the coupling helices, as the main structural element for the NBD-TMD coupling. Surprisingly, we find that in the absence of the NBDs the coupling helices can be conformationally decoupled from the rest of the TMDs, despite their covalent connection. That is, the structural integrity of the coupling helices and their tight coupling to the core of the TMDs rely on the contacts provided by the NBDs. Based on the conformational and dynamical analysis of the simulation trajectories, we propose that the core coupling elements in the maltose transporter involve contributions from several structural motifs located at the NBD-TMD interface, namely, the EAA loops from the TMDs, and the Q-loop and the ENI motifs from the NBDs. These three structural motifs in small ABC importers show a high degree of correlation in motion and mediate the necessary conformational coupling between the core of TMDs and the helical subdomains of NBDs. A comprehensive analysis of the structurally known ABC transporters shows a high degree of conservation of the identified 3-motif coupling elements only in the subfamily of small ABC importers, suggesting a distinct mode of NBD-TMD coupling from the other two major ABC transporter folds, namely large ABC importers and ABC exporters.

Structural basis of complex formation between mitochondrial anion channel VDAC1 and Hexokinase-II.

Complex formation between hexokinase-II (HKII) and the mitochondrial VDAC1 is crucial to cell growth and survival. We hypothesize that HKII first inserts into the outer membrane of mitochondria (OMM) and then interacts with VDAC1 on the cytosolic leaflet of OMM to form a binary complex. To systematically investigate this process, we devised a hybrid approach. First, we describe membrane binding of HKII with molecular dynamics (MD) simulations employing a membrane mimetic model with enhanced lipid diffusion capturing membrane insertion of its H-anchor. The insertion depth of the H-anchor was then used to derive positional restraints in subsequent millisecond-scale Brownian dynamics (BD) simulations to preserve the membrane-bound pose of HKII during the formation of the HKII/VDAC1 binary complex. Multiple BD-derived structural models for the complex were further refined and their structural stability probed with additional MD simulations, resulting in one stable complex. A major feature in the complex is the partial (not complete) blockade of VDAC1's permeation pathway, a result supported by our comparative electrophysiological measurements of the channel in the presence and absence of HKII. We also show how VDAC1 phosphorylation disrupts HKII binding, a feature that is verified by our electrophysiology recordings and has implications in mitochondria-mediated cell death.

Rationalizing the generation of broad spectrum antibiotics with the addition of a positive charge.

Antibiotic resistance of Gram-negative bacteria is largely attributed to the low permeability of their outer membrane (OM). Recently, we disclosed the eNTRy rules, a key lesson of which is that the introduction of a primary amine enhances OM permeation in certain contexts. To understand the molecular basis for this finding, we perform an extensive set of molecular dynamics (MD) simulations and free energy calculations comparing the permeation of aminated and amine-free antibiotic derivatives through the most abundant OM porin of E. coli, OmpF. To improve sampling of conformationally flexible drugs in MD simulations, we developed a novel, Monte Carlo and graph theory based algorithm to probe more efficiently the rotational and translational degrees of freedom visited during the permeation of the antibiotic molecule through OmpF. The resulting pathways were then used for free-energy calculations, revealing a lower barrier against the permeation of the aminated compound, substantiating its greater OM permeability. Further analysis revealed that the amine facilitates permeation by enabling the antibiotic to align its dipole to the luminal electric field of the porin and form favorable electrostatic interactions with specific, highly-conserved charged residues. The importance of these interactions in permeation was further validated with experimental mutagenesis and whole cell accumulation assays. Overall, this study provides insights on the importance of the primary amine for antibiotic permeation into Gram-negative pathogens that could help the design of future antibiotics. We also offer a new computational approach for calculating free-energy of processes where relevant molecular conformations cannot be efficiently captured.

Structural and functional diversity calls for a new classification of ABC transporters.

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs.

Structural Insights into the Lipid A Transport Pathway in MsbA.

MsbA is an essential ATP-binding cassette transporter in Gram-negative bacteria that transports lipid A and lipopolysaccharide from the cytoplasmic leaflet to the periplasmic leaflet of the inner membrane. Here we report the X-ray structure of MsbA from Salmonella typhimurium at 2.8-Å resolution in an inward-facing conformation after cocrystallization with lipid A and using a stabilizing facial amphiphile. The structure displays a large amplitude opening in the transmembrane portal, which is likely required for lipid A to pass from its site of synthesis into the protein-enclosed transport pathway. Putative lipid A density is observed further inside the transmembrane cavity, consistent with a trap and flip model. Additional electron density attributed to lipid A is observed near an outer surface cleft at the periplasmic ends of the transmembrane helices. These findings provide new structural insights into the lipid A transport pathway through comparative analysis with existing MsbA structures.

Dimer opening of the nucleotide binding domains of ABC transporters after ATP hydrolysis.

ABC transporters constitute one of the most abundant membrane transporter families. The most common feature shared in the family is the highly conserved nucleotide binding domains (NBDs) that drive the transport process through binding and hydrolysis of ATP. Molecular dynamics simulations are used to investigate the effect of ATP hydrolysis in the NBDs. Starting with the ATP-bound, closed dimer of MalK, four simulation systems with all possible combinations of ATP or ADP-P(i) bound to the two nucleotide binding sites are constructed and simulated with equilibrium molecular dynamics for approximately 70 ns each. The results suggest that the closed form of the NBD dimer can only be maintained with two bound ATP molecules; in other words, hydrolysis of one ATP can lead to the opening of the dimer interface of the NBD dimer. Furthermore, we observed that the opening is an immediate effect of hydrolysis of ATP into ADP and P(i) rather than the dissociation of hydrolysis products. In addition, the opening is mechanistically triggered by the dissociation of the LSGGQ motif from the bound nucleotide. A metastable ADP-P(i) bound conformational state is consistently observed before the dimer opening in all the simulation systems.

Probing key elements of teixobactin-lipid II interactions in membranes.

Teixobactin (Txb) is a recently discovered antibiotic against Gram-positive bacteria that induces no detectable resistance. The bactericidal mechanism is believed to be the inhibition of cell wall biosynthesis by Txb binding to lipid II and lipid III. Txb binding specificity likely arises from targeting of the shared lipid component, the pyrophosphate moiety. Despite synthesis and functional assessment of numerous chemical analogs of Txb, and consequent identification of the Txb pharmacophore, the detailed structural information of Txb-substrate binding is still lacking. Here, we use molecular modeling and microsecond-scale molecular dynamics simulations to capture the formation of Txb-lipid II complexes at a membrane surface. Two dominant binding conformations were observed, both showing characteristic lipid II phosphate binding by the Txb backbone amides near the C-terminal cyclodepsipeptide (d-Thr8-Ile11) ring. Additionally, binding by Txb also involved the side chain hydroxyl group of Ser7, as well as a secondary phosphate binding provided by the side chain of l-allo-enduracididine. Interestingly, those conformations differ by swapping two groups of hydrogen bond donors that coordinate the two phosphate moieties of lipid II, resulting in opposite orientations of lipid II binding. In addition, residues d-allo-Ile5 and Ile6 serve as the membrane anchors in both Txb conformations, regardless of the detailed phosphate binding interactions near the cyclodepsipeptide ring. The role of hydrophobic residues in Txb activity is primarily for its membrane insertion, and subsidiarily to provide non-polar interactions with the lipid II tail. Based on the Txb-lipid II interactions captured in their complexes, as well as their partitioning depths into the membrane, we propose that the bactericidal mechanism of Txb is to arrest cell wall synthesis by selectively inhibiting the transglycosylation of peptidoglycan, while possibly leaving the transpeptidation step unaffected. The observed "pyrophosphate caging" mechanism of lipid II inhibition appears to be similar to some lantibiotics, but different from that of vancomycin or bacitracin.

Microscopic view of lipids and their diverse biological functions.

Biological membranes and their diverse lipid constituents play key roles in a broad spectrum of cellular and physiological processes. Characterization of membrane-associated phenomena at a microscopic level is therefore essential to our fundamental understanding of such processes. Due to the semi-fluid and dynamic nature of lipid bilayers, and their complex compositions, detailed characterization of biological membranes at an atomic scale has been refractory to experimental approaches. Computational modeling and simulation offer a highly complementary toolset with sufficient spatial and temporal resolutions to fill this gap. Here, we review recent molecular dynamics studies focusing on the diversity of lipid composition of biological membranes, or aiming at the characterization of lipid-protein interaction, with the overall goal of dissecting how lipids impact biological roles of the cellular membranes.

Mitochondrial VDAC1: A Key Gatekeeper as Potential Therapeutic Target.

Mitochondria are the key source of ATP that fuels cellular functions, and they are also central in cellular signaling, cell division and apoptosis. Dysfunction of mitochondria has been implicated in a wide range of diseases, including neurodegenerative and cardiac diseases, and various types of cancer. One of the key proteins that regulate mitochondrial function is the voltage-dependent anion channel 1 (VDAC1), the most abundant protein on the outer membrane of mitochondria. VDAC1 is the gatekeeper for the passages of metabolites, nucleotides, and ions; it plays a crucial role in regulating apoptosis due to its interaction with apoptotic and anti-apoptotic proteins, namely members of the Bcl-2 family of proteins and hexokinase. Therefore, regulation of VDAC1 is crucial not only for metabolic functions of mitochondria, but also for cell survival. In fact, multiple lines of evidence have confirmed the involvement of VDAC1 in several diseases. Consequently, modulation or dysregulation of VDAC1 function can potentially attenuate or exacerbate pathophysiological conditions. Understanding the role of VDAC1 in health and disease could lead to selective protection of cells in different tissues and diverse diseases. The purpose of this review is to discuss the role of VDAC1 in the pathogenesis of diseases and as a potentially effective target for therapeutic management of various pathologies.