Discovery of Inhibitors of the Lipopolysaccharide Transporter MsbA: From a Screening Hit to Potent Wild-Type Gram-Negative Activity.

The dramatic increase in the prevalence of multi-drug resistant Gram-negative bacterial infections and the simultaneous lack of new classes of antibiotics is projected to result in approximately 10 million deaths per year by 2050. We report on efforts to target the Gram-negative ATP-binding cassette (ABC) transporter MsbA, an essential inner membrane protein that transports lipopolysaccharide from the inner leaflet to the periplasmic face of the inner membrane. We demonstrate the improvement of a high throughput screening hit into compounds with on-target single digit micromolar (µM) minimum inhibitory concentrations against wild-type uropathogenic Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae. A 2.98 Å resolution X-ray crystal structure of MsbA complexed with an inhibitor revealed a novel mechanism for inhibition of an ABC transporter. The identification of a fully encapsulated membrane binding site in Gram-negative bacteria led to unique physicochemical property requirements for wild-type activity.

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.

Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab.

Cluster of differentiation 20 (CD20) is a B cell membrane protein that is targeted by monoclonal antibodies for the treatment of malignancies and autoimmune disorders but whose structure and function are unknown. Rituximab (RTX) has been in clinical use for two decades, but how it activates complement to kill B cells remains poorly understood. We obtained a structure of CD20 in complex with RTX, revealing CD20 as a compact double-barrel dimer bound by two RTX antigen-binding fragments (Fabs), each of which engages a composite epitope and an extensive homotypic Fab:Fab interface. Our data suggest that RTX cross-links CD20 into circular assemblies and lead to a structural model for complement recruitment. Our results further highlight the potential relevance of homotypic Fab:Fab interactions in targeting oligomeric cell-surface markers.

Development of Photocrosslinking Probes Based on Huwentoxin-IV to Map the Site of Interaction on Nav1.7.

Voltage-gated sodium (Nav) channels respond to changes in the membrane potential of excitable cells through the concerted action of four voltage-sensor domains (VSDs). Subtype Nav1.7 plays an important role in the propagation of signals in pain-sensing neurons and is a target for the clinical development of novel analgesics. Certain inhibitory cystine knot (ICK) peptides produced by venomous animals potently modulate Nav1.7; however, the molecular mechanisms underlying their selective binding and activity remain elusive. This study reports on the design of a library of photoprobes based on the potent spider toxin Huwentoxin-IV and the determination of the toxin binding interface on VSD2 of Nav1.7 through a photocrosslinking and tandem mass spectrometry approach. Our Huwentoxin-IV probes selectively crosslink to extracellular loop S1-S2 and helix S3 of VSD2 in a chimeric channel system. Our results provide a strategy that will enable mapping of sites of interaction of other ICK peptides on Nav channels.

A Flexible and Scalable High-Throughput Platform for Recombinant Membrane Protein Production.

Integral membrane proteins (MP) are implicated in many disease processes and are the primary targets of numerous marketed drugs. Despite recent advances in the areas of MP solubilization, stabilization, and reconstitution, it remains a time-consuming task to identify the combination of constructs and purification conditions that will enable MP structure-function studies outside of the lipid bilayer. In this chapter, we describe a strategy for rapidly identifying and optimizing the solubilization and purification conditions for nearly any recombinant MP, based on the use of a noninvasive fluorescent probe (His-Glow) that specifically binds to the common hexahistidine affinity tag of expressed targets. This His-Glow approach permits fluorescent size-exclusion chromatography (FSEC) without the need for green fluorescent protein (GFP) fusion. A two-stage detergent screening strategy is employed at the solubilization stage, whereby appropriate detergent families are identified first, followed by optimization within these families. Screening up to 96 unique combinations of solubilization conditions and constructs can be achieved in less than 24 h. At the outset of each new project, we screen six different detergents for each construct and the subsequent implementation of a simple thermostability challenge further aids in the identification of constructs and conditions suitable for large-scale production. Our strategy streamlines the parallel optimization of appropriate production conditions for multiple MP targets to rapidly enable downstream biochemical, immunization, or structural studies.

Massive antibody discovery used to probe structure-function relationships of the essential outer membrane protein LptD.

Outer membrane proteins (OMPs) in Gram-negative bacteria dictate permeability of metabolites, antibiotics, and toxins. Elucidating the structure-function relationships governing OMPs within native membrane environments remains challenging. We constructed a diverse library of >3000 monoclonal antibodies to assess the roles of extracellular loops (ECLs) in LptD, an essential OMP that inserts lipopolysaccharide into the outer membrane of Escherichia coli. Epitope binning and mapping experiments with LptD-loop-deletion mutants demonstrated that 7 of the 13 ECLs are targeted by antibodies. Only ECLs inaccessible to antibodies were required for the structure or function of LptD. Our results suggest that antibody-accessible loops evolved to protect key extracellular regions of LptD, but are themselves dispensable. Supporting this hypothesis, no α-LptD antibody interfered with essential functions of LptD. Our experimental workflow enables structure-function studies of OMPs in native cellular environments, provides unexpected insight into LptD, and presents a method to assess the therapeutic potential of antibody targeting.

Structural Basis of Nav1.7 Inhibition by a Gating-Modifier Spider Toxin.

Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and toxin modulation remains ill-defined. Protoxin-II (ProTx2) from the Peruvian green velvet tarantula is an inhibitor cystine-knot peptide and selective antagonist of the human Nav1.7 channel. Here, we visualize ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7 using X-ray crystallography and cryoelectron microscopy. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule to antagonize S4 gating-charge movement through an electrostatic mechanism. ProTx2 has trapped activated and deactivated states of VSD2, revealing a remarkable ∼10 Å translation of the S4 helix, providing a structural framework for activation gating in voltage-gated ion channels. Finally, our results deliver key templates to design selective Nav channel antagonists.

Disrupting Gram-Negative Bacterial Outer Membrane Biosynthesis through Inhibition of the Lipopolysaccharide Transporter MsbA.

There is a critical need for new antibacterial strategies to counter the growing problem of antibiotic resistance. In Gram-negative bacteria, the outer membrane (OM) provides a protective barrier against antibiotics and other environmental insults. The outer leaflet of the outer membrane is primarily composed of lipopolysaccharide (LPS). Outer membrane biogenesis presents many potentially compelling drug targets as this pathway is absent in higher eukaryotes. Most proteins involved in LPS biosynthesis and transport are essential; however, few compounds have been identified that inhibit these proteins. The inner membrane ABC transporter MsbA carries out the first essential step in the trafficking of LPS to the outer membrane. We conducted a biochemical screen for inhibitors of MsbA and identified a series of quinoline compounds that kill Escherichia coli through inhibition of its ATPase and transport activity, with no loss of activity against clinical multidrug-resistant strains. Identification of these selective inhibitors indicates that MsbA is a viable target for new antibiotics, and the compounds we identified serve as useful tools to further probe the LPS transport pathway in Gram-negative bacteria.

Structural basis for dual-mode inhibition of the ABC transporter MsbA.

The movement of core-lipopolysaccharide across the inner membrane of Gram-negative bacteria is catalysed by an essential ATP-binding cassette transporter, MsbA. Recent structures of MsbA and related transporters have provided insights into the molecular basis of active lipid transport; however, structural information about their pharmacological modulation remains limited. Here we report the 2.9 Å resolution structure of MsbA in complex with G907, a selective small-molecule antagonist with bactericidal activity, revealing an unprecedented mechanism of ABC transporter inhibition. G907 traps MsbA in an inward-facing, lipopolysaccharide-bound conformation by wedging into an architecturally conserved transmembrane pocket. A second allosteric mechanism of antagonism occurs through structural and functional uncoupling of the nucleotide-binding domains. This study establishes a framework for the selective modulation of ABC transporters and provides rational avenues for the design of new antibiotics and other therapeutics targeting this protein family.

A targeted boost-and-sort immunization strategy using Escherichia coli BamA identifies rare growth inhibitory antibodies.

Outer membrane proteins (OMPs) in Gram-negative bacteria are essential for a number of cellular functions including nutrient transport and drug efflux. Escherichia coli BamA is an essential component of the OMP ß-barrel assembly machinery and a potential novel antibacterial target that has been proposed to undergo large (~15 Å) conformational changes. Here, we explored methods to isolate anti-BamA monoclonal antibodies (mAbs) that might alter the function of this OMP and ultimately lead to bacterial growth inhibition. We first optimized traditional immunization approaches but failed to identify mAbs that altered cell growth after screening >3000 hybridomas. We then developed a "targeted boost-and-sort" strategy that combines bacterial cell immunizations, purified BamA protein boosts, and single hybridoma cell sorting using amphipol-reconstituted BamA antigen. This unique workflow improves the discovery efficiency of FACS + mAbs by >600-fold and enabled the identification of rare anti-BamA mAbs with bacterial growth inhibitory activity in the presence of a truncated lipopolysaccharide layer. These mAbs represent novel tools for dissecting the BamA-mediated mechanism of ß-barrel folding and our workflow establishes a new template for the efficient discovery of novel mAbs against other highly dynamic membrane proteins.

A Novel Inhibitor of the LolCDE ABC Transporter Essential for Lipoprotein Trafficking in Gram-Negative Bacteria.

The outer membrane is an essential structural component of Gram-negative bacteria that is composed of lipoproteins, lipopolysaccharides, phospholipids, and integral ß-barrel membrane proteins. A dedicated machinery, called the Lol system, ensures proper trafficking of lipoproteins from the inner to the outer membrane. The LolCDE ABC transporter is the inner membrane component, which is essential for bacterial viability. Here, we report a novel pyrrolopyrimidinedione compound, G0507, which was identified in a phenotypic screen for inhibitors of Escherichia coli growth followed by selection of compounds that induced the extracytoplasmic σE stress response. Mutations in lolC, lolD, and lolE conferred resistance to G0507, suggesting LolCDE as its molecular target. Treatment of E. coli cells with G0507 resulted in accumulation of fully processed Lpp, an outer membrane lipoprotein, in the inner membrane. Using purified protein complexes, we found that G0507 binds to LolCDE and stimulates its ATPase activity. G0507 still binds to LolCDE harboring a Q258K substitution in LolC (LolCQ258K), which confers high-level resistance to G0507 in vivo but no longer stimulates ATPase activity. Our work demonstrates that G0507 has significant promise as a chemical probe to dissect lipoprotein trafficking in Gram-negative bacteria.

Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions.

Voltage-gated Kv1.3 and Ca2+-dependent KCa3.1 are the most prevalent K+ channels expressed by human and rat T cells. Despite the preferential upregulation of Kv1.3 over KCa3.1 on autoantigen-experienced effector memory T cells, whether Kv1.3 is required for their induction and function is unclear. Here we show, using Kv1.3-deficient rats, that Kv1.3 is involved in the development of chronically activated antigen-specific T cells. Several immune responses are normal in Kv1.3 knockout (KO) rats, suggesting that KCa3.1 can compensate for the absence of Kv1.3 under these specific settings. However, experiments with Kv1.3 KO rats and Kv1.3 siRNA knockdown or channel-specific inhibition of human T cells show that maximal T-cell responses against autoantigen or repeated tetanus toxoid stimulations require both Kv1.3 and KCa3.1. Finally, our data also suggest that T-cell dependency on Kv1.3 or KCa3.1 might be irreversibly modulated by antigen exposure.

Voltage-gated sodium channels viewed through a structural biology lens.

Voltage-gated sodium (Nav) channels initiate and propagate action potentials in excitable cells, and are frequently dysregulated or mutated in human disease. Despite decades of intense physiological and biophysical research, eukaryotic Nav channels have so far eluded high-resolution structure determination because of their biochemical complexity. Recently, simpler bacterial voltage-gated sodium (BacNav) channels have provided templates to understand the structural basis of voltage-dependent activation, inactivation, ion selectivity, and drug block in eukaryotic Nav and related voltage-gated calcium (Cav) channels. Further breakthroughs employing BacNav channels have also enabled visualization of bound small molecule modulators that can guide the rational design of next generation therapeutics. This review will highlight the emerging structural biology of BacNav channels and its contribution to our understanding of the gating, ion selectivity, and pharmacological regulation of eukaryotic Nav (and Cav) channels.

Structural basis of Nav1.7 inhibition by an isoform-selective small-molecule antagonist.

Voltage-gated sodium (Nav) channels propagate action potentials in excitable cells. Accordingly, Nav channels are therapeutic targets for many cardiovascular and neurological disorders. Selective inhibitors have been challenging to design because the nine mammalian Nav channel isoforms share high sequence identity and remain recalcitrant to high-resolution structural studies. Targeting the human Nav1.7 channel involved in pain perception, we present a protein-engineering strategy that has allowed us to determine crystal structures of a novel receptor site in complex with isoform-selective antagonists. GX-936 and related inhibitors bind to the activated state of voltage-sensor domain IV (VSD4), where their anionic aryl sulfonamide warhead engages the fourth arginine gating charge on the S4 helix. By opposing VSD4 deactivation, these compounds inhibit Nav1.7 through a voltage-sensor trapping mechanism, likely by stabilizing inactivated states of the channel. Residues from the S2 and S3 helices are key determinants of isoform selectivity, and bound phospholipids implicate the membrane as a modulator of channel function and pharmacology. Our results help to elucidate the molecular basis of voltage sensing and establish structural blueprints to design selective Nav channel antagonists.

Structural analysis of bacterial ABC transporter inhibition by an antibody fragment.

Bacterial ATP-binding cassette (ABC) importers play critical roles in nutrient acquisition and are potential antibacterial targets. However, structural bases for their inhibition are poorly defined. These pathways typically rely on substrate binding proteins (SBPs), which are essential for substrate recognition, delivery, and transporter function. We report the crystal structure of a Staphylococcus aureus SBP for Mn(II), termed MntC, in complex with FabC1, a potent antibody inhibitor of the MntABC pathway. This pathway is essential and highly expressed during S. aureus infection and facilitates the import of Mn(II), a critical cofactor for enzymes that detoxify reactive oxygen species (ROS). Structure-based functional studies indicate that FabC1 sterically blocks a structurally conserved surface of MntC, preventing its interaction with the MntB membrane importer and increasing wild-type S. aureus sensitivity to oxidative stress by more than 10-fold. The results define an SBP blocking mechanism as the basis for ABC importer inhibition by an engineered antibody fragment.

Inhibitory mechanism of an allosteric antibody targeting the glucagon receptor.

Elevated glucagon levels and increased hepatic glucagon receptor (GCGR) signaling contribute to hyperglycemia in type 2 diabetes. We have identified a monoclonal antibody that inhibits GCGR, a class B G-protein coupled receptor (GPCR), through a unique allosteric mechanism. Receptor inhibition is mediated by the binding of this antibody to two distinct sites that lie outside of the glucagon binding cleft. One site consists of a patch of residues that are surface-exposed on the face of the extracellular domain (ECD) opposite the ligand-binding cleft, whereas the second binding site consists of residues in the αA helix of the ECD. A docking model suggests that the antibody does not occlude the ligand-binding cleft. We solved the crystal structure of GCGR ECD containing a naturally occurring G40S mutation and found a shift in the register of the αA helix that prevents antibody binding. We also found that alterations in the αA helix impact the normal function of GCGR. We present a model for the allosteric inhibition of GCGR by a monoclonal antibody that may form the basis for the development of allosteric modulators for the treatment of diabetes and other class B GPCR-related diseases.

Histone deacetylase (HDAC) inhibitor kinetic rate constants correlate with cellular histone acetylation but not transcription and cell viability.

Histone deacetylases (HDACs) are critical in the control of gene expression, and dysregulation of their activity has been implicated in a broad range of diseases, including cancer, cardiovascular, and neurological diseases. HDAC inhibitors (HDACi) employing different zinc chelating functionalities such as hydroxamic acids and benzamides have shown promising results in cancer therapy. Although it has also been suggested that HDACi with increased isozyme selectivity and potency may broaden their clinical utility and minimize side effects, the translation of this idea to the clinic remains to be investigated. Moreover, a detailed understanding of how HDACi with different pharmacological properties affect biological functions in vitro and in vivo is still missing. Here, we show that a panel of benzamide-containing HDACi are slow tight-binding inhibitors with long residence times unlike the hydroxamate-containing HDACi vorinostat and trichostatin-A. Characterization of changes in H2BK5 and H4K14 acetylation following HDACi treatment in the neuroblastoma cell line SH-SY5Y revealed that the timing and magnitude of histone acetylation mirrored both the association and dissociation kinetic rates of the inhibitors. In contrast, cell viability and microarray gene expression analysis indicated that cell death induction and changes in transcriptional regulation do not correlate with the dissociation kinetic rates of the HDACi. Therefore, our study suggests that determining how the selective and kinetic inhibition properties of HDACi affect cell function will help to evaluate their therapeutic utility.

Molecular basis for negative regulation of the glucagon receptor.

Members of the class B family of G protein-coupled receptors (GPCRs) bind peptide hormones and have causal roles in many diseases, ranging from diabetes and osteoporosis to anxiety. Although peptide, small-molecule, and antibody inhibitors of these GPCRs have been identified, structure-based descriptions of receptor antagonism are scarce. Here we report the mechanisms of glucagon receptor inhibition by blocking antibodies targeting the receptor's extracellular domain (ECD). These studies uncovered a role for the ECD as an intrinsic negative regulator of receptor activity. The crystal structure of the ECD in complex with the Fab fragment of one antibody, mAb1, reveals that this antibody inhibits glucagon receptor by occluding a surface extending across the entire hormone-binding cleft. A second antibody, mAb23, blocks glucagon binding and inhibits basal receptor activity, indicating that it is an inverse agonist and that the ECD can negatively regulate receptor activity independent of ligand binding. Biochemical analyses of receptor mutants in the context of a high-resolution ECD structure show that this previously unrecognized inhibitory activity of the ECD involves an interaction with the third extracellular loop of the receptor and suggest that glucagon-mediated structural changes in the ECD accompany receptor activation. These studies have implications for the design of drugs to treat class B GPCR-related diseases, including the potential for developing novel allosteric regulators that target the ECDs of these receptors.

Crystal structure of the ectodomain complex of the CGRP receptor, a class-B GPCR, reveals the site of drug antagonism.

Dysregulation of the calcitonin gene-related peptide (CGRP), a potent vasodilator, is directly implicated in the pathogenesis of migraine. CGRP binds to and signals through the CGRP receptor (CGRP-R), a heterodimer containing the calcitonin receptor-like receptor (CLR), a class B GPCR, and RAMP1, a receptor activity-modifying protein. We have solved the crystal structure of the CLR/RAMP1 N-terminal ectodomain heterodimer, revealing how RAMPs bind to and potentially modulate the activities of the CLR GPCR subfamily. We also report the structures of CLR/RAMP1 in complex with the clinical receptor antagonists olcegepant (BIBN4096BS) and telcagepant (MK0974). Both drugs act by blocking access to the peptide-binding cleft at the interface of CLR and RAMP1. These structures illustrate, for the first time, how small molecules bind to and modulate the activity of a class B GPCR, and highlight the challenges of designing potent receptor antagonists for the treatment of migraine and other class B GPCR-related diseases.