Negamycin induces translational stalling and miscoding by binding to the small subunit head domain of the Escherichia coli ribosome.

Negamycin is a natural product with broad-spectrum antibacterial activity and efficacy in animal models of infection. Although its precise mechanism of action has yet to be delineated, negamycin inhibits cellular protein synthesis and causes cell death. Here, we show that single point mutations within 16S rRNA that confer resistance to negamycin are in close proximity of the tetracycline binding site within helix 34 of the small subunit head domain. As expected from its direct interaction with this region of the ribosome, negamycin was shown to displace tetracycline. However, in contrast to tetracycline-class antibiotics, which serve to prevent cognate tRNA from entering the translating ribosome, single-molecule fluorescence resonance energy transfer investigations revealed that negamycin specifically stabilizes near-cognate ternary complexes within the A site during the normally transient initial selection process to promote miscoding. The crystal structure of the 70S ribosome in complex with negamycin, determined at 3.1 Å resolution, sheds light on this finding by showing that negamycin occupies a site that partially overlaps that of tetracycline-class antibiotics. Collectively, these data suggest that the small subunit head domain contributes to the decoding mechanism and that small-molecule binding to this domain may either prevent or promote tRNA entry by altering the initial selection mechanism after codon recognition and before GTPase activation.

Crystal structure of A. aeolicus LpxC with bound product suggests alternate deacetylation mechanism.

UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is the first committed step to form lipid A, an essential component of the outer membrane of Gram-negative bacteria. As it is essential for the survival of many pathogens, LpxC is an attractive target for antibacterial therapeutics. Herein, we report the product-bound co-crystal structure of LpxC from the acheal Aquifex aeolicus solved to 1.6 Å resolution. We identified interactions by hydroxyl and hydroxymethyl substituents of the product glucosamine ring that may enable new insights to exploit waters in the active site for structure-based design of LpxC inhibitors with novel scaffolds. By using this product structure, we have performed quantum mechanical modeling on the substrate in the active site. Based on our results and published experimental data, we propose a new mechanism that may lead to a better understanding of LpxC catalysis and inhibition.

Synergy of streptogramin antibiotics occurs independently of their effects on translation.

Streptogramin antibiotics are divided into types A and B, which in combination can act synergistically. We compared the molecular interactions of the streptogramin combinations Synercid (type A, dalfopristin; type B, quinupristin) and NXL 103 (type A, flopristin; type B, linopristin) with the Escherichia coli 70S ribosome by X-ray crystallography. We further analyzed the activity of the streptogramin components individually and in combination. The streptogramin A and B components in Synercid and NXL 103 exhibit synergistic antimicrobial activity against certain pathogenic bacteria. However, in transcription-coupled translation assays, only combinations that include dalfopristin, the streptogramin A component of Synercid, show synergy. Notably, the diethylaminoethylsulfonyl group in dalfopristin reduces its activity but is the basis for synergy in transcription-coupled translation assays before its rapid hydrolysis from the depsipeptide core. Replacement of the diethylaminoethylsulfonyl group in dalfopristin by a nonhydrolyzable group may therefore be beneficial for synergy. The absence of general streptogramin synergy in transcription-coupled translation assays suggests that the synergistic antimicrobial activity of streptogramins can occur independently of the effects of streptogramin on translation.

Overexpression of Pseudomonas aeruginosa LpxC with its inhibitors in an acrB-deficient Escherichia coli strain.

In Gram-negative bacteria, the cell wall is surrounded by an outer membrane, the outer leaflet of which is comprised of charged lipopolysaccharide (LPS) molecules. Lipid A, a component of LPS, anchors this molecule to the outer membrane. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent metalloamidase that catalyzes the first committed step of biosynthesis of Lipid A, making it a promising target for antibiotic therapy. Formation of soluble aggregates of Pseudomonas aeruginosa LpxC protein when overexpressed in Escherichia coli has limited the availability of high quality protein for X-ray crystallography. Expression of LpxC in the presence of an inhibitor dramatically increased protein solubility, shortened crystallization time and led to a high-resolution crystal structure of LpxC bound to the inhibitor. However, this approach required large amounts of compound, restricting its use. To reduce the amount of compound needed, an overexpression strain of E. coli was created lacking acrB, a critical component of the major efflux pump. By overexpressing LpxC in the efflux deficient strain in the presence of LpxC inhibitors, several structures of P. aeruginosa LpxC in complex with different compounds were solved to accelerate structure-based drug design.

Caged mono- and divalent ligands for light-assisted disruption of PDZ domain-mediated interactions.

We report a general method for light-assisted control of interactions of PDZ domain binding motifs with their cognate domains by the incorporation of a photolabile caging group onto the essential C-terminal carboxylate binding determinant of the motif. The strategy was implemented and validated for both simple monovalent and biomimetic divalent ligands, which have recently been established as powerful tools for acute perturbation of native PDZ domain-dependent interactions in live cells.

Biomimetic divalent ligands for the acute disruption of synaptic AMPAR stabilization.

The interactions of the AMPA receptor (AMPAR) auxiliary subunit Stargazin with PDZ domain-containing scaffold proteins such as PSD-95 are critical for the synaptic stabilization of AMPARs. To investigate these interactions, we have developed biomimetic competing ligands that are assembled from two Stargazin-derived PSD-95/DLG/ZO-1 (PDZ) domain-binding motifs using 'click' chemistry. Characterization of the ligands in vitro and in a cellular FRET-based model revealed an enhanced affinity for the multiple PDZ domains of PSD-95 compared to monovalent peptides. In cultured neurons, the divalent ligands competed with transmembrane AMPAR regulatory protein (TARP) for the intracellular membrane-associated guanylate kinase resulting in increased lateral diffusion and endocytosis of surface AMPARs, while showing strong inhibition of synaptic AMPAR currents. This provides evidence for a model in which the TARP-containing AMPARs are stabilized at the synapse by engaging in multivalent interactions. In light of the prevalence of PDZ domain clusters, these new biomimetic chemical tools could find broad application for acutely perturbing multivalent complexes.

Sulfonylpiperidines as novel, antibacterial inhibitors of Gram-positive thymidylate kinase (TMK).

Thymidylate kinase (TMK) is an essential enzyme for DNA synthesis in bacteria, phosphorylating deoxythymidine monophosphate (dTMP) to deoxythymidine diphosphate (dTDP), and thus is a potential new antibacterial drug target. Previously, we have described the first potent and selective inhibitors of Gram-positive TMK, leading to in vivo validation of the target. Here, a structure-guided design approach based on the initial series led to the discovery of novel sulfonylpiperidine inhibitors of TMK. Formation of hydrogen bonds with Arg48 in Staphylococcus aureus TMK was key to obtaining excellent enzyme affinity, as verified by protein crystallography. Replacement of a methylene linker in the series by a sulfonamide was accomplished with retention of binding conformation. Further optimization of logD yielded phenol derivative 11, a potent inhibitor of TMK showing excellent MICs against a broad spectrum of Gram-positive bacteria and >10(5) selectivity versus the human TMK homologue.

Mapping the structure and conformational movements of proteins with transition metal ion FRET.

Visualizing conformational dynamics in proteins has been difficult, and the atomic-scale motions responsible for the behavior of most allosteric proteins are unknown. Here we report that fluorescence resonance energy transfer (FRET) between a small fluorescent dye and a nickel ion bound to a dihistidine motif can be used to monitor small structural rearrangements in proteins. This method provides several key advantages over classical FRET, including the ability to measure the dynamics of close-range interactions, the use of small probes with short linkers, a low orientation dependence, and the ability to add and remove unique tunable acceptors. We used this 'transition metal ion FRET' approach along with X-ray crystallography to determine the structural changes of the gating ring of the mouse hyperpolarization-activated cyclic nucleotide-regulated ion channel HCN2. Our results suggest a general model for the conformational switch in the cyclic nucleotide-binding site of cyclic nucleotide-regulated ion channels.

Structural analysis of WbpE from Pseudomonas aeruginosa PAO1: a nucleotide sugar aminotransferase involved in O-antigen assembly.

In recent years, the opportunistic pathogen Pseudomonas aeruginosa has emerged as a major source of hospital-acquired infections. Effective treatment has proven increasingly difficult due to the spread of multidrug resistant strains and thus requires a deeper understanding of the biochemical mechanisms of pathogenicity. The central carbohydrate of the P. aeruginosa PAO1 (O5) B-band O-antigen, ManNAc(3NAc)A, has been shown to be critical for virulence and is produced in a stepwise manner by five enzymes in the Wbp pathway (WbpA, WbpB, WbpE, WbpD, and WbpI). Herein, we present the crystal structure of the aminotransferase WbpE from P. aeruginosa PAO1 in complex with the cofactor pyridoxal 5'-phosphate (PLP) and product UDP-GlcNAc(3NH(2))A as the external aldimine at 1.9 A resolution. We also report the structures of WbpE in complex with PMP alone as well as the PLP internal aldimine and show that the dimeric structure of WbpE observed in the crystal structure is confirmed by analytical ultracentrifugation. Analysis of these structures reveals that the active site of the enzyme is composed of residues from both subunits. In particular, we show that a key residue (Arg229), which has previously been implicated in direct interactions with the alpha-carboxylate moiety of alpha-ketoglutarate, is also uniquely positioned to bestow specificity for the 6''-carboxyl group of GlcNAc(3NH(2))A through a salt bridge. This finding is intriguing because while an analogous basic residue is present in WbpE homologues that do not process 6''-carboxyl-modified saccharides, recent structural studies reveal that this side chain is retracted to accommodate a neutral C6'' atom. This work represents the first structural analysis of a nucleotide sugar aminotransferase with a bound product modified at the C2'', C3'', and C6'' positions and provides insight into a novel target for treatment of P. aeruginosa infection.

Voltage-dependent opening of HCN channels: Facilitation or inhibition by the phytoestrogen, genistein, is determined by the activation status of the cyclic nucleotide gating ring.

Investigation of the mechanistic bases and physiological importance of cAMP regulation of HCN channels has exploited an arginine to glutamate mutation in the nucleotide-binding fold, an approach critically dependent on the mutation selectively lowering the channel's nucleotide affinity. In apparent conflict with this, in intact Xenopus oocytes, HCN and HCN-RE channels exhibit qualitatively and quantitatively distinct responses to the tyrosine kinase inhibitor, genistein -- the estrogenic isoflavonoid strongly depolarizes the activation mid-point of HCN1-R538E, but not HCN1 channels (+9.8 mV + or - 0.9 versus +2.2 mV + or - 0.6) and hyperpolarizes gating of HCN2 (-4.8 mV + or - 1.0) but depolarizes gating of HCN2-R591E (+13.2 mV + or - 2.1). However, excised patch recording, X-ray crystallography and modeling reveal that this is not due to either a fundamental effect of the mutation on channel gating per se or of genistein acting as a mutation-sensitive partial agonist at the cAMP site. Rather, we find that genistein equivalently moves both HCN and HCN-RE channels closer to the open state (rendering the channels inherently easier to open but at a cost of decreasing the coupling energy of cAMP) and that the anomaly reflects a balance of these energetic effects with the isoform-specific inhibition of activation by the nucleotide gating ring and relief of this by endogenous cAMP. These findings have specific implications with regard to findings based on HCN-RE channels and kinase antagonists and general implications with respect to interpretation of drug effects in mutant channel backgrounds.

Structural basis for modulation and agonist specificity of HCN pacemaker channels.

The family of hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels are crucial for a range of electrical signalling, including cardiac and neuronal pacemaker activity, setting resting membrane electrical properties and dendritic integration. These nonselective cation channels, underlying the I(f), I(h) and I(q) currents of heart and nerve cells, are activated by membrane hyperpolarization and modulated by the binding of cyclic nucleotides such as cAMP and cGMP. The cAMP-mediated enhancement of channel activity is largely responsible for the increase in heart rate caused by beta-adrenergic agonists. Here we have investigated the mechanism underlying this modulation by studying a carboxy-terminal fragment of HCN2 containing the cyclic nucleotide-binding domain (CNBD) and the C-linker region that connects the CNBD to the pore. X-ray crystallographic structures of this C-terminal fragment bound to cAMP or cGMP, together with equilibrium sedimentation analysis, identify a tetramerization domain and the mechanism for cyclic nucleotide specificity, and suggest a model for ligand-dependent channel modulation. On the basis of amino acid sequence similarity to HCN channels, the cyclic nucleotide-gated, and eag- and KAT1-related families of channels are probably related to HCN channels in structure and mechanism.

A conserved tripeptide in CNG and HCN channels regulates ligand gating by controlling C-terminal oligomerization.

Cyclic nucleotides directly enhance the opening of the tetrameric CNG and HCN channels, although the mechanism remains unclear. We examined why HCN and certain CNG subunits form functional homomeric channels, whereas other CNG subunits only function in heteromeric channels. The "defect" in the CNGA4 subunit that prevents its homomeric expression was localized to its C-linker, which connects the transmembrane domain to the binding domain and contains a tripeptide that decreases the efficacy of ligand gating. Remarkably, replacement of the homologous HCN tripeptide with the CNGA4 sequence transformed cAMP into an inverse agonist that inhibits HCN channel opening. Using analytical ultracentrifugation, we identified the structural basis for this gating switch: whereas cAMP normally enhances the assembly of HCN C-terminal domains into a tetrameric gating ring, inclusion of the CNGA4 tripeptide reversed this action so that cAMP now causes gating ring disassembly. Thus, ligand gating depends on the dynamic oligomerization of C-terminal binding domains.

ETX2514 is a broad-spectrum ß-lactamase inhibitor for the treatment of drug-resistant Gram-negative bacteria including Acinetobacter baumannii.

Multidrug-resistant (MDR) bacterial infections are a serious threat to public health. Among the most alarming resistance trends is the rapid rise in the number and diversity of ß-lactamases, enzymes that inactivate ß-lactams, a class of antibiotics that has been a therapeutic mainstay for decades. Although several new ß-lactamase inhibitors have been approved or are in clinical trials, their spectra of activity do not address MDR pathogens such as Acinetobacter baumannii. This report describes the rational design and characterization of expanded-spectrum serine ß-lactamase inhibitors that potently inhibit clinically relevant class A, C and D ß-lactamases and penicillin-binding proteins, resulting in intrinsic antibacterial activity against Enterobacteriaceae and restoration of ß-lactam activity in a broad range of MDR Gram-negative pathogens. One of the most promising combinations is sulbactam-ETX2514, whose potent antibacterial activity, in vivo efficacy against MDR A. baumannii infections and promising preclinical safety demonstrate its potential to address this significant unmet medical need.

Antibacterial inhibitors of Gram-positive thymidylate kinase: structure-activity relationships and chiral preference of a new hydrophobic binding region.

Thymidylate kinase (TMK), an essential enzyme in bacterial DNA biosynthesis, is an attractive therapeutic target for the development of novel antibacterial agents, and we continue to explore TMK inhibitors with improved potency, protein binding, and pharmacokinetic potential. A structure-guided design approach was employed to exploit a previously unexplored region in Staphylococcus aureus TMK via novel interactions. These efforts produced compound 39, with 3 nM IC50 against S. aureus TMK and 2 µg/mL MIC against methicillin-resistant S. aureus (MRSA). This compound exhibits a striking inverted chiral preference for binding relative to earlier compounds and also has improved physical properties and pharmacokinetics over previously published compounds. An example of this new series was efficacious in a murine S. aureus infection model, suggesting that compounds like 39 are options for further work toward a new Gram-positive antibiotic by maintaining a balance of microbiological potency, low clearance, and low protein binding that can result in lower efficacious doses.

In vivo validation of thymidylate kinase (TMK) with a rationally designed, selective antibacterial compound.

There is an urgent need for new antibacterials that pinpoint novel targets and thereby avoid existing resistance mechanisms. We have created novel synthetic antibacterials through structure-based drug design that specifically target bacterial thymidylate kinase (TMK), a nucleotide kinase essential in the DNA synthesis pathway. A high-resolution structure shows compound TK-666 binding partly in the thymidine monophosphate substrate site, but also forming new induced-fit interactions that give picomolar affinity. TK-666 has potent, broad-spectrum Gram-positive microbiological activity (including activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus), bactericidal action with rapid killing kinetics, excellent target selectivity over the human ortholog, and low resistance rates. We demonstrate in vivo efficacy against S. aureus in a murine infected-thigh model. This work presents the first validation of TMK as a compelling antibacterial target and provides a rationale for pursuing novel clinical candidates for treating Gram-positive infections through TMK.

Discovery of selective and potent inhibitors of gram-positive bacterial thymidylate kinase (TMK).

Thymidylate kinase (TMK) is an essential enzyme in bacterial DNA synthesis. The deoxythymidine monophosphate (dTMP) substrate binding pocket was targeted in a rational-design, structure-supported effort, yielding a unique series of antibacterial agents showing a novel, induced-fit binding mode. Lead optimization, aided by X-ray crystallography, led to picomolar inhibitors of both Streptococcus pneumoniae and Staphylococcus aureus TMK. MICs < 1 µg/mL were achieved against methicillin-resistant S. aureus (MRSA), S. pneumoniae, and vancomycin-resistant Enterococcus (VRE). Log D adjustments yielded single diastereomers 14 (TK-666) and 46, showing a broad antibacterial spectrum against Gram-positive bacteria and excellent selectivity against the human thymidylate kinase ortholog.

Crystal structure and catalytic mechanism of PglD from Campylobacter jejuni.

The carbohydrate 2, 4-diacetamido-2, 4, 6-trideoxy-alpha-D-glucopyranose (BacAc(2)) is found in a variety of eubacterial pathogens. In Campylobacter jejuni, PglD acetylates the C4 amino group on UDP-2-acetamido-4-amino-2, 4, 6-trideoxy-alpha-D-glucopyranose (UDP-4-amino-sugar) to form UDP-BacAc(2). Sequence analysis predicts PglD to be a member of the left-handed beta helix family of enzymes. However, poor sequence homology between PglD and left-handed beta helix enzymes with existing structural data precludes unambiguous identification of the active site. The co-crystal structures of PglD in the presence of citrate, acetyl coenzyme A, or the UDP-4-amino-sugar were solved. The biological assembly is a trimer with one active site formed between two protomers. Residues lining the active site were identified, and results from functional assays on alanine mutants suggest His-125 is critical for catalysis, whereas His-15 and His-134 are involved in substrate binding. These results are discussed in the context of implications for proteins homologous to PglD in other pathogens.

C-terminal movement during gating in cyclic nucleotide-modulated channels.

Activation of cyclic nucleotide-modulated channels such as CNG and HCN channels is promoted by ligand-induced conformational changes in their C-terminal regions. The primary intersubunit interface of these C termini includes two salt bridges per subunit, formed between three residues (one positively charged and two negatively charged amino acids) that we term the SB triad. We previously hypothesized that the SB triad is formed in the closed channel and breaks when the channel opens. Here we tested this hypothesis by dynamically manipulating the SB triad in functioning CNGA1 channels. Reversing the charge at positions Arg-431 and Glu-462, two of the SB triad residues, by either mutation or application of charged reagents increased the favorability of channel opening. To determine how a charge reversal mutation in the SB triad structurally affects the channel, we solved the crystal structure of the HCN2 C-terminal region with the equivalent E462R mutation. The backbone structure of this mutant was very similar to that of wild type, but the SB triad was rearranged such that both salt bridges did not always form simultaneously, suggesting a mechanism for the increased ease of opening of the mutant channels. To prevent movement in the SB triad, we tethered two components of the SB triad region together with cysteine-reactive cross-linkers. Preventing normal movement of the SB triad region with short cross-linkers inhibited channel opening, whereas longer cross-linkers did not. These results support our hypothesis that the SB triad forms in the closed channel and indicate that this region expands as the channel opens.

In vitro biosynthesis of UDP-N,N'-diacetylbacillosamine by enzymes of the Campylobacter jejuni general protein glycosylation system.

In Campylobacter jejuni 2,4-diacetamido-2,4,6-trideoxy-alpha-d-glucopyranose, termed N,N'-diacetylbacillosamine (Bac2,4diNAc), is the first carbohydrate in the glycoprotein N-linked heptasaccharide. With uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) as a starting point, two enzymes of the general protein glycosylation (Pgl) pathway in C. jejuni (PglF and PglE) have recently been shown to modify this sugar nucleotide to form UDP-2-acetamido-4-amino-2,4,6-trideoxy-alpha-d-glycopyranose (UDP-4-amino-sugar) [Schoenhofen, I. C., et al. (2006) J. Biol. Chem. 281, 723-732]. PglD has been proposed to catalyze the final step in N,N'-diacetylbacillosamine synthesis by N-acetylation of the UDP-4-amino-sugar at the C4 position. We have cloned, overexpressed, and purified PglD from the pgl locus of C. jejuni NCTC 11168 and identified it as the acetyltransferase that modifies the UDP-4-amino-sugar to form UDP-N,N'-diacetylbacillosamine, utilizing acetyl-coenzyme A as the acetyl group donor. The UDP-N,N'-diacetylbacillosamine product was purified from the reaction by reverse phase C18 HPLC and the structure determined by NMR analysis. Additionally, the full-length PglF was overexpressed and purified in the presence of detergent as a GST fusion protein, allowing for derivation of kinetic parameters. We found that the UDP-4-amino-sugar was readily synthesized from UDP-GlcNAc in a coupled reaction using PglF and PglE. We also demonstrate the in vitro biosynthesis of the complete heptasaccharide lipid-linked donor by coupling the action of eight enzymes (PglF, PglE, PglD, PglC, PglA, PglJ, PglH, and PglI) in the Pgl pathway in a single reaction vessel.