Author Correction: A new antibiotic selectively kills Gram-negative pathogens.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A new antibiotic selectively kills Gram-negative pathogens.

The current need for novel antibiotics is especially acute for drug-resistant Gram-negative pathogens1,2. These microorganisms have a highly restrictive permeability barrier, which limits the penetration of most compounds3,4. As a result, the last class of antibiotics that acted against Gram-negative bacteria was developed in the 1960s2. We reason that useful compounds can be found in bacteria that share similar requirements for antibiotics with humans, and focus on Photorhabdus symbionts of entomopathogenic nematode microbiomes. Here we report a new antibiotic that we name darobactin, which was obtained using a screen of Photorhabdus isolates. Darobactin is coded by a silent operon with little production under laboratory conditions, and is ribosomally synthesized. Darobactin has an unusual structure with two fused rings that form post-translationally. The compound is active against important Gram-negative pathogens both in vitro and in animal models of infection. Mutants that are resistant to darobactin map to BamA, an essential chaperone and translocator that folds outer membrane proteins. Our study suggests that bacterial symbionts of animals contain antibiotics that are particularly suitable for development into therapeutics.

Structural basis of AcrIF24 as an anti-CRISPR protein and transcriptional suppressor.

Anti-CRISPR (Acr) proteins are encoded by phages to inactivate CRISPR-Cas systems of bacteria and archaea and are used to enhance the CRISPR toolbox for genome editing. Here we report the structure and mechanism of AcrIF24, an Acr protein that inhibits the type I-F CRISPR-Cas system from Pseudomonas aeruginosa. AcrIF24 is a homodimer that associates with two copies of the surveillance complex (Csy) and prevents the hybridization between CRISPR RNA and target DNA. Furthermore, AcrIF24 functions as an anti-CRISPR-associated (Aca) protein to repress the transcription of the acrIF23-acrIF24 operon. Alone or in complex with Csy, AcrIF24 is capable of binding to the acrIF23-acrIF24 promoter DNA with nanomolar affinity. The structure of a Csy-AcrIF24-promoter DNA complex at 2.7 Å reveals the mechanism for transcriptional suppression. Our results reveal that AcrIF24 functions as an Acr-Aca fusion protein, and they extend understanding of the diverse mechanisms used by Acr proteins.

Divergence and Convergence: Complexity Emerges in Crystal Engineering from an 8-mer DNA.

Biology provides plenty of examples on achieving complicated structures out of minimal numbers of building blocks. In contrast, structural complexity of designed molecular systems is achieved by increasing the numbers of component molecules. In this study, the component DNA strand assembles into a highly complex crystal structure via an unusual path of divergence and convergence. This assembly path suggests a route to minimalists for increasing structural complexity. The original purpose of this study is to engineer DNA crystals with high resolution, which is the primary motivation and a key objective for structural DNA nanotechnology. Despite great efforts in the last 40 years, engineered DNA crystals have not yet consistently reached resolution better than 2.5 Å, limiting their potential uses. Our research has shown that small, symmetrical building blocks generally lead to high resolution crystals. Herein, by following this principle, we report an engineered DNA crystal with unprecedented high resolution (2.17 Å) assembled from one single DNA component: an 8-base-long DNA strand. This system has three unique characteristics: (1) It has a very complex architecture, (2) the same DNA strand forms two different structural motifs, both of which are incorporated into the final crystal, and (3) the component DNA molecule is only an 8-base-long DNA strand, which is, arguably, the smallest DNA motif for DNA nanostructures to date. This high resolution opens the possibility of using these DNA crystals to precisely organize guest molecules at the Å level, which could stimulate a range of new investigations.

Engineering DNA Crystals toward Studying DNA-Guest Molecule Interactions.

Sequence-selective recognition of DNA duplexes is important for a wide range of applications including regulating gene expression, drug development, and genome editing. Many small molecules can bind DNA duplexes with sequence selectivity. It remains as a challenge how to reliably and conveniently obtain the detailed structural information on DNA-molecule interactions because such information is critically needed for understanding the underlying rules of DNA-molecule interactions. If those rules were understood, we could design molecules to recognize DNA duplexes with a sequence preference and intervene in related biological processes, such as disease treatment. Here, we have demonstrated that DNA crystal engineering is a potential solution. A molecule-binding DNA sequence is engineered to self-assemble into highly ordered DNA crystals. An X-ray crystallographic study of molecule-DNA cocrystals reveals the structural details on how the molecule interacts with the DNA duplex. In this approach, the DNA will serve two functions: (1) being part of the molecule to be studied and (2) forming the crystal lattice. It is conceivable that this method will be a general method for studying drug/peptide-DNA interactions. The resulting DNA crystals may also find use as separation matrices, as hosts for catalysts, and as media for material storage.

Point Mutations in TbpA Abrogate Human Transferrin Binding in Neisseria gonorrhoeae.

TonB-dependent transporters (TDTs) are essential proteins for metal acquisition, an important step in the growth and pathogenesis of many pathogens, including Neisseria gonorrhoeae, the causative agent of gonorrhea. There is currently no available vaccine for gonorrhea; TDTs are being investigated as vaccine candidates because they are highly conserved and expressed in vivo. Transferrin binding protein A (TbpA) is an essential virulence factor in the initiation of experimental infection in human males and functions by acquiring iron upon binding to host transferrin (human transferrin [hTf]). The loop 3 helix (L3H) is a helix finger that inserts into the hTf C-lobe and is required for hTf binding and subsequent iron acquisition. This study identified and characterized the first TbpA single-point substitutions resulting in significantly decreased hTf binding and iron acquisition, suggesting that the helix structure is more important than charge for hTf binding and utilization. The tbpA D355P ΔtbpB and tbpA A356P ΔtbpB mutants demonstrated significantly reduced hTf binding and impaired iron uptake from Fe-loaded hTf; however, only the tbpA A356P ΔtbpB mutant was able to grow when hTf was the sole source of iron. The expression of tbpB was able to restore function in all tbpA mutants. These results implicate both D355 and A356 in the key binding, extraction, and uptake functions of gonococcal TbpA.

The assembly of ß-barrel membrane proteins by BAM and SAM.

Gram-negative bacteria, mitochondria, and chloroplasts all possess an outer membrane populated with a host of ß-barrel outer-membrane proteins (ßOMPs). These ßOMPs play crucial roles in maintaining viability of their hosts, and therefore, it is essential to understand the biogenesis of this class of membrane proteins. In recent years, significant structural and functional advancements have been made toward elucidating this process, which is mediated by the ß-barrel assembly machinery (BAM) in Gram-negative bacteria, and by the sorting and assembly machinery (SAM) in mitochondria. Structures of both BAM and SAM have now been reported, allowing a comparison and dissection of the two machineries, with other studies reporting on functional aspects of each. Together, these new insights provide compelling support for the proposed budding mechanism, where each nascent ßOMP forms a hybrid-barrel intermediate with BAM/SAM in route to its biogenesis into the membrane. Here, we will review these recent studies and highlight their contributions toward understanding ßOMP biogenesis in Gram-negative bacteria and in mitochondria. We will also weigh the evidence supporting each of the two leading mechanistic models for how BAM/SAM function, and offer an outlook on future studies within the field.

Structural insight into the role of the Ton complex in energy transduction.

In Gram-negative bacteria, outer membrane transporters import nutrients by coupling to an inner membrane protein complex called the Ton complex. The Ton complex consists of TonB, ExbB, and ExbD, and uses the proton motive force at the inner membrane to transduce energy to the outer membrane via TonB. Here, we structurally characterize the Ton complex from Escherichia coli using X-ray crystallography, electron microscopy, double electron-electron resonance (DEER) spectroscopy, and crosslinking. Our results reveal a stoichiometry consisting of a pentamer of ExbB, a dimer of ExbD, and at least one TonB. Electrophysiology studies show that the Ton subcomplex forms pH-sensitive cation-selective channels and provide insight into the mechanism by which it may harness the proton motive force to produce energy.

Exploring Unconventional SAM Analogues To Build Cell-Potent Bisubstrate Inhibitors for Nicotinamide N-Methyltransferase.

Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide and has been associated with various diseases. Herein, we report the first cell-potent NNMT bisubstrate inhibitor II399, demonstrating a Ki of 5.9 nM in a biochemical assay and a cellular IC50 value of 1.9 µM. The inhibition mechanism and cocrystal structure confirmed II399 engages both the substrate and cofactor binding pockets. Computational modeling and binding data reveal a balancing act between enthalpic and entropic components that lead to II399's low nM binding affinity. Notably, II399 is 1 000-fold more selective for NNMT than closely related methyltransferases. We expect that II399 would serve as a valuable probe to elucidate NNMT biology. Furthermore, this strategy provides the first case of introducing unconventional SAM mimics, which can be adopted to develop cell-potent inhibitors for other SAM-dependent methyltransferases.

C-terminal kink formation is required for lateral gating in BamA.

In Gram-negative bacteria, the outer membrane contains primarily ß-barrel transmembrane proteins and lipoproteins. The insertion and assembly of ß-barrel outer-membrane proteins (OMPs) is mediated by the ß-barrel assembly machinery (BAM) complex, the core component of which is the 16-stranded transmembrane ß-barrel BamA. Recent studies have indicated a possible role played by the seam between the first and last ß-barrel strands of BamA in the OMP insertion process through lateral gating and a destabilized membrane region. In this study, we have determined the stability and dynamics of the lateral gate through over 12.5 µs of equilibrium simulations and 4 µs of free-energy calculations. From the equilibrium simulations, we have identified a persistent kink in the C-terminal strand and observed spontaneous lateral-gate separation in a mimic of the native bacterial outer membrane. Free-energy calculations of lateral gate opening revealed a significantly lower barrier to opening in the C-terminal kinked conformation; mutagenesis experiments confirm the relevance of C-terminal kinking to BamA structure and function.

The POTRA domains of Toc75 exhibit chaperone-like function to facilitate import into chloroplasts.

Protein trafficking across membranes is an essential function in cells; however, the exact mechanism for how this occurs is not well understood. In the endosymbionts, mitochondria and chloroplasts, the vast majority of proteins are synthesized in the cytoplasm as preproteins and then imported into the organelles via specialized machineries. In chloroplasts, protein import is accomplished by the TOC (translocon on the outer chloroplast membrane) and TIC (translocon on the inner chloroplast membrane) machineries in the outer and inner envelope membranes, respectively. TOC mediates initial recognition of preproteins at the outer membrane and includes a core membrane channel, Toc75, and two receptor proteins, Toc33/34 and Toc159, each containing GTPase domains that control preprotein binding and translocation. Toc75 is predicted to have a ß-barrel fold consisting of an N-terminal intermembrane space (IMS) domain and a C-terminal 16-stranded ß-barrel domain. Here we report the crystal structure of the N-terminal IMS domain of Toc75 from Arabidopsis thaliana, revealing three tandem polypeptide transport-associated (POTRA) domains, with POTRA2 containing an additional elongated helix not observed previously in other POTRA domains. Functional studies show an interaction with the preprotein, preSSU, which is mediated through POTRA2-3. POTRA2-3 also was found to have chaperone-like activity in an insulin aggregation assay, which we propose facilitates preprotein import. Our data suggest a model in which the POTRA domains serve as a binding site for the preprotein as it emerges from the Toc75 channel and provide a chaperone-like activity to prevent misfolding or aggregation as the preprotein traverses the intermembrane space.

Structure and Stoichiometry of the Ton Molecular Motor.

The Ton complex is a molecular motor that uses the proton gradient at the inner membrane of Gram-negative bacteria to generate force and movement, which are transmitted to transporters at the outer membrane, allowing the entry of nutrients into the periplasmic space. Despite decades of investigation and the recent flurry of structures being reported by X-ray crystallography and cryoEM, the mode of action of the Ton molecular motor has remained elusive, and the precise stoichiometry of its subunits is still a matter of debate. This review summarizes the latest findings on the Ton system by presenting the recently reported structures and related reports on the stoichiometry of the fully assembled complex.

Structural and functional insights into the role of BamD and BamE within the ß-barrel assembly machinery in Neisseria gonorrhoeae.

The ß-barrel assembly machinery (BAM) is a conserved multicomponent protein complex responsible for the biogenesis of ß-barrel outer membrane proteins (OMPs) in Gram-negative bacteria. Given its role in the production of OMPs for survival and pathogenesis, BAM represents an attractive target for the development of therapeutic interventions, including drugs and vaccines against multidrug-resistant bacteria such as Neisseria gonorrhoeae The first structure of BamA, the central component of BAM, was from N. gonorrhoeae, the etiological agent of the sexually transmitted disease gonorrhea. To aid in pharmaceutical targeting of BAM, we expanded our studies to BamD and BamE within BAM of this clinically relevant human pathogen. We found that the presence of BamD, but not BamE, is essential for gonococcal viability. However, BamE, but not BamD, was cell-surface-displayed under native conditions; however, in the absence of BamE, BamD indeed becomes surface-exposed. Loss of BamE altered cell envelope composition, leading to slower growth and an increase in both antibiotic susceptibility and formation of membrane vesicles containing greater amounts of vaccine antigens. Both BamD and BamE are expressed in diverse gonococcal isolates, under host-relevant conditions, and throughout different phases of growth. The solved structures of Neisseria BamD and BamE share overall folds with Escherichia coli proteins but contain differences that may be important for function. Together, these studies highlight that, although BAM is conserved across Gram-negative bacteria, structural and functional differences do exist across species, which may be leveraged in the development of species-specific therapeutics in the effort to combat multidrug resistance.

Structural insight into the biogenesis of ß-barrel membrane proteins.

ß-barrel membrane proteins are essential for nutrient import, signalling, motility and survival. In Gram-negative bacteria, the ß-barrel assembly machinery (BAM) complex is responsible for the biogenesis of ß-barrel membrane proteins, with homologous complexes found in mitochondria and chloroplasts. Here we describe the structure of BamA, the central and essential component of the BAM complex, from two species of bacteria: Neisseria gonorrhoeae and Haemophilus ducreyi. BamA consists of a large periplasmic domain attached to a 16-strand transmembrane ß-barrel domain. Three structural features shed light on the mechanism by which BamA catalyses ß-barrel assembly. First, the interior cavity is accessible in one BamA structure and conformationally closed in the other. Second, an exterior rim of the ß-barrel has a distinctly narrowed hydrophobic surface, locally destabilizing the outer membrane. And third, the ß-barrel can undergo lateral opening, suggesting a route from the interior cavity in BamA into the outer membrane.

Structure of the agonist-bound neurotensin receptor.

Neurotensin (NTS) is a 13-amino-acid peptide that functions as both a neurotransmitter and a hormone through the activation of the neurotensin receptor NTSR1, a G-protein-coupled receptor (GPCR). In the brain, NTS modulates the activity of dopaminergic systems, opioid-independent analgesia, and the inhibition of food intake; in the gut, NTS regulates a range of digestive processes. Here we present the structure at 2.8 Å resolution of Rattus norvegicus NTSR1 in an active-like state, bound to NTS(8-13), the carboxy-terminal portion of NTS responsible for agonist-induced activation of the receptor. The peptide agonist binds to NTSR1 in an extended conformation nearly perpendicular to the membrane plane, with the C terminus oriented towards the receptor core. Our findings provide, to our knowledge, the first insight into the binding mode of a peptide agonist to a GPCR and may support the development of non-peptide ligands that could be useful in the treatment of neurological disorders, cancer and obesity.

Structural basis for iron piracy by pathogenic Neisseria.

Neisseria are obligate human pathogens causing bacterial meningitis, septicaemia and gonorrhoea. Neisseria require iron for survival and can extract it directly from human transferrin for transport across the outer membrane. The transport system consists of TbpA, an integral outer membrane protein, and TbpB, a co-receptor attached to the cell surface; both proteins are potentially important vaccine and therapeutic targets. Two key questions driving Neisseria research are how human transferrin is specifically targeted, and how the bacteria liberate iron from transferrin at neutral pH. To address these questions, we solved crystal structures of the TbpA-transferrin complex and of the corresponding co-receptor TbpB. We characterized the TbpB-transferrin complex by small-angle X-ray scattering and the TbpA-TbpB-transferrin complex by electron microscopy. Our studies provide a rational basis for the specificity of TbpA for human transferrin, show how TbpA promotes iron release from transferrin, and elucidate how TbpB facilitates this process.

Structural Insights into Substrate Recognition and Catalysis in Outer Membrane Protein B (OmpB) by Protein-lysine Methyltransferases from Rickettsia.

Rickettsia belong to a family of Gram-negative obligate intracellular infectious bacteria that are the causative agents of typhus and spotted fever. Outer membrane protein B (OmpB) occurs in all rickettsial species, serves as a protective envelope, mediates host cell adhesion and invasion, and is a major immunodominant antigen. OmpBs from virulent strains contain multiple trimethylated lysine residues, whereas the avirulent strain contains mainly monomethyllysine. Two protein-lysine methyltransferases (PKMTs) that catalyze methylation of recombinant OmpB at multiple sites with varying sequences have been identified and overexpressed. PKMT1 catalyzes predominantly monomethylation, whereas PKMT2 catalyzes mainly trimethylation. Rickettsial PKMT1 and PKMT2 are unusual in that their primary substrate appears to be limited to OmpB, and both are capable of methylating multiple lysyl residues with broad sequence specificity. Here we report the crystal structures of PKMT1 from Rickettsia prowazekii and PKMT2 from Rickettsia typhi, both the apo form and in complex with its cofactor S-adenosylmethionine or S-adenosylhomocysteine. The structure of PKMT1 in complex with S-adenosylhomocysteine is solved to a resolution of 1.9 Å. Both enzymes are dimeric with each monomer containing an S-adenosylmethionine binding domain with a core Rossmann fold, a dimerization domain, a middle domain, a C-terminal domain, and a centrally located open cavity. Based on the crystal structures, residues involved in catalysis, cofactor binding, and substrate interactions were examined using site-directed mutagenesis followed by steady state kinetic analysis to ascertain their catalytic functions in solution. Together, our data reveal new structural and mechanistic insights into how rickettsial methyltransferases catalyze OmpB methylation.

Unusual Synergism of Transferrin and Citrate in the Regulation of Ti(IV) Speciation, Transport, and Toxicity.

Human serum transferrin (sTf) is a protein that mediates the transport of iron from blood to cells. Assisted by the synergistic anion carbonate, sTf transports Fe(III) by binding the metal ion in a closed conformation. Previous studies suggest sTf's role as a potential transporter of other metals such as titanium. Ti is a widely used metal in colorants, foods, and implants. A substantial amount of Ti is leached into blood from these implants. However, the fate of the leached Ti and its transport into the cells is not known. Understanding Ti interaction with sTf assumes a greater significance with our ever increasing exposure to Ti in the form of implants. On the basis of in vitro studies, it was speculated that transferrin can bind Ti(IV) assisted by a synergistic anion. However, the role and identity of the synergistic anion(s) and the conformational state in which sTf binds Ti(IV) are not known. Here we have solved the first X-ray crystal structure of a Ti(IV)-bound sTf. We find that sTf binds Ti(IV) in an open conformation with both carbonate and citrate as synergistic anions at the metal binding sites, an unprecedented role for citrate. Studies with cell lines suggest that Ti(IV)-sTf is transported into cells and that sTf and citrate regulate the metal's blood speciation and attenuate its cytotoxic property. Our results provide the first glimpse into the citrate-transferrin synergism in the regulation of Ti(IV) bioactivity and offers insight into the future design of Ti(IV)-based anticancer drugs.

Fitting the Pieces of the ß-Barrel Assembly Machinery Complex.

ß-Barrel membrane proteins are found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria; however, exactly how they are folded and inserted remains unknown. Over the past decade, both functional and structural studies have greatly contributed to addressing this elusive mechanism. It is known that a conserved core machinery is required for each organelle, though the overall composition varies significantly. The vast majority of studies that aimed to understand the biogenesis of ß-barrel membrane proteins has been conducted in Gram-negative bacteria. Here, it is the task of a multicomponent complex known as the ß-barrel assembly machinery (BAM) complex to fold and insert new ß-barrel membrane proteins into the outer membrane. In this review, we will discuss recent discoveries with the goal of utilizing all reported structural and functional studies to piece together a current structural model for the fully assembled BAM complex.

Beyond the Crystal Structure: Insight into the Function and Vaccine Potential of TbpA Expressed by Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, is not preventable by vaccination and is rapidly developing resistance to antibiotics. However, the transferrin (Tf) receptor system, composed of TbpA and TbpB, is an ideal target for novel therapeutics and vaccine development. Using a three-dimensional structure of gonococcal TbpA, we investigated two hypotheses, i.e., that loop-derived antibodies can interrupt ligand-receptor interactions in the native bacterium and that the loop 3 helix is a critical functional domain. Preliminary loop-derived antibodies, as well as optimized second-generation antibodies, demonstrated similar modest ligand-blocking effects on the gonococcal surface but different effects in Escherichia coli. Mutagenesis of loop 3 helix residues was employed, generating 11 mutants. We separately analyzed the mutants' abilities to (i) bind Tf and (ii) internalize Tf-bound iron in the absence of the coreceptor TbpB. Single residue mutations resulted in up to 60% reductions in ligand binding and up to 85% reductions in iron utilization. All strains were capable of growing on Tf as the sole iron source. Interestingly, in the presence of TbpB, only a 30% reduction in Tf-iron utilization was observed, indicating that the coreceptor can compensate for TbpA impairment. Complete deletion of the loop 3 helix of TbpA eliminated the abilities to bind Tf, internalize iron, and grow with Tf as the sole iron source. Our studies demonstrate that while the loop 3 helix is a key functional domain, its function does not exclusively rely on any single residue.