Gene amplifications cause high-level resistance against albicidin in gram-negative bacteria.

Antibiotic resistance is a continuously increasing concern for public healthcare. Understanding resistance mechanisms and their emergence is crucial for the development of new antibiotics and their effective use. The peptide antibiotic albicidin is such a promising candidate that, as a gyrase poison, shows bactericidal activity against a wide range of gram-positive and gram-negative bacteria. Here, we report the discovery of a gene amplification-based mechanism that imparts an up to 1000-fold increase in resistance levels against albicidin. RNA sequencing and proteomics data show that this novel mechanism protects Salmonella Typhimurium and Escherichia coli by increasing the copy number of STM3175 (YgiV), a transcription regulator with a GyrI-like small molecule binding domain that traps albicidin with high affinity. X-ray crystallography and molecular docking reveal a new conserved motif in the binding groove of the GyrI-like domain that can interact with aromatic building blocks of albicidin. Phylogenetic studies suggest that this resistance mechanism is ubiquitous in gram-negative bacteria, and our experiments confirm that STM3175 homologs can confer resistance in pathogens such as Vibrio vulnificus and Pseudomonas aeruginosa.

An ultra-stable gold-coordinated protein cage displaying reversible assembly.

Symmetrical protein cages have evolved to fulfil diverse roles in nature, including compartmentalization and cargo delivery1, and have inspired synthetic biologists to create novel protein assemblies via the precise manipulation of protein-protein interfaces. Despite the impressive array of protein cages produced in the laboratory, the design of inducible assemblies remains challenging2,3. Here we demonstrate an ultra-stable artificial protein cage, the assembly and disassembly of which can be controlled by metal coordination at the protein-protein interfaces. The addition of a gold (I)-triphenylphosphine compound to a cysteine-substituted, 11-mer protein ring triggers supramolecular self-assembly, which generates monodisperse cage structures with masses greater than 2 MDa. The geometry of these structures is based on the Archimedean snub cube and is, to our knowledge, unprecedented. Cryo-electron microscopy confirms that the assemblies are held together by 120 S-Aui-S staples between the protein oligomers, and exist in two chiral forms. The cage shows extreme chemical and thermal stability, yet it readily disassembles upon exposure to reducing agents. As well as gold, mercury(II) is also found to enable formation of the protein cage. This work establishes an approach for linking protein components into robust, higher-order structures, and expands the design space available for supramolecular assemblies to include previously unexplored geometries.

Reengineering of an Artificial Protein Cage for Efficient Packaging of Active Enzymes.

Protein cages that readily encapsulate active enzymes of interest present useful nanotools for delivery and catalysis, wherein those with programmable disassembly characteristics serve as particularly attractive platforms. Here, a general guest packaging system based on an artificial protein cage, TRAP-cage, the disassembly of which can be induced by the addition of reducing agents, is established. In this system, TRAP-cage with SpyCatcher moieties in the lumen is prepared using genetic modification of the protein building block and assembled into a cage structure with either monovalent gold ions or molecular crosslinkers. The resulting protein cage can efficiently capture guest proteins equipped with a SpyTag by simply mixing them in an aqueous solution. This post-assembly loading system, which circumvents the exposure of guests to thiol-reactive crosslinkers, enables the packaging of enzymes possessing a catalytic cysteine or a metal cofactor while retaining their catalytic activity.

Pentapeptide repeat protein QnrB1 requires ATP hydrolysis to rejuvenate poisoned gyrase complexes.

DNA gyrase, a type II topoisomerase found predominantly in bacteria, is the target for a variety of 'poisons', namely natural product toxins (e.g. albicidin, microcin B17) and clinically important synthetic molecules (e.g. fluoroquinolones). Resistance to both groups can be mediated by pentapeptide repeat proteins (PRPs). Despite long-term studies, the mechanism of action of these protective PRPs is not known. We show that a PRP, QnrB1 provides specific protection against fluoroquinolones, which strictly requires ATP hydrolysis by gyrase. QnrB1 binds to the GyrB protein and stimulates ATPase activity of the isolated N-terminal ATPase domain of GyrB (GyrB43). We probed the QnrB1 binding site using site-specific incorporation of a photoreactive amino acid and mapped the crosslinks to the GyrB43 protein. We propose a model in which QnrB1 binding allosterically promotes dissociation of the fluoroquinolone molecule from the cleavage complex.

Artificial Protein Cage with Unusual Geometry and Regularly Embedded Gold Nanoparticles.

Artificial protein cages have great potential in a number of areas including cargo capture and delivery and as artificial vaccines. Here, we investigate an artificial protein cage whose assembly is triggered by gold nanoparticles. Using biochemical and biophysical methods we were able to determine both the mechanical properties and the gross compositional features of the cage which, combined with mathematical models and biophysical data, allowed the structure of the cage to be predicted. The accuracy of the overall geometrical prediction was confirmed by the cryo-EM structure determined to sub-5 Å resolution. This showed the cage to be nonregular but similar to a dodecahedron, being constructed from 12 11-membered rings. Surprisingly, the structure revealed that the cage also contained a single, small gold nanoparticle at each 3-fold axis meaning that each cage acts as a synthetic framework for regular arrangement of 20 gold nanoparticles in a three-dimensional lattice.

Artificial Protein Cage Delivers Active Protein Cargos to the Cell Interior.

Artificial protein cages have potential as programmable, protective carriers of fragile macromolecules to cells. While natural cages and VLPs have been extensively exploited, the use of artificial cages to deliver active proteins to cells has not yet been shown. TRAP-cage is an artificial protein cage with an unusual geometry and extremely high stability, which can be triggered to break apart in the presence of cellular reducing agents. Here, we demonstrate that TRAP-cage can be filled with a protein cargo and decorated with a cell-penetrating peptide, allowing it to enter cells. Tracking of both the TRAP-cage and the cargo shows that the protein of interest can be successfully delivered intracellularly in the active form. These results provide a valuable proof of concept for the further development of TRAP-cage as a delivery platform.

A bacteriophage mimic of the bacterial nucleoid-associated protein Fis.

We report the identification and characterization of a bacteriophage λ-encoded protein, NinH. Sequence homology suggests similarity between NinH and Fis, a bacterial nucleoid-associated protein (NAP) involved in numerous DNA topology manipulations, including chromosome condensation, transcriptional regulation and phage site-specific recombination. We find that NinH functions as a homodimer and is able to bind and bend double-stranded DNA in vitro. Furthermore, NinH shows a preference for a 15 bp signature sequence related to the degenerate consensus favored by Fis. Structural studies reinforced the proposed similarity to Fis and supported the identification of residues involved in DNA binding which were demonstrated experimentally. Overexpression of NinH proved toxic and this correlated with its capacity to associate with DNA. NinH is the first example of a phage-encoded Fis-like NAP that likely influences phage excision-integration reactions or bacterial gene expression.

Three-Dimensional Protein Cage Array Capable of Active Enzyme Capture and Artificial Chaperone Activity.

Development of protein cages for encapsulation of active enzyme cargoes and their subsequent arrangement into a controllable three-dimensional array is highly desirable. However, cargo capture is typically challenging because of difficulties in achieving reversible assembly/disassembly of protein cages in mild conditions. Herein we show that by using an unusual ferritin cage protein that undergoes triggerable assembly under mild conditions, we can achieve reversible filling with protein cargoes including an active enzyme. We demonstrate that these filled cages can be arrayed in three-dimensional crystal lattices and have an additional chaperone-like effect, increasing both thermostability and enzymatic activity of the encapsulated enzyme.

Reciprocal Nucleopeptides as the Ancestral Darwinian Self-Replicator.

Even the simplest organisms are too complex to have spontaneously arisen fully formed, yet precursors to first life must have emerged ab initio from their environment. A watershed event was the appearance of the first entity capable of evolution: the Initial Darwinian Ancestor. Here, we suggest that nucleopeptide reciprocal replicators could have carried out this important role and contend that this is the simplest way to explain extant replication systems in a mathematically consistent way. We propose short nucleic acid templates on which amino-acylated adapters assembled. Spatial localization drives peptide ligation from activated precursors to generate phosphodiester-bond-catalytic peptides. Comprising autocatalytic protein and nucleic acid sequences, this dynamical system links and unifies several previous hypotheses and provides a plausible model for the emergence of DNA and the operational code.

The Three S's for Aptamer-Mediated Control of DNA Nanostructure Dynamics: Shape, Self-Complementarity, and Spatial Flexibility.

DNA aptamers are ideal tools to enable modular control of the dynamics of DNA nanostructures. For molecular recognition, they have a particular advantage over antibodies in that they can be integrated into DNA nanostructures in a bespoke manner by base pairing or nucleotide extension without any complex bioconjugation strategy. Such simplicity will be critical upon considering advanced therapeutic and diagnostic applications of DNA nanostructures. However, optimizing DNA aptamers for functional control of the dynamics of DNA nanostructure can be challenging. Herein, we present three considerations-shape, self-complementarity, and spatial flexibility-that should be paramount upon optimizing aptamer functionality. These lessons, learnt from the growing number of aptamer-nanostructure reports thus far, will be helpful for future studies in which aptamers are used to control the dynamics of nucleic acid nanostructures.

An aptamer-enabled DNA nanobox for protein sensing.

DNA nanostructures can show dynamic responses to molecular triggers for a wide variety of applications. While DNA sequence signal triggers are now well-established, there is a critical need for a broader diversity of molecular triggers to drive dynamic responses in DNA nanostructures. DNA aptamers are ideal; they can both seamlessly integrate into DNA nanostructure scaffolds and transduce molecular recognition into functional responses. Here, we report construction and optimization of a DNA origami nanobox locked by a pair of DNA double strands where one strand is a DNA aptamer targeting the malaria biomarker protein Plasmodium falciparum lactate dehydrogenase. The protein acts as the key which enables box opening. We observe highly specific protein-mediated box opening by both transmission electron microscopy and fluorescence. Aptamer-enabled DNA boxes have significant potential for enabling direct responses to proteins and other biomolecules in nanoscale diagnostics, drug delivery and sensing devices.

Virus-Templated Near-Amorphous Iron Oxide Nanotubes.

We present a simple synthesis of iron oxide nanotubes, grown under very mild conditions from a solution containing Fe(II) and Fe(III), on rod-shaped tobacco mosaic virus templates. Their well-defined shape and surface chemistry suggest that these robust bionanoparticles are a versatile platform for synthesis of small, thin mineral tubes, which was achieved efficiently. Various characterization tools were used to explore the iron oxide in detail: Electron microscopy (SEM, TEM), magnetometry (SQUID-VSM), diffraction (XRD, TEM-SAED), electron spectroscopies (EELS, EDX, XPS), and X-ray absorption (XANES with EXAFS analysis). They allowed determination of the structure, crystallinity, magnetic properties, and composition of the tubes. The protein surface of the viral templates was crucial to nucleate iron oxide, exhibiting analogies to biomineralization in natural compartments such as ferritin cages.

Probing structural dynamics of an artificial protein cage using high-speed atomic force microscopy.

A cysteine-substituted mutant of the ring-shaped protein TRAP (trp-RNA binding attenuation protein) can be induced to self-assemble into large, monodisperse hollow spherical cages in the presence of 1.4 nm diameter gold nanoparticles. In this study we use high-speed atomic force microscopy (HS-AFM) to probe the dynamics of the structural changes related to TRAP interactions with the gold nanoparticle as well as the disassembly of the cage structure. The dynamic aggregation of TRAP protein in the presence of gold nanoparticles was observed, including oligomeric rearrangements, consistent with a role for gold in mediating intermolecular disulfide bond formation. We were also able to observe that the TRAP-cage is composed of multiple, closely packed TRAP rings in an apparently regular arrangement. A potential role for inter-ring disulfide bonds in forming the TRAP-cage was shown by the fact that ring-ring interactions were reversed upon the addition of reducing agent dithiothreitol. A dramatic disassembly of TRAP-cages was observed using HS-AFM after the addition of dithiothreitol. To the best of our knowledge, this is the first report to show direct high-resolution imaging of the disassembly process of a large protein complex in real time.

Squaring up to DNA: pentapeptide repeat proteins and DNA mimicry.

Pentapeptide repeats are a class of proteins characterized by the presence of multiple repeating sequences five amino acids in length. The sequences fold into a right-handed ß-helix with a roughly square-shaped cross section. Pentapeptide repeat proteins include a number of examples which are thought to function as structural mimics of DNA and act to competitively bind to the type II topoisomerase DNA gyrase, an important antibacterial target. DNA gyrase-targeting pentapeptide repeat proteins can both inhibit DNA gyrase-a potentially useful therapeutic property-and contribute to resistance to quinolone antibacterials (by acting to prevent them forming a lethal complex with the DNA and enzyme). Pentapeptide repeat proteins are therefore of wide interest not only because of their unusual structure, function, and potential as an antibacterial target, but also because knowledge of their mechanism of action may lead to both a greater understanding of the details of DNA gyrase function as well as being a useful template for the design of new DNA gyrase inhibitors. However, many puzzling aspects as to how these DNA mimics function and indeed even their ability to act as DNA mimics itself remains open to question. This review summarizes the current state of knowledge regarding pentapeptide repeat proteins, focusing on those that are thought to mimic DNA, and speculates on potential structure-function relationships which may account for their differing specificities.

An artificial protein cage made from a 12-membered ring.

Artificial protein cages have great potential in diverse fields including as vaccines and drug delivery vehicles. TRAP-cage is an artificial protein cage notable for the way in which the interface between its ring-shaped building blocks can be modified such that the conditions under which cages disassemble can be controlled. To date, TRAP-cages have been constructed from homo-11mer rings, i.e., hendecamers. This is interesting as convex polyhedra with identical regular faces cannot be formed from hendecamers. TRAP-cage overcomes this limitation due to intrinsic flexibility, allowing slight deformation to absorb any error. The resulting TRAP-cage made from 24 TRAP 11mer rings is very close to regular with only very small errors necessary to allow the cage to form. The question arises as to the limits of the error that can be absorbed by a protein structure in this way before the formation of an apparently regular convex polyhedral becomes impossible. Here we use a naturally occurring TRAP variant consisting of twelve identical monomers (i.e., a dodecamer) to probe these limits. We show that it is able to form an apparently regular protein cage consisting of twelve TRAP rings. Comparison of the cryo-EM structure of the new cage with theoretical models and related cages gives insight into the rules of cage formation and allows us to predict other cages that may be formed given TRAP-rings consisting of different numbers of monomers.

Gold nanoparticle-induced formation of artificial protein capsids.

Gold nanoparticles are generally considered to be biologically inactive. However, in this study we show that the addition of 1.4 nm diameter gold nanoparticle induces the remodeling of the ring-shaped protein TRAP into a hollow, capsid-like configuration. This structural remodeling is dependent upon the presence of cysteine residues on the TRAP surface as well as the specific type of gold nanoparticle. The results reveal an apparent novel catalytic role of gold nanoparticles.

Production of Metallic Alloy Nanowires and Particles Templated Using Tomato Mosaic Virus (ToMV).

We demonstrate a simple, low-energy method whereby tomato mosaic virus (ToMV) particles can be used to template the production of nanowires and particles consisting of alloys of gold (Au), platinum (Pt) and palladium (Pd) in various combinations. Selective nanowire growth within the inner channel of the particles was achieved using the polymeric capping agent polyvinylpyrrolidone (PVPK30) and the reducing agent ascorbic acid. The reaction conditions also resulted in the deposition of alloy nanoparticles on the external surface of the rods in addition to the nanowire structures within the internal cavity. The resulting materials were characterized using a variety of electron microscopic and spectroscopic techniques, which revealed both the structural and chemical composition of the alloys within the nanomaterials.

Complementary charge-driven encapsulation of functional protein by engineered protein cages in cellulo.

Charge-driven inclusion complex formation in live cells was examined using a degradation-prone fluorescent protein and a series of protein cages. The results show that sufficiently strong host-guest ionic interaction and an intact shell-like structure are crucial for the protective guest encapsulation.

CRAFTing Delivery of Membrane Proteins into Protocells using Nanodiscs.

For the successful generative engineering of functional artificial cells, a convenient and controllable means of delivering membrane proteins into membrane lipid bilayers is necessary. Here we report a delivery system that achieves this by employing membrane protein-carrying nanodiscs and the calcium-dependent fusion of phosphatidylserine lipid membranes. We show that lipid nanodiscs can fuse a transported lipid bilayer with the lipid bilayers of small unilamellar vesicles (SUVs) or giant unilamellar vesicles (GUVs) while avoiding recipient vesicles aggregation. This is triggered by a simple, transient increase in calcium concentration, which results in efficient and rapid fusion in a one-pot reaction. Furthermore, nanodiscs can be loaded with membrane proteins that can be delivered into target SUV or GUV membranes in a detergent-independent fashion while retaining their functionality. Nanodiscs have a proven ability to carry a wide range of membrane proteins, control their oligomeric state, and are highly adaptable. Given this, our approach may be the basis for the development of useful tools that will allow bespoke delivery of membrane proteins to protocells, equipping them with the cell-like ability to exchange material across outer/subcellular membranes.

Molecular mechanism of topoisomerase poisoning by the peptide antibiotic albicidin.

The peptide antibiotic albicidin is a DNA topoisomerase inhibitor with low-nanomolar bactericidal activity towards fluoroquinolone-resistant Gram-negative pathogens. However, its mode of action is poorly understood. We determined a 2.6 Å resolution cryoelectron microscopy structure of a ternary complex between Escherichia coli topoisomerase DNA gyrase, a 217 bp double-stranded DNA fragment and albicidin. Albicidin employs a dual binding mechanism where one end of the molecule obstructs the crucial gyrase dimer interface, while the other intercalates between the fragments of cleaved DNA substrate. Thus, albicidin efficiently locks DNA gyrase, preventing it from religating DNA and completing its catalytic cycle. Two additional structures of this trapped state were determined using synthetic albicidin analogues that demonstrate improved solubility, and activity against a range of gyrase variants and E. coli topoisomerase IV. The extraordinary promiscuity of the DNA-intercalating region of albicidins and their excellent performance against fluoroquinolone-resistant bacteria holds great promise for the development of last-resort antibiotics.