Sortase-Modified Cholera Toxoids Show Specific Golgi Localization.

Cholera toxoid is an established tool for use in cellular tracing in neuroscience and cell biology. We use a sortase labeling approach to generate site-specific N-terminally modified variants of both the A2-B5 heterohexamer and B5 pentamer forms of the toxoid. Both forms of the toxoid are endocytosed by GM1-positive mammalian cells, and while the heterohexameric toxoid was principally localized in the ER, the B5 pentamer showed an unexpectedly specific localization in the medial/trans-Golgi. This study suggests a future role for specifically labeled cholera toxoids in live-cell imaging beyond their current applications in neuronal tracing and labeling of lipid rafts in fixed cells.

The Mutagenic Plasticity of the Cholera Toxin B-Subunit Surface Residues: Stability and Affinity.

Mastering selective molecule trafficking across human cell membranes poses a formidable challenge in healthcare biotechnology while offering the prospect of breakthroughs in drug delivery, gene therapy, and diagnostic imaging. The cholera toxin B-subunit (CTB) has the potential to be a useful cargo transporter for these applications. CTB is a robust protein that is amenable to reengineering for diverse applications; however, protein redesign has mostly focused on modifications of the N- and C-termini of the protein. Exploiting the full power of rational redesign requires a detailed understanding of the contributions of the surface residues to protein stability and binding activity. Here, we employed Rosetta-based computational saturation scans on 58 surface residues of CTB, including the GM1 binding site, to analyze both ligand-bound and ligand-free structures to decipher mutational effects on protein stability and GM1 affinity. Complimentary experimental results from differential scanning fluorimetry and isothermal titration calorimetry provided melting temperatures and GM1 binding affinities for 40 alanine mutants among these positions. The results showed that CTB can accommodate diverse mutations while maintaining its stability and ligand binding affinity. These mutations could potentially allow modification of the oligosaccharide binding specificity to change its cellular targeting, alter the B-subunit intracellular routing, or impact its shelf-life and in vivo half-life through changes to protein stability. We anticipate that the mutational space maps presented here will serve as a cornerstone for future CTB redesigns, paving the way for the development of innovative biotechnological tools.

Quantitative N- or C-Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d-Aminopeptidase.

Quantitative and selective labelling of proteins is widely used in both academic and industrial laboratories, and catalytic labelling of proteins using transpeptidases, such as sortases, has proved to be a popular strategy for such selective modification. A major challenge for this class of enzymes is that the majority of procedures require an excess of the labelling reagent or, alternatively, activated substrates rather than simple commercially sourced peptides. We report the use of a coupled enzyme strategy which enables quantitative N- and C-terminal labelling of proteins using unactivated labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence-specific degradation of the peptide by-product, shifting the equilibrium to favor product formation, which greatly enhances the reaction efficiency. Subsequent optimisation of the reaction allows N-terminal labelling of proteins using essentially equimolar ratios of peptide label to protein and C-terminal labelling with only a small excess. Minimizing the amount of substrate required for quantitative labelling has the potential to improve industrial processes and facilitate the use of transpeptidation as a method for protein labelling.

Combined Application of Orthogonal Sortases and Depsipeptide Substrates for Dual Protein Labeling.

Staphylococcus aureus sortase A is a transpeptidase that has been extensively exploited for site-specific modification of proteins and was originally used to attach a labeling reagent containing an LPXTG recognition sequence to a protein or peptide with an N-terminal glycine. Sortase mutants with other recognition sequences have also been reported, but in all cases, the reversibility of the transpeptidation reaction limits the efficiency of sortase-mediated labeling reactions. For the wildtype sortase, depsipeptide substrates, in which the scissile peptide bond is replaced with an ester, allow effectively irreversible sortase-mediated labeling as the alcohol byproduct is a poor competing nucleophile. In this paper, the use of depsipeptide substrates for evolved sortase variants is reported. Substrate specificities of three sortases have been investigated allowing identification of an orthogonal pair of enzymes accepting LPEToG and LPESoG depsipeptides, which have been applied to dual N-terminal labeling of a model protein mutant containing a second, latent N-terminal glycine residue. The method provides an efficient orthogonal site-specific labeling technique that further expands the biochemical protein labeling toolkit.

Membrane Fusion Mediated by Non-covalent Binding of Re-engineered Cholera Toxin Assemblies to Glycolipids.

Membrane fusion is essential for the transport of macromolecules and viruses across membranes. While glycan-binding proteins (lectins) often initiate cellular adhesion, subsequent fusion events require additional protein machinery. No mechanism for membrane fusion arising from simply a protein binding to membrane glycolipids has been described thus far. Herein, we report that a biotinylated protein derived from cholera toxin becomes a fusogenic lectin upon cross-linking with streptavidin. This novel reengineered protein brings about hemifusion and fusion of vesicles as demonstrated by mixing of fluorescently labeled lipids between vesicles as well as content mixing of liposomes filled with fluorescently labeled dextran. Exclusion of the complex at vesicle-vesicle interfaces could also be observed, indicating the formation of hemifusion diaphragms. Discovery of this fusogenic lectin complex demonstrates that new emergent properties can arise from simple changes in protein architecture and provides insights into new mechanisms of lipid-driven fusion.

Bioorthogonal, Bifunctional Linker for Engineering Synthetic Glycoproteins.

Post-translational glycosylation of proteins results in complex mixtures of heterogeneous protein glycoforms. Glycoproteins have many potential applications from fundamental studies of glycobiology to potential therapeutics, but generating homogeneous recombinant glycoproteins using chemical or chemoenzymatic reactions to mimic natural glycoproteins or creating homogeneous synthetic neoglycoproteins is a challenging synthetic task. In this work, we use a site-specific bioorthogonal approach to produce synthetic homogeneous glycoproteins. We develop a bifunctional, bioorthogonal linker that combines oxime ligation and strain-promoted azide-alkyne cycloaddition chemistry to functionalize reducing sugars and glycan derivatives for attachment to proteins. We demonstrate the utility of this minimal length linker by producing neoglycoprotein inhibitors of cholera toxin in which derivatives of the disaccharide lactose and GM1os pentasaccharide are attached to a nonbinding variant of the cholera toxin B-subunit that acts as a size- and valency-matched multivalent scaffold. The resulting neoglycoproteins decorated with GM1 ligands inhibit cholera toxin B-subunit adhesion with a picomolar IC50.

Synthesis of cholera toxin B subunit glycoconjugates using site-specific orthogonal oxime and sortase ligation reactions.

The chemoenzymatic synthesis of a series of dual N- and C-terminal-functionalized cholera toxin B subunit (CTB) glycoconjugates is described. Mucin 1 peptides bearing different levels of Tn antigen glycosylation [MUC1(Tn)] were prepared via solid-phase peptide synthesis. Using sortase-mediated ligation, the MUC1(Tn) epitopes were conjugated to the C-terminus of CTB in a well-defined manner allowing for high-density display of the MUC1(Tn) epitopes. This work explores the challenges of using sortase-mediated ligation in combination with glycopeptides and the practical considerations to obtain high levels of conjugation. Furthermore, we describe methods to combine two orthogonal labeling methodologies, oxime- and sortase-mediated ligation, to expand the biochemical toolkit and produce dual N- and C-terminal-labeled conjugates.

The N-terminal substrate specificity of the SurE peptide cyclase.

SurE is a standalone peptide cyclase essential for the production of surugamide antibiotics. Although SurE catalyses the cyclisation of varied nonribosomal peptides in vivo, its substrate specificity is poorly understood. To address this issue, an on-resin SurE cyclisation assay was developed and in combination with SNAC thioesters and kinetic measurements was used to define the chemical space of the N-terminal substrate residue.

In-Depth Characterization of a Re-Engineered Cholera Toxin Manufacturing Process Using Growth-Decoupled Production in Escherichia coli.

Non-toxic derivatives of the cholera toxin are extensively used in neuroscience, as neuronal tracers to reveal the location of cells in the central nervous system. They are, also, being developed as vaccine components and drug-delivery vehicles. Production of cholera-toxin derivatives is often non-reproducible; the quality and quantity require extensive fine-tuning to produce them in lab-scale settings. In our studies, we seek a resolution to this problem, by expanding the molecular toolbox of the Escherichia coli expression system with suitable production, purification, and offline analytics, to critically assess the quality of a probe or drug delivery, based on a non-toxic derivative of the cholera toxin. We present a re-engineered Cholera Toxin Complex (rCTC), wherein its toxic A1 domain was replaced with Maltose Binding Protein (MBP), as a model for an rCTC-based targeted-delivery vehicle. Here, we were able to improve the rCTC production by 11-fold (168 mg/L vs. 15 mg/L), in comparison to a host/vector combination that has been previously used (BL21(DE3) pTRBAB5-G1S). This 11-fold increase in the rCTC production capability was achieved by (1) substantial vector backbone modifications, (2) using Escherichia coli strains capable of growth-decoupling (V strains), (3) implementing a well-tuned fed-batch production protocol at a 1 L scale, and (4) testing the stability of the purified product. By an in-depth characterization of the production process, we revealed that secretion of rCTC across the E. coli Outer Membrane (OM) is processed by the Type II secretion-system general secretory pathway (gsp-operon) and that cholera toxin B-pentamerization is, likely, the rate-limiting step in complex formation. Upon successful manufacturing, we have validated the biological activity of rCTC, by measuring its binding affinity to its carbohydrate receptor GM1 oligosaccharide (Kd = 40 nM), or binding to Jurkat cells (93 pM) and delivering the cargo (MBP) in a retrograde fashion to the cell.

Challenges in the use of sortase and other peptide ligases for site-specific protein modification.

Site-specific protein modification is a widely-used biochemical tool. However, there are many challenges associated with the development of protein modification techniques, in particular, achieving site-specificity, reaction efficiency and versatility. The engineering of peptide ligases and their substrates has been used to address these challenges. This review will focus on sortase, peptidyl asparaginyl ligases (PALs) and variants of subtilisin; detailing how their inherent specificity has been utilised for site-specific protein modification. The review will explore how the engineering of these enzymes and substrates has led to increased reaction efficiency mainly due to enhanced catalytic activity and reduction of reversibility. It will also describe how engineering peptide ligases to broaden their substrate scope is opening up new opportunities to expand the biochemical toolkit, particularly through the development of techniques to conjugate multiple substrates site-specifically onto a protein using orthogonal peptide ligases.

Piggybacking on the Cholera Toxin: Identification of a CTB-Binding Protein as an Approach for Targeted Delivery of Proteins to Motor Neurons.

A significant unmet need exists for the delivery of biologic drugs such as polypeptides or nucleic acids to the central nervous system for the treatment and understanding of neurodegenerative diseases. Naturally occurring bacterial toxins have been considered as tools to meet this need. However, due to the complexity of tethering macromolecular drugs to toxins and the inherent dangers of working with large quantities of recombinant toxins, no such route has been successfully exploited. Developing a method where a bacterial toxin's nontoxic targeting subunit can be assembled with a drug immediately prior to in vivo administration has the potential to circumvent some of these issues. Using a phage-display screen, we identified two antibody mimetics, anticholera toxin Affimer (ACTA)-A2 and ACTA-C6 that noncovalently associate with the nonbinding face of the cholera toxin B-subunit. In a first step toward the development of a nonviral motor neuron drug-delivery vehicle, we show that Affimers can be selectively delivered to motor neurons in vivo.

A Standalone ß-Ketoreductase Acts Concomitantly with Biosynthesis of the Antimycin Scaffold.

Antimycins are anticancer compounds produced by a hybrid nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) pathway. The biosynthesis of these compounds is well characterized, with the exception of the standalone ß-ketoreductase enzyme AntM that is proposed to catalyze the reduction of the C8 carbonyl of the antimycin scaffold. Inactivation of antM and structural characterization suggested that rather than functioning as a post-PKS tailoring enzyme, AntM acts upon the terminal biosynthetic intermediate while it is tethered to the PKS acyl carrier protein. Mutational analysis identified two amino acid residues (Tyr185 and Phe223) that are proposed to serve as checkpoints controlling substrate access to the AntM active site. Aromatic checkpoint residues are conserved in uncharacterized standalone ß-ketoreductases, indicating that they may also act concomitantly with synthesis of the scaffold. These data provide novel mechanistic insights into the functionality of standalone ß-ketoreductases and will enable their reprogramming for combinatorial biosynthesis.

The Desotamide Family of Antibiotics.

Microbial natural products underpin the majority of antimicrobial compounds in clinical use and the discovery of new effective antibacterial treatments is urgently required to combat growing antimicrobial resistance. Non-ribosomal peptides are a major class of natural products to which many notable antibiotics belong. Recently, a new family of non-ribosomal peptide antibiotics were discovered-the desotamide family. The desotamide family consists of desotamide, wollamide, surugamide, ulleungmycin and noursamycin/curacomycin, which are cyclic peptides ranging in size between six and ten amino acids in length. Their biosynthesis has attracted significant attention because their highly functionalised scaffolds are cyclised by a recently identified standalone cyclase. Here, we provide a concise review of the desotamide family of antibiotics with an emphasis on their biosynthesis.

A versatile cholera toxin conjugate for neuronal targeting and tracing.

Tracing of neurons plays an essential role in elucidating neural networks in the brain and spinal cord. Cholera toxin B subunit (CTB) is already widely used as a tracer although its use is limited by the need for immunohistochemical detection. A new construct incorporating non-canonical azido amino acids (azido-CTB) offers a novel way to expand the range and flexibility of this neuronal tracer. Azido-CTB can be detected rapidly in vivo following intramuscular tongue injection by 'click' chemistry, eliminating the need for antibodies. Cadmium selenide/zinc sulfide (CdSe/ZnS) core/shell nanoparticles were attached to azido-CTB by strain-promoted alkyne-azide cycloaddition to make a nano-conjugate. Following tongue injections the complex was detected in vivo in the brainstem by light microscopy and electron microscopy via silver enhancement. This method does not require membrane permeabilization and so ultrastructure is maintained. Azido-CTB offers new possibilities to enhance the utility of CTB as a neuronal tracer and delivery vehicle by modification using 'click' chemistry.

A 'catch-and-release' receptor for the cholera toxin.

Stimuli-responsive receptors for the recognition unit of the cholera toxin (CTB) have been prepared by attaching multiple copies of its natural carbohydrate ligand, the GM1 oligosaccharide, to a thermoresponsive polymer scaffold. Below their lower critical solution temperature (LCST), polymers complex CTB with nanomolar affinity. When heated above their LCST, polymers undergo a reversible coil to globule transition which renders a proportion of the carbohydrate recognition motifs inaccessible to CTB. This thermally-modulated decrease in the avidity of the material for the protein has been used to reversibly capture CTB from solution, enabling its convenient isolation from a complex mixture.

Control of conformation in α-helix mimicking aromatic oligoamide foldamers through interactions between adjacent side-chains.

The design, synthesis and structural characterization of non-natural oligomers that adopt well-defined conformations, so called foldamers, is a key objective in developing biomimetic 3D functional architectures. For the aromatic oligoamide foldamer family, use of interactions between side-chains to control conformation is underexplored. The current manuscript addresses this objective through the design, synthesis and conformational analyses of model dimers derived from 3-O-alkylated para-aminobenzoic acid monomers. The O-alkyl groups on these foldamers are capable of adopting syn- or anti-conformers through rotation around the Ar-CO/NH axes. In the syn-conformation this allows the foldamer to act as a topographical mimic of the α-helix whereby the O-alkyl groups mimic the spatial orientation of the i and i + 4 side-chains from the α-helix. Using molecular modelling and 2D NMR analyses, this work illustrates that covalent links and hydrogen-bonding interactions between side-chains can bias the conformation in favour of the α-helix mimicking syn-conformer, offering insight that may be more widely applied to control secondary structure in foldamers.

A catalytic protein-proteomimetic complex: using aromatic oligoamide foldamers as activators of RNase S.

Foldamers are abiotic molecules that mimic the ability of bio-macromolecules to adopt well-defined and organised secondary, tertiary or quaternary structure. Such templates have enabled the generation of defined architectures which present structurally defined surfaces that can achieve molecular recognition of diverse and complex targets. Far less explored is whether this mimicry of nature can extend to more advanced functions of biological macromolecules such as the generation and activation of catalytic function. In this work, we adopt a novel replacement strategy whereby a segment of protein structure (the S-peptide from RNase S) is replaced by a foldamer that mimics an α-helix. The resultant prosthetic replacement forms a non-covalent complex with the S-protein leading to restoration of catalytic function, despite the absence of a key catalytic residue. Thus this functional protein-proteomimetic complex provides proof that significant segments of protein can be replaced with non-natural building blocks that may, in turn, confer advantageous properties.

Directed Assembly of Homopentameric Cholera Toxin B-Subunit Proteins into Higher-Order Structures Using Coiled-Coil Appendages.

The self-assembly of proteins into higher order structures is ubiquitous in living systems. It is also an essential process for the bottom-up creation of novel molecular architectures and devices for synthetic biology. However, the complexity of protein-protein interaction surfaces makes it challenging to mimic natural assembly processes in artificial systems. Indeed, many successful computationally designed protein assemblies are prescreened for "designability", limiting the choice of components. Here, we report a simple and pragmatic strategy to assemble chosen multisubunit proteins into more complex structures. A coiled-coil domain appended to one face of the pentameric cholera toxin B-subunit (CTB) enabled the ordered assembly of tubular supra-molecular complexes. Analysis of a tubular structure determined by X-ray crystallography has revealed a hierarchical assembly process that displays features reminiscent of the polymorphic assembly of polyomavirus proteins. The approach provides a simple and straightforward method to direct the assembly of protein building blocks which present either termini on a single face of an oligomer. This scaffolding approach can be used to generate bespoke supramolecular assemblies of functional proteins. Additionally, structural resolution of the scaffolded assemblies highlight "native-state" forced protein-protein interfaces, which may prove useful as starting conformations for future computational design.

A trans-Acting Cyclase Offloading Strategy for Nonribosomal Peptide Synthetases.

The terminal step in the biosynthesis of nonribosomal peptides is the hydrolytic release and, frequently, macrocyclization of an aminoacyl-S-thioester by an embedded thioesterase. The surugamide biosynthetic pathway is composed of two nonribosomal peptide synthetase (NRPS) assembly lines in which one produces surugamide A, which is a cyclic octapeptide, and the other produces surugamide F, a linear decapeptide. The terminal module of each system lacks an embedded thioesterase, which led us to question how the peptides are released from the assembly line (and cyclized in the case of surugamide A). We characterized a cyclase belonging to the ß-lactamase superfamily in vivo, established that it is a trans-acting release factor for both compounds, and verified this functionality in vitro with a thioester mimic of linear surugamide A. Using bioinformatics, we estimate that ∼11% of filamentous Actinobacteria harbor an NRPS system lacking an embedded thioesterase and instead employ a trans-acting cyclase. This study improves the paradigmatic understanding of how nonribosomal peptides are released from the terminal peptidyl carrier protein and adds a new dimension to the synthetic biology toolkit.

Identification of a cyanobacterial aldehyde dehydrogenase that produces retinoic acid in vitro.

Retinoic acid signalling is generally considered to be of animal origin. Recently, retinoic acid has been identified in cyanobacteria, yet no mechanism for its production has been identified. Here, we characterise for the first time a cyanobacterial aldehyde dehydrogenase that produces retinoic acid in vitro. Our computational studies suggest that the cyanobacterial aldehyde dehydrogenase resembles an ancestor of both eukaryotic aldehyde dehydrogenase 1 and aldehyde dehydrogenase 2. The Chlorogloeopsis fritschii aldehyde dehydrogenase described here may find applications in synthetic production of retinoic acid as well as contributing to our understanding of retinoid synthesis in cyanobacteria.