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.

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.

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.

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.

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.

Double quick, double click reversible peptide "stapling".

The development of constrained peptides for inhibition of protein-protein interactions is an emerging strategy in chemical biology and drug discovery. This manuscript introduces a versatile, rapid and reversible approach to constrain peptides in a bioactive helical conformation using BID and RNase S peptides as models. Dibromomaleimide is used to constrain BID and RNase S peptide sequence variants bearing cysteine (Cys) or homocysteine (hCys) amino acids spaced at i and i + 4 positions by double substitution. The constraint can be readily removed by displacement of the maleimide using excess thiol. This new constraining methodology results in enhanced α-helical conformation (BID and RNase S peptide) as demonstrated by circular dichroism and molecular dynamics simulations, resistance to proteolysis (BID) as demonstrated by trypsin proteolysis experiments and retained or enhanced potency of inhibition for Bcl-2 family protein-protein interactions (BID), or greater capability to restore the hydrolytic activity of the RNAse S protein (RNase S peptide). Finally, use of a dibromomaleimide functionalized with an alkyne permits further divergent functionalization through alkyne-azide cycloaddition chemistry on the constrained peptide with fluorescein, oligoethylene glycol or biotin groups to facilitate biophysical and cellular analyses. Hence this methodology may extend the scope and accessibility of peptide stapling.

Affimer proteins are versatile and renewable affinity reagents.

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications.

Sortase-mediated labelling of lipid nanodiscs for cellular tracing.

Lipid nanodiscs have broad applications in membrane protein assays, biotechnology and materials science. Chemical modification of the nanodiscs to expand their functional attributes is generally desirable for all of these uses. We present a method for site-selective labelling of the N-terminus of the nanodisc's membrane scaffold protein (MSP) using the Sortase A protein. Labelling of the MSP was achieved when assembled within the lipid nanodisc architecture, demonstrating that this method can be used as a retrofit approach to modification of preformed nanodiscs before or during application. We label the MSP with a fluorescent fluorescein moiety and use them to image nanodisc uptake into HeLa cells. The Sortase A labelling method could be employed as a general approach to labelling nanodiscs with application-specific functionalities.

Confirmation of a Protein-Protein Interaction in the Pantothenate Biosynthetic Pathway by Using Sortase-Mediated Labelling.

High-throughput studies have been widely used to identify protein-protein interactions; however, few of these candidate interactions have been confirmed in vitro. We have used a combination of isothermal titration calorimetry and fluorescence anisotropy to screen candidate interactions within the pantothenate biosynthetic pathway. In particular, we observed no interaction between the next enzyme in the pathway, pantothenate synthetase (PS), and aspartate decarboxylase, but did observe an interaction between PS and the putative Nudix hydrolase, YfcD. Confirmation of the interaction by fluorescence anisotropy was dependent upon labelling an adventitiously formed glycine on the protein N-terminal affinity purification tag by using Sortase. Subsequent formation of the protein-protein complex led to apparent restriction of the dynamics of this tag, thus suggesting that this approach could be generally applied to a subset of other protein-protein interaction complexes.

Chemical generation and modification of peptides containing multiple dehydroalanines.

Chemical formation of dehydroalanine has been widely used for the post-translational modification of proteins and peptides, however methods to incorporate multiple dehydroalanine residues into a single peptide have not been defined. We report the use of methyl 2,5-dibromovalerate which can be used to cleanly carry out this transformation.

Evaluation of the interaction between phosphohistidine analogues and phosphotyrosine binding domains.

We have investigated the interaction of peptides containing phosphohistidine analogues and their homologues with the prototypical phosphotyrosine binding SH2 domain from the eukaryotic cell signalling protein Grb2 by using a combination of isothermal titration calorimetry and a fluorescence anisotropy competition assay. These investigations demonstrated that the triazole class of phosphohistidine analogues are capable of binding too, suggesting that phosphohistidine could potentially be detected by this class of proteins in vivo.

Depsipeptide substrates for sortase-mediated N-terminal protein ligation.

Technologies that allow the efficient chemical modification of proteins under mild conditions are widely sought after. Sortase-mediated peptide ligation provides a strategy for modifying the N or C terminus of proteins. This protocol describes the use of depsipeptide substrates (containing an ester linkage) with sortase A (SrtA) to completely modify proteins carrying a single N-terminal glycine residue under mild conditions in 4-6 h. The SrtA-mediated ligation reaction is reversible, so most labeling protocols that use this enzyme require a large excess of both substrate and sortase to produce high yields of ligation product. In contrast, switching to depsipeptide substrates effectively renders the reaction irreversible, allowing complete labeling of proteins with a small excess of substrate and catalytic quantities of sortase. Herein we describe the synthesis of depsipeptide substrates that contain an ester linkage between a threonine and glycolic acid residue and an N-terminal FITC fluorophore appended via a thiourea linkage. The synthesis of the depsipeptide substrate typically takes 2-3 d.

Prospects for stable analogues of phosphohistidine.

Phosphorylation is a ubiquitous protein post-translational modification, and the importance of phosphorylation of serine, threonine and tyrosine is well established. What is lesser known is that almost all heteroatom-containing amino acids can be phosphorylated and, among these, histidine, aspartate and cysteine have well established roles in bacterial signalling pathways. The first of these, phosphohistidine, is the most unusual in that it is labile under many conditions used to study proteins in vitro and can exist as two different isomers. In the present short review, we highlight the chemical challenges that this modification presents and the manner in which chemical synthesis has been used to identify and mimic the modification in proteins.

Triazole phosphohistidine analogues compatible with the Fmoc-strategy.

Phosphorylation of histidine is essential for bacterial two-component signalling; its importance to modulation of eukaryotic protein function remains undefined. Until recently, no immunochemical probes of this post-translational modification existed, however triazole phosphonate analogues of this modified amino acid have now been applied to the generation of site-specific antibodies. The protecting group strategy used in the original report is incompatible with standard protocols for Fmoc-solid phase peptide synthesis. In this paper, we report the application of P(III) chemistry to generate the complementary dibenzyl and di-tert-butyl phosphonate esters. These forms of the triazole analogue are fully compatible with standard Fmoc-SPPS and are therefore ideal for wider application by the chemical and biochemical community.

Characterization of the evolutionarily conserved iron-sulfur cluster of sirohydrochlorin ferrochelatase from Arabidopsis thaliana.

Sirohaem is a cofactor of nitrite and sulfite reductases, essential for assimilation of nitrogen and sulfur. Sirohaem is synthesized from the central tetrapyrrole intermediate uroporphyrinogen III by methylation, oxidation and ferrochelation reactions. In Arabidopsis thaliana, the ferrochelation step is catalysed by sirohydrochlorin ferrochelatase (SirB), which, unlike its counterparts in bacteria, contains an [Fe-S] cluster. We determined the cluster to be a [4Fe-4S] type, which quickly oxidizes to a [2Fe-2S] form in the presence of oxygen. We also identified the cluster ligands as four conserved cysteine residues located at the C-terminus. A fifth conserved cysteine residue, Cys(135), is not involved in ligating the cluster directly, but influences the oxygen-sensitivity of the [4Fe-4S] form, and possibly the affinity for the substrate metal. Substitution mutants of the enzyme lacking the Fe-S cluster or Cys(135) retain the same specific activity in vitro and dimeric quaternary structure as the wild-type enzyme. The mutant variants also rescue a defined Escherichia coli sirohaem-deficient mutant. However, the mutant enzymes cannot complement Arabidopsis plants with a null AtSirB mutation, which exhibits post-germination arrest. These observations suggest an important physiological role for the Fe-S cluster in Planta, highlighting the close association of iron, sulfur and tetrapyrrole metabolism.

Fmoc-chemistry of a stable phosphohistidine analogue.

We report the synthesis of the phosphohistidine analogue, Fmoc-4-diethylphosphonotriazolylalanine 5 and its incorporation into peptides. Our synthesis of 5 has enabled us to demonstrate that the analogue is compatible with Fmoc-solid phase peptide synthesis (SPPS) conditions. Standard cleavage conditions yield the diethyl phosphonate-protected peptide, however this can be subsequently deprotected using trimethylsilyl bromide to yield the free phosphonic acid-containing peptides.

Oligobenzamide proteomimetic inhibitors of the p53-hDM2 protein-protein interaction.

Oligobenzamide inhibitors of the p53-hDM2 protein-protein interaction are described.