Amino acid variability at W194 of Staphylococcus aureus sortase A alters nucleophile specificity.

Bacterial sortases are a family of cysteine transpeptidases in Gram-positive bacteria of which sortase A (SrtA) enzymes are responsible for ligating proteins to the peptidoglycan layer of the cell surface. Engineered versions of sortases are also used in sortase-mediated ligation (SML) strategies for a variety of protein engineering applications. Although a versatile tool, substrate recognition by Staphylococcus aureus SrtA (saSrtA), the most commonly utilized enzyme in SML, is stringent and relies on an LPXTG pentapeptide motif.

Sortases: structure, mechanism, and implications for protein engineering.

Sortase enzymes are critical cysteine transpeptidases on the surface of bacteria that attach proteins to the cell wall and are involved in the construction of bacterial pili. Due to their ability to recognize specific substrates and covalently ligate a range of reaction partners, sortases are widely used in protein engineering applications via sortase-mediated ligation (SML) strategies. In this review, we discuss recent structural studies elucidating key aspects of sortase specificity and the catalytic mechanism.

A unique binding mode of P1' Leu-containing target sequences for sortase A results in alternative cleavage.

Sortase enzymes are cysteine transpeptidases that attach environmental sensors, toxins, and other proteins to the cell surface in Gram-positive bacteria. The recognition motif for many sortases is the cell wall sorting signal (CWSS), LPXTG, where X = any amino acid. Recent work from ourselves and others has described recognition of additional amino acids at a number of positions in the CWSS, specifically at the Thr (or P1) and Gly (or P1') positions.

Structures of Streptococcus pyogenes class A sortase in complex with substrate and product mimics provide key details of target recognition.

The cell wall is a critical extracellular barrier for bacteria and many other organisms. In bacteria, this structural layer consists of peptidoglycan, which maintains cell shape and structural integrity and provides a scaffold for displaying various protein factors. To attach proteins to the cell wall, Gram-positive bacteria utilize sortase enzymes, which are cysteine transpeptidases that recognize and cleave a specific sorting signal, followed by ligation of the sorting signal-containing protein to the peptidoglycan precursor lipid II (LII).

Structural and biochemical analyses of selectivity determinants in chimeric Streptococcus Class A sortase enzymes.

Sequence variation in related proteins is an important characteristic that modulates activity and selectivity. An example of a protein family with a large degree of sequence variation is that of bacterial sortases, which are cysteine transpeptidases on the surface of gram-positive bacteria. Class A sortases are responsible for attachment of diverse proteins to the cell wall to facilitate environmental adaption and interaction.

Sortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in proteins.

A significant number of proteins possess sizable intrinsically disordered regions (IDRs). Due to the dynamic nature of IDRs, NMR spectroscopy is often the tool of choice for characterizing these segments. However, the application of NMR to IDRs is often hindered by their instability, spectral overlap and resonance assignment difficulties.

Sequence variation in the β7-β8 loop of bacterial class A sortase enzymes alters substrate selectivity.

Gram-positive bacteria contain sortase enzymes on their cell surfaces that catalyze transpeptidation reactions critical for proper cellular function. In vitro, sortases are used in sortase-mediated ligation (SML) reactions for a variety of protein engineering applications. Historically, sortase A from Staphylococcus aureus (saSrtA) has been the enzyme of choice to catalyze SML reactions.

Efficient Sortase-Mediated Ligation Using a Common C-Terminal Fusion Tag.

Sortase-mediated ligation is a powerful method for generating site-specifically modified proteins. However, this process is limited by the inherent reversibility of the ligation reaction. To address this, here we report the continued development and optimization of an experimentally facile strategy for blocking reaction reversibility.

Plant Villin Headpiece Domain Demonstrates a Novel Surface Charge Pattern and High Affinity for F-Actin.

Plants utilize multiple isoforms of villin, an F-actin regulating protein with an N-terminal gelsolin-like core and a distinct C-terminal headpiece domain. Unlike their vertebrate homologues, plant villins have a much longer linker polypeptide connecting the core and headpiece. Moreover, the linker-headpiece connection region in plant villins lacks sequence homology to the vertebrate villin sequences.

Expanding the Scope of Sortase-Mediated Ligations by Using Sortase Homologues.

Sortase-catalyzed transacylation reactions are widely used for the construction of non-natural protein derivatives. However, the most commonly used enzyme for these strategies (sortase A from Staphylococcus aureus) is limited by its narrow substrate scope. To expand the range of substrates compatible with sortase-mediated reactions, we characterized the in vitro substrate preferences of eight sortase A homologues.

Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation.

Strategies for site-specific protein modification are highly desirable for the construction of conjugates containing non-genetically-encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non-natural moieties. The transpeptidation reaction catalyzed by bacterial sortases is a prominent strategy for protein derivatization that possesses these features.

Recent advances in sortase-catalyzed ligation methodology.

The transpeptidation reaction catalyzed by bacterial sortases continues to see increasing use in the construction of novel protein derivatives. In addition to growth in the number of applications that rely on sortase, this field has also seen methodology improvements that enhance reaction performance and scope. In this opinion, we present an overview of key developments in the practice and implementation of sortase-based strategies, including applications relevant to structural biology.

Enhancing the efficiency of sortase-mediated ligations through nickel-peptide complex formation.

A modified sortase A recognition motif containing a masked Ni(2+)-binding peptide was employed to boost the efficiency of sortase-catalyzed ligation reactions. Deactivation of the Ni(2+)-binding peptide using a Ni(2+) additive improved reaction performance at low to equimolar ratios of the glycine amine nucleophile and sortase substrate. The success of this approach was demonstrated with both peptide and protein substrates.

Sustained Delivery of Chemokine CXCL12 from Chemically Modified Silk Hydrogels.

A delivery platform was developed using silk-based hydrogels, and sustained delivery of the cationic chemokine CXCL12 at therapeutically relevant doses is demonstrated. Hydrogels were prepared from plain silk and silk that had been chemically modified with sulfonic acid groups. CXCL12 was mixed with the silk solution prior to gelation, resulting in 100% encapsulation efficiency, and both hydrated and lyophilized gels were compared.

Development of an influenza virus protein array using Sortagging technology.

Protein array technology is an emerging tool that enables high-throughput screening of protein-protein or protein-lipid interactions and identification of immunodominant antigens during the course of a bacterial or viral infection. In this work, we developed an Influenza virus protein array using the sortase-mediated transpeptidation reaction known as "Sortagging". LPETG-tagged Influenza virus proteins from bacterial and eukaryotic cellular extracts were immobilized at their carboxyl-termini onto a preactivated amine-glass slide coated with a Gly3 linker.

Sortase A as a tool for high-yield histatin cyclization.

Cyclic peptides are highly valued tools in biomedical research. In many cases, they show higher receptor affinity, enhanced biological activity, and improved serum stability. Technical difficulties in producing cyclic peptides, especially larger ones, in appreciable yields have precluded a prolific use in biomedical research.

Characterization and structural studies of the Plasmodium falciparum ubiquitin and Nedd8 hydrolase UCHL3.

Like their human hosts, Plasmodium falciparum parasites rely on the ubiquitin-proteasome system for survival. We previously identified PfUCHL3, a deubiquitinating enzyme, and here we characterize its activity and changes in active site architecture upon binding to ubiquitin. We find strong evidence that PfUCHL3 is essential to parasite survival.

Site-specific N- and C-terminal labeling of a single polypeptide using sortases of different specificity.

The unique reactivity of two sortase enzymes, SrtA(staph) from Staphylococcus aureus and SrtA(strep) from Streptococcus pyogenes, is exploited for site-specific labeling of a single polypeptide with different labels at its N and C termini. SrtA(strep) is used to label the protein's C terminus at an LPXTG site with a fluorescently labeled dialanine nucleophile. Selective N-terminal labeling of proteins containing N-terminal glycine residues is achieved using SrtA(staph) and LPXT derivatives.

Chemoselective tryptophan labeling with rhodium carbenoids at mild pH.

Significant improvements have been made to a previously reported tryptophan modification method using rhodium carbenoids in aqueous solution, allowing the reaction to proceed at pH 6-7. This technique is based on the discovery that N-(tert-butyl)hydroxylamine promotes indole modification with rhodium carbenoids over a broad pH range (2-7). This methodology was demonstrated on peptide and protein substrates, generally yielding 40-60% conversion with excellent tryptophan chemoselectivity.

Site-specific protein labeling via sortase-mediated transpeptidation.

Creation of functional protein bioconjugates demands methods for attaching a diverse array of probes to target proteins with high specificity, under mild conditions. The sortase A transpeptidase enzyme from Staphylococcus aureus catalyzes the cleavage of a short 5-aa recognition sequence (LPXTG) with the concomitant formation of an amide linkage between an oligoglycine peptide and the target protein. By functionalizing the oligoglycine peptide, it is possible to incorporate reporters into target proteins in a site-specific fashion.

A straight path to circular proteins.

Folding and stability are parameters that control protein behavior. The possibility of conferring additional stability on proteins has implications for their use in vivo and for their structural analysis in the laboratory. Cyclic polypeptides ranging in size from 14 to 78 amino acids occur naturally and often show enhanced resistance toward denaturation and proteolysis when compared with their linear counterparts.

Lipid modification of proteins through sortase-catalyzed transpeptidation.

A general chemoenzymatic method for the site-specific attachment of lipids to protein substrates is described. Sortase A is used to append short lipid-modified oligoglycine peptides to the C terminus of protein substrates bearing a five amino acid sortase A recognition sequence (LPETG). We demonstrate the attachment of a range of hydrophobic modifications in excellent yield (60-90%), including a simple step for removing the sortase enzyme postreaction.

Sortagging: a versatile method for protein labeling.

Genetically encoded reporter constructs that yield fluorescently labeled fusion proteins are a powerful tool for observing cell biological phenomena, but they have limitations. Sortagging (sortase-mediated transpeptidation) is a versatile chemoenzymatic system for site-specific labeling of proteins with small (<2 kDa) probes. Sortagging combines the precision of a genetically encoded tag with the specificity of an enzymatic reaction and the ease and chemical versatility of peptide synthesis.

Transition metal catalyzed methods for site-selective protein modification.

The broad utility of protein bioconjugates has created a need for new and diverse strategies for site-selective protein modification. In particular, chemical reactions that target alternative amino acid side chains or unnatural functional groups are emerging as a valuable complement to more commonly used lysine- and cysteine-based strategies. Considering their widespread use in organic synthesis, reactions catalyzed by transition metals could provide a particularly powerful set of transformations for the continued expansion of the bioconjugation toolkit.

Selective tryptophan modification with rhodium carbenoids in aqueous solution.

A new transition metal-based reaction has been developed for the selective modification of tryptophan residues on protein substrates. After activation of vinyl-substituted diazo compounds by Rh2(OAc)4, the resulting metallocarbenoid intermediates were found to modify indoles in aqueous media despite competing reactions with water. Both N- and 2-substituted indole products were observed in the reaction.