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. We also highlight select recent applications of SML, including examples where fundamental studies of sortase structure and function have informed the continued development of these enzymes as tools for protein engineering.

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). This mechanism is the subject of considerable interest as a target for therapeutic intervention and as a tool for protein engineering, where sortases have enabled sortase-mediated ligation or sortagging strategies. Despite these uses, there remains an incomplete understanding of the stereochemistry of substrate recognition and ligation product formation. Here, we solved the first structures of sortase A from Streptococcus pyogenes bound to two substrate sequences, LPATA and LPATS. In addition, we synthesized a mimetic of the product of sortase-mediated ligation involving LII (LPAT-LII) and solved the complex structure in two ligand conformations. These structures were further used as the basis for molecular dynamics simulations to probe sortase A-ligand dynamics and to construct a model of the acyl-enzyme intermediate, thus providing a structural view of multiple key states in the catalytic mechanism. Overall, this structural information provides new insights into the recognition of the sortase substrate motif and LII ligation partner and will support the continued development of sortases for protein engineering applications.

Sequence variation in the ß7-ß8 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. However, the stringent specificity of saSrtA for the LPXTG sequence motif limits its uses. Here, we describe the impact on substrate selectivity of a structurally conserved loop with a high degree of sequence variability in all classes of sortases. We investigate the contribution of this ß7-ß8 loop by designing and testing chimeric sortase enzymes. Our chimeras utilize natural sequence variation of class A sortases from eight species engineered into the SrtA sequence from Streptococcus pneumoniae. While some of these chimeric enzymes mimic the activity and selectivity of the WT protein from which the loop sequence was derived (e.g., that of saSrtA), others results in chimeric Streptococcus pneumoniae SrtA enzymes that are able to accommodate a range of residues in the final position of the substrate motif (LPXTX). Using mutagenesis, structural comparisons, and sequence analyses, we identify three interactions facilitated by ß7-ß8 loop residues that appear to be broadly conserved or converged upon in class A sortase enzymes. These studies provide the foundation for a deeper understanding of sortase target selectivity and can expand the sortase toolbox for future SML applications.

Engineered Sortases in Peptide and Protein Chemistry.

The transpeptidase sortase A of Staphylococcus aureus (Sa-SrtA) is a valuable tool in protein chemistry. The native enzyme anchors surface proteins containing a highly conserved LPxTG sorting motif to a terminal glycine residue of the peptidoglycan layer in Gram-positive bacteria. This reaction is exploited for sortase-mediated ligation (SML), allowing the site-specific linkage of synthetic peptides and recombinant proteins by a native peptide bond. However, the moderate catalytic efficiency and specificity of Sa-SrtA fueled the development of new biocatalysts for SML, including the screening of sortase A variants form microorganisms other than S. aureus and the directed protein evolution of the Sa-SrtA enzyme itself. Novel display platforms and screening formats were developed to isolate sortases with altered properties from mutant libraries. This yielded sortases with strongly enhanced catalytic activity and enzymes recognizing new sorting motifs as substrates. This minireview focuses on recent advances in the field of directed sortase evolution and applications of these tailor-made enzymes in biochemistry.

Stable and selective permeable hydrogel microcapsules for high-throughput cell cultivation and enzymatic analysis.

BACKGROUND: Miniaturization of biochemical reaction volumes within artificial microcompartments has been the key driver for directed evolution of several catalysts in the past two decades. Typically, single cells are co-compartmentalized within water-in-oil emulsion droplets with a fluorogenic substrate whose conversion allows identification of catalysts with improved performance. However, emulsion droplet-based technologies prevent cell proliferation to high density and preclude the feasibility of biochemical reactions that require the exchange of small molecule substrates. Here, we report on the development of a high-throughput screening method that addresses these shortcomings and that relies on a novel selective permeable polymer hydrogel microcapsule. RESULTS: Hollow-core polyelectrolyte-coated chitosan alginate microcapsules (HC-PCAMs) with selective permeability were successfully constructed by jet break-up and layer-by-layer (LBL) technology. We showed that HC-PCAMs serve as miniaturized vessels for single cell encapsulation, enabling cell growth to high density and cell lysis to generate monoclonal cell lysate compartments suitable for high-throughput analysis using a large particle sorter (COPAS). The feasibility of using HC-PCAMs as reaction compartments which exchange small molecule substrates was demonstrated using the transpeptidation reaction catalyzed by the bond-forming enzyme sortase F from P. acnes. The polyelectrolyte shell surrounding microcapsules allowed a fluorescently labelled peptide substrate to enter the microcapsule and take part in the transpeptidation reaction catalyzed by the intracellularly expressed sortase enzyme retained within the capsule upon cell lysis. The specific retention of fluorescent transpeptidation products inside microcapsules enabled the sortase activity to be linked with a fluorescent readout and allowed clear separation of microcapsules expressing the wild type SrtF from those expressing the inactive variant. CONCLUSION: A novel polymer hydrogel microcapsule-based method, which allows for high-throughput analysis based on encapsulation of single cells has been developed. The method has been validated for the transpeptidation activity of sortase enzymes and represents a powerful tool for screening of libraries of sortases, other bond-forming enzymes, as well as of binding affinities in directed evolution experiments. Moreover, selective permeable microcapsules encapsulating microcolonies provide a new and efficient means for preparing novel caged biocatalyst and biosensor agents.

The Streptococcos suis sortases SrtB and SrtF are essential for disease in pigs.

The porcine pathogen Streptococcus suis colonizes the upper respiratory tracts of pigs, potentially causing septicaemia, meningitis and death, thus placing a severe burden on the agricultural industry worldwide. It is also a zoonotic pathogen that is known to cause systemic infections and meningitis in humans. Understanding how S. suis colonizes and interacts with its hosts is relevant for future strategies of drug and vaccine development. As with other Gram-positive bacteria, S. suis utilizes enzymes known as sortases to attach specific proteins bearing cell wall sorting signals to its surface, where they can play a role in host-pathogen interactions. The surface proteins of bacteria are often important in adhesion to and invasion of host cells. In this study, markerless in-frame deletion mutants of the housekeeping sortase srtA and the two pilus-associated sortases, srtB and srtF, were generated and their importance in S. suis infections was investigated. We found that all three of these sortases are essential to disease in pigs, concluding that their cognate-sorted proteins may also be useful in protecting pigs against infection.

Light-Controlled Chemoenzymatic Immobilization of Proteins towards Engineering of Bioactive Papers.

Efficient and reliable methods for the generation of bioactive papers are of growing interest in relation to point-of-care testing devices that do not require extensive analytical equipment. Herein, we report the immobilization of functional proteins on paper fibers using a modular chemoenzymatic approach. The synthetic strategy relies on a combination of highly efficient spatially controllable photo-triggered cycloaddition followed by site-specific sortase A-catalyzed transamidation. This site-directed and regiospecific method has allowed unidirectional and covalent immobilization of several proteins displaying different functional properties, with ramifications for application in paper-based diagnostics.

Delivery of antibody mimics into mammalian cells via anthrax toxin protective antigen.

Antibody mimics have significant scientific and therapeutic utility for the disruption of protein-protein interactions inside cells; however, their delivery to the cell cytosol remains a major challenge. Here we show that protective antigen (PA), a component of anthrax toxin, efficiently transports commonly used antibody mimics to the cytosol of mammalian cells when conjugated to the N-terminal domain of LF (LFN). In contrast, a cell-penetrating peptide (CPP) was not able to deliver any of these antibody mimics into the cell cytosol. The refolding and binding of a transported tandem monobody to Bcr-Abl (its protein target) in chronic myeloid leukemia cells were confirmed by co-immunoprecipitation. We also observed inhibition of Bcr-Abl kinase activity and induction of apoptosis caused by the monobody. In a separate case, we show disruption of key interactions in the MAPK signaling pathway after PA-mediated delivery of an affibody binder that targets hRaf-1. We show for the first time that PA can deliver bioactive antibody mimics to disrupt intracellular protein-protein interactions. This technology adds a useful tool to expand the applications of these modern agents to the intracellular milieu.

Phenotypic surface properties (aggregation, adhesion and biofilm formation) and presence of related genes in beneficial vaginal lactobacilli.

AIMS: To evaluate the phenotypic expression of auto-aggregation, adhesion to mucin and biofilm formation of lactobacilli isolated from human vagina and the presence of related genes. METHODS AND RESULTS: Seven different strains of three Lactobacillus species (Lactobacillus gasseri, Lactobacillus rhamnosus and Lactobacillus reuteri) were evaluated. The auto-aggregation property was determined by spectrophotometric assay and flow cytometry. Adhesion and biofilm formation were assayed by crystal violet staining. The presence of the genes encoding sortases, pilin subunits and surface proteins was evaluated by polymerase chain reactions. The two Lact. reuteri strains assayed showed high auto-aggregation, adhesion to mucin and biofilm formation ability. In these strains, the genes encoding three adhesion proteins were identified. In Lact. rhamnosus CRL (Centro de Referencia para Lactobacilos Culture Collection) 1332, pilus-encoding genes were detected. In all Lact. rhamnosus strains assayed, two genes encoding for other surface proteins related to adhesion and biofilm formation were detected. CONCLUSIONS: The vaginal lactobacilli assayed exhibited phenotypic and genetic characteristics that were specific for each strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on auto-aggregation, adhesion and biofilm formation of vaginal Lactobacillus strains by phenotypic and genetic assays.

Structural and functional insights of sortases and their interactions with antivirulence compounds.

Sortase proteins play a crucial role as integral membrane proteins in anchoring bacterial surface proteins by recognizing them through a Cell-Wall Sorting (CWS) motif and cleaving them at specific sites before initiating pilus assembly. Both sortases and their substrate proteins are major virulence factors in numerous Gram-positive pathogens, making them attractive targets for antimicrobial intervention. Recognizing the significance of virulence proteins, a comprehensive exploration of their structural and functional characteristics is essential to enhance our understanding of pilus assembly in diverse Gram-positive bacteria. Therefore, this review article discusses the structural features of different classes of sortases and pilin proteins, primarily serving as substrates for sortase-assembled pili. Moreover, it thoroughly examines the molecular-level interactions between sortases and their inhibitors, providing insights from both structural and functional perspectives. In essence, this review article will provide a contemporary and complete understanding of both sortase pathways and various strategies to target them effectively to counteract the virulence.

An idea to explore: Engaging high school students in structure-function studies of bacterial sortase enzymes and inhibitors - A comprehensive computational experimental pipeline.

High school science fairs provide an exceptional opportunity for students to gain experience with scientific research, and participation has positive outcomes with respect to chosen careers in the sciences. However, it can be challenging to engage high school students in university-level research outside of formal internship programs. Here, we describe an experimental pipeline for a computational structural biology project that engages high school students. Students are involved at every step of the investigation and utilize freely available software to dock inhibitors onto protein homologues, and then analyze the resulting complexes. Bacterial sortases are transpeptidases on the cell surface of Gram-positive bacteria and are a potential target for the development of antibiotics. Students modeled inhibitors bound to sortases from several organisms, asking questions about affinity and selectivity. Their project was ranked in the top 10% at both regional and state science fairs. This project design is easily adaptable to countless other protein systems and provides a pipeline for collaborative high school student/university professor inquiry.

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. These enzymes are also used in protein engineering applications for sortase-mediated ligations (SML) or sortagging of protein targets. We previously investigated SrtA from Streptococcus pneumoniae, identifying a number of putative ß7-ß8 loop-mediated interactions that affected in vitro enzyme function. We identified residues that contributed to the ability of S. pneumoniae SrtA to recognize several amino acids at the P1' position of the substrate motif, underlined in LPXTG, in contrast to the strict P1' Gly recognition of SrtA from Staphylococcus aureus. However, motivated by the lack of a structural model for the active, monomeric form of S. pneumoniae SrtA, here, we expanded our studies to other Streptococcus SrtA proteins. We solved the first monomeric structure of S. agalactiae SrtA which includes the C-terminus, and three others of ß7-ß8 loop chimeras from S. pyogenes and S. agalactiae SrtA. These structures and accompanying biochemical data support our previously identified ß7-ß8 loop-mediated interactions and provide additional insight into their role in Class A sortase substrate selectivity. A greater understanding of individual SrtA sequence and structural determinants of target selectivity may also facilitate the design or discovery of improved sortagging tools.

Discerning the catalytic mechanism of Staphylococcus aureus sortase A with QM/MM free energy calculations.

Sortases are key virulence factors in Gram-positive bacteria. These enzymes embed surface proteins in the cell wall through a transpeptidation reaction that involves recognizing a penta-peptide "sorting signal" in a target protein, cleaving it, and covalently attaching it to a second substrate that is later inserted into the cell wall. Although well studied, several aspects of the mechanism by which sortases perform these functions remains unclear. In particular, experiments have revealed two potential sorting signal binding motifs: a "Threonine-Out" (Thr-Out) structure in which the catalytically critical threonine residues protrudes into solution, and a "Threonine-In" (Thr-In) configuration in which this residue inserts into the binding site. To determine which of these is the biologically relevant state, we have performed a series of conventional and hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations of the Staphylococcus aureus sortase A (SrtA) enzyme bound to a sorting signal substrate. Through the use of multi-dimensional metadynamics, our simulations were able to both map the acylation mechanism of SrtA in the Thr-In and Thr-Out states, as well as determine the free energy minima and barriers along these reactions. Results indicate that in both states the catalytic mechanisms are similar, however the free energy barriers are lower in the Thr-In configuration, suggesting that Thr-In is the catalytically relevant state. This has important implications for advancing our understanding of the mechanisms of sortase enzymes, as well we for future structure based drug design efforts aimed at inhibiting sortase function in vivo.

Site-specific immobilization of protein layers on gold surfaces via orthogonal sortases.

We report a site-specific, sortase-mediated ligation to immobilize proteins layer-by-layer on a gold surface. Recombinant fluorescent proteins with a Sortase A recognition tag at the C-terminus were immobilized on peptide-modified gold surfaces. We used two sortases with different substrate specificities (Streptococcus pyogenes Sortase A and Staphylococcus aureus Sortase A) to immobilize layers of GFP and mCherry site-specifically on the gold surface. Surfaces were characterized using fluorescence and atomic force microscopy after immobilizing each layer of protein. Fluorescent micrographs showed that both protein immobilization on the modified gold surface and protein oligomerization are sortase-dependent. AFM images showed that either homogenous protein monolayers or layers of protein oligomers can be generated using appropriately tagged substrate proteins. Using Sortase A variants with orthogonal peptide substrate specificities, site-specific immobilization of appropriately tagged GFP onto a layer of immobilized mCherry was achieved without disruption of the underlying protein layer.