Structure and Function of the Glycosylphosphatidylinositol Transamidase, a Transmembrane Complex Catalyzing GPI Anchoring of Proteins.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a ubiquitous posttranslational modification in eukaryotic cells. GPI-anchored proteins (GPI-APs) play critical roles in enzymatic, signaling, regulatory, and adhesion processes. Over 20 enzymes are involved in GPI synthesis, attachment to client proteins, and remodeling after attachment. The GPI transamidase (GPI-T), a large complex located in the endoplasmic reticulum membrane, catalyzes the attachment step by replacing a C-terminal signal peptide of proproteins with GPI. In the last three decades, extensive research has been conducted on the mechanism of the transamidation reaction, the components of the GPI-T complex, the role of each subunit, and the substrate specificity. Two recent studies have reported the three-dimensional architecture of GPI-T, which represent the first structures of the pathway. The structures provide detailed mechanisms for assembly that rationalizes previous biochemical results and subunit-dependent stability data. While the structural data confirm the catalytic role of PIGK, which likely uses a caspase-like mechanism to cleave the proproteins, they suggest that unlike previously proposed, GPAA1 is not a catalytic subunit. The structures also reveal a shared cavity for GPI binding. Somewhat unexpectedly, PIGT, a single-pass membrane protein, plays a crucial role in GPI recognition. Consistent with the assembly mechanisms and the active site architecture, most of the disease mutations occur near the active site or the subunit interfaces. Finally, the catalytic dyad is located ~22 Å away from the membrane interface of the GPI-binding site, and this architecture may confer substrate specificity through topological matching between the substrates and the elongated active site. The research conducted thus far sheds light on the intricate processes involved in GPI anchoring and paves the way for further mechanistic studies of GPI-T.

Sortase A-Based Post-translational Modifications on Encapsulin Nanocompartments.

Protein-based encapsulin nanocompartments, known for their well-defined structures and versatile functionalities, present promising opportunities in the fields of biotechnology and nanomedicine. In this investigation, we effectively developed a sortase A-mediated protein ligation system in Escherichia coli to site-specifically attach target proteins to encapsulin, both internally and on its surfaces without any further in vitro steps. We explored the potential applications of fusing sortase enzyme and a protease for post-translational ligation of encapsulin to a green fluorescent protein and anti-CD3 scFv. Our results demonstrated that this system could attach other proteins to the nanoparticles' exterior surfaces without adversely affecting their folding and assembly processes. Additionally, this system enabled the attachment of proteins inside encapsulins which varied shapes and sizes of the nanoparticles due to cargo overload. This research developed an alternative enzymatic ligation method for engineering encapsulin nanoparticles to facilitate the conjugation process.

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.

Molecular basis for dual functions in pilus assembly modulated by the lid of a pilus-specific sortase.

The biphasic assembly of Gram-positive pili begins with the covalent polymerization of distinct pilins catalyzed by a pilus-specific sortase, followed by the cell wall anchoring of the resulting polymers mediated by the housekeeping sortase. In Actinomyces oris, the pilus-specific sortase SrtC2 not only polymerizes FimA pilins to assemble type 2 fimbriae with CafA at the tip, but it can also act as the anchoring sortase, linking both FimA polymers and SrtC1-catalyzed FimP polymers (type 1 fimbriae) to peptidoglycan when the housekeeping sortase SrtA is inactive. To date, the structure-function determinants governing the unique substrate specificity and dual enzymatic activity of SrtC2 have not been illuminated. Here, we present the crystal structure of SrtC2 solved to 2.10-Å resolution. SrtC2 harbors a canonical sortase fold and a lid typical for class C sortases and additional features specific to SrtC2. Structural, biochemical, and mutational analyses of SrtC2 reveal that the extended lid of SrtC2 modulates its dual activity. Specifically, we demonstrate that the polymerizing activity of SrtC2 is still maintained by alanine-substitution, partial deletion, and replacement of the SrtC2 lid with the SrtC1 lid. Strikingly, pilus incorporation of CafA is significantly reduced by these mutations, leading to compromised polymicrobial interactions mediated by CafA. In a srtA mutant, the partial deletion of the SrtC2 lid reduces surface anchoring of FimP polymers, and the lid-swapping mutation enhances this process, while both mutations diminish surface anchoring of FimA pili. Evidently, the extended lid of SrtC2 enables the enzyme the cell wall-anchoring activity in a substrate-selective fashion.

Sortase-Catalyzed Protein Domain Inversion.

Topological transformations and permutations of proteins have attracted significant interest as strategies to generate new protein functionalities or stability. These efforts have mainly been inspired by naturally occurring post-translational modifications, such as head-to-tail cyclization, circular permutation, or lasso-like entanglement. Such approaches can be realized experimentally via genetic encoding, in the case of circular permutation, or via enzymatic processing, in the case of cyclization. Notably, these previously described strategies leave the polypeptide backbone orientation unaltered. Here we describe an unnatural protein permutation, the protein domain inversion, whereby a C-terminal portion of a protein is enzymatically inverted from the canonical N-to-C to a C-to-C configuration with respect to the N-terminal part of the protein. The closest conceptually analogous biological process is perhaps the inversion of DNA segments as catalyzed by recombinases. We achieve these inversions using an engineered sortase A, a widely used transpeptidase. Our reactions proceed efficiently under mild conditions at 4-25 °C and are compatible with entirely heterologously-produced protein substrates.

Structure-based virtual screening and molecular dynamics approaches to identify new inhibitors of Staphylococcus aureus sortase A.

Staphylococcus aureus is a prevalent Gram-positive bacteria leading cause of a wide range of human pathologies. Moreover, antibiotic résistance of pathogenesis bacteria is one of the worldwide health problems. In Gram-positive bacteria, the enzyme of SrtA, is responsible for the anchoring of surface-exposed proteins to the cell wall peptidoglycan. Because of its critical role in Gram-positive bacterial pathogenesis, SrtA is an attractive target for anti-virulence during drug development. To date, some SrtA inhibitors have been discovered most of them being derived from flavonoid compounds, like Myricetin. In order to provide potential hit molecules against SrtA for clinical use, we obtained a total of 293 compounds by performing in silico shape-based screening of compound libraries against Myristin as a reference structure. Employing molecular docking and scoring functions, the top 3 compounds Apigenin, Efloxate, and Compound 8261032 were screened by comparing their docking scores with Myricetin. Furthermore, MD simulations and MM-PBSA binding energy calculation studies revealed that only Compound 8261032 strongly binds to the catalytic core of the SrtA enzyme than Myricetin, and stable behavior was consistently observed in the docking complex. Compound 8261032 showed a good number of hydrogen bonds with SrtA and higher MM-PBSA binding energy when compared to all three molecules. Also, it makes strength interactions with Arg139 and His62, which are critical for SrtA biological activity. This study showed that the development of this inhibitor could be a fundamental strategy against resistant bacteria, but further studies in vitro are needed to confirm this claim.Communicated by Ramaswamy H. Sarma.

Empowering Site-Specific Bioconjugations In Vitro and In Vivo: Advances in Sortase Engineering and Sortase-Mediated Ligation.

Sortase-mediated ligation (SML) has emerged as a powerful and versatile methodology for site-specific protein conjugation, functionalization/labeling, immobilization, and design of biohybrid molecules and systems. However, the broader application of SML faces several challenges, such as limited activity and stability, dependence on calcium ions, and reversible reactions caused by nucleophilic side-products. Over the past decade, protein engineering campaigns and particularly directed evolution, have been extensively employed to overcome sortase limitations, thereby expanding the potential application of SML in multiple directions, including therapeutics, biorthogonal chemistry, biomaterials, and biosensors. This review provides an overview of achieved advancements in sortase engineering and highlights recent progress in utilizing SML in combination with other state-of-the-art chemical and biological methodologies. The aim is to encourage scientists to employ sortases in their conjugation experiments.

Discovery of potential scaffolds for glutaminyl cyclase inhibitors: Virtual screening, synthesis, and evaluation.

Glutaminyl cyclase (QC) plays a crucial role in the early stages of Alzheimer's disease (AD), thus inhibition of QC may be a promising strategy for the treatment of early AD. Therefore, QC inhibitors with novel chemical scaffolds may contribute to the development of additional anti-AD agents. We conducted a virtual screening of 3 million compounds from the Chemdiv and Enamine databases, to discover potential scaffolds for QC inhibitors. Three scaffolds, 120974, 147706, and 141449, were selected from this structure-based virtual screening through a combination of pharmacophore modeling, a receptor-ligand pharmacophore model, and the GALAHAD model, and furtherly filtered by chelation with zinc ion and docking properties. Consequently, three compounds, 1, 2, and 3, were designed and synthesized based on these three scaffolds, respectively. The IC50 of compounds 1 and 3 against QC were 14.19 ± 4.21 and 4.34 ± 0.35 µM, respectively. Our results indicate that the new scaffolds selected using a virtual screening process exhibit potential as novel QC inhibitors.

Crystal structure of the pilus-specific sortase from early colonizing oral Streptococcus sanguinis captures an active open-lid conformation.

Dental plaque is a complex microbial biofilm community of many species and a major cause of oral infections and infectious endocarditis. Plaque development begins when primary colonizers attach to oral tissues and undergo coaggregation. Primary colonizers facilitate cellular attachment and inter-bacterial interactions through sortase-dependent pili (or fimbriae) extending out from their cell surface. Consequently, the sortase enzyme is viewed as a potential drug target for controlling biofilm formation and avoiding infection. Streptococcus sanguinis is a primary colonizing bacterium whose pili consist of three different pilin subunits that are assembled together by the pilus-specific (C-type) SsaSrtC sortase. Here, we report on the crystal structure determination of the recombinant wild-type and active-site mutant forms of SsaSrtC. Interestingly, the SsaSrtC structure exhibits an open-lid conformation, although a conserved DPX motif is lacking in the lid. Based on molecular docking and structural analysis, we identified the substrate-binding residues essential for pilin recognition and pilus assembly. We also demonstrated that while recombinant SsaSrtC is enzymatically active toward the five-residue LPNTG sorting motif peptide of the pilins, this activity is significantly reduced by the presence of zinc. We further showed that rutin and α-crocin are potential candidate inhibitors of the SsaSrtC sortase via structure-based virtual screening and inhibition assays. The structural knowledge gained from our study will provide the means to develop new approaches that target pilus-mediated attachment, thereby preventing oral biofilm growth and infection.

Arginyltransferase: A Personal and Historical Perspective.

In the late 1960s and early 1970s, characterization of arginylation has been spearheaded via biochemical studies that enabled the first characterization of ATE1 and its substrate specificity. This chapter summarized the recollections and insights from the era of research that followed from the original discovery of arginylation and led up to the identification of the arginylation enzyme.

Assaying ATE1 Activity in Yeast by ß-Gal Degradation.

In the 1980s, it was found that addition of N-terminal Arg to proteins induces their ubiquitination and degradation by the N-end rule pathway. While this mechanism applies only to the proteins which also have other features of the N-degron (including a closely adjacent Lys that is accessible for ubiquitination), several test substrates have been found to follow this mechanism very efficiently after ATE1-dependent arginylation. Such property enabled researchers to test ATE1 activity in cells indirectly by assaying for the degradation of such arginylation-dependent substrates. The most commonly used substrate for this assay is E. coli beta-galactosidase (beta-Gal) because its level can be easily measured using standardized colorimetric assays. Here, we describe this method, which has served as a quick and easy way to characterize ATE1 activity during identification of arginyltransferases in different species.

Assaying Arginylation Activity in Cell Lysates Using a Fluorescent Reporter.

Here, we describe an antibody-based method to evaluate the enzymatic activity of arginyltransferase1 (Ate1). The assay is based on the arginylation of a reporter protein, which contains the N-terminal peptide of beta-actin, a known endogenous substrate of Ate1, and a C-terminal GFP. The arginylation level of the reporter protein is determined  on an immunoblot with an antibody specific for the arginylated N-terminus, while the total amount of substrate is evaluated with anti-GFP antibody. This method can be used to conveniently and accurately examine the Ate1 activity in yeast and mammalian cell lysates. Moreover, the effect of mutation on Ate1 critical residues and effect of stress and other factors on Ate1 activity can also be successfully determined with this method.

High-Throughput Arginylation Assay in Microplate Format.

Here, we describe the biochemical assay for ATE1-mediated arginylation in microplate format, which can be applied to high-throughput screens for the identification of small molecule inhibitors and activators of ATE1, high-volume analysis of AE1 substrates, and other similar applications. Originally, we have applied this screen to a library of 3280 compounds and identified 2 compounds which specifically affect ATE1-regulated processes in vitro and in vivo. The assay is based on in vitro ATE1-mediated arginylation of beta-actin's N-terminal peptide, but it can also be applied using other ATE1 substrates.

Assaying for Arginyltransferase Activity and Specificity by Peptide Arrays.

Here, we describe arginylation assays performed on peptide arrays immobilized on cellulose membranes via chemical synthesis. In this assay, it is possible to simultaneously compare arginylation activity on hundreds of peptide substrates to analyze the specificity of arginyltransferase ATE1 toward its target site(s) and the amino acid sequence context. This assay was successfully employed in prior studies to dissect the arginylation consensus site and enable predictions of arginylated proteins encoded in eukaryotic genomes.

Reconstitution of the Arginyltransferase (ATE1) Iron-Sulfur Cluster.

As global regulators of eukaryotic homeostasis, arginyltransferases (ATE1s) have essential functions within the cell. Thus, the regulation of ATE1 is paramount. It was previously postulated that ATE1 was a hemoprotein and that heme was an operative cofactor responsible for enzymatic regulation and inactivation. However, we have recently shown that ATE1 instead binds an iron-sulfur ([Fe-S]) cluster that appears to function as an oxygen sensor to regulate ATE1 activity. As this cofactor is oxygen-sensitive, purification of ATE1 in the presence of O2 results in cluster decomposition and loss. Here, we describe an anoxic chemical reconstitution protocol to assemble the [Fe-S] cluster cofactor in Saccharomyces cerevisiae ATE1 (ScATE1) and Mus musculus ATE1 isoform 1 (MmATE1-1).

N-Terminal Arginylation Pull-down Analysis Using the R-Catcher Tool.

Protein arginylation is a unique and under-explored posttranslational modification, which governs many biological functions and the fate of affected proteins. Since ATE1 was discovered in 1963, a central tenet of protein arginylation is that arginylated proteins are destined for proteolysis. However, recent studies have shown that protein arginylation controls not only the half-life of a protein but also various signaling pathways. Here, we introduce a novel molecular tool to elucidate protein arginylation. This new tool, termed R-catcher, is derived from the ZZ domain of p62/sequestosome-1, an N-recognin of the N-degron pathway. The ZZ domain, which has been shown to strongly bind N-terminal arginine, has been modified at specific residues to increase specificity and affinity for N-terminal arginine. R-catcher is a powerful analysis tool allowing researchers to capture the cellular arginylation patterns under various stimuli and conditions, thereby identifying potential therapeutic targets in numerous diseases.

Sortase E-mediated site-specific immobilization of green fluorescent protein and xylose dehydrogenase on gold nanoparticles.

Sortase, a bacterial transpeptidase enzyme, is an attractive tool for protein engineering due to its ability to break a peptide bond at a specific site and then reform a new bond with an incoming nucleophile. Here, we present the immobilization of two recombinant proteins, enhanced green fluorescent protein (eGFP) and xylose dehydrogenase (XylB) over triglycine functionalized PEGylated gold nanoparticles (AuNPs) using C. glutamicum sortase E. For the first time, we used a new class of sortase from a non-pathogenic organism for sortagging. The site-specific conjugation of proteins with LAHTG-tagged sequences on AuNPs via covalent cross-linking was successfully detected by surface-enhanced Raman scattering (SERS) and UV-vis spectral analysis. The sortagging was initially validated by an eGFP model protein and later with the xylose dehydrogenase enzyme. The catalytic activity, stability, and reusability of the immobilized XylB were studied with the bioconversion of xylose to xylonic acid. When compared to the free enzyme, the immobilized XylB was able to retain 80% of its initial activity after four sequential cycles and exhibited no significant variations in instability after each cycle for about 72 h. These findings suggest that C. glutamicum sortase could be useful for immobilizing site-specific proteins/enzymes in biotransformation applications for value-added chemical production.

Harnessing sortase A transpeptidation for advanced targeted therapeutics and vaccine engineering.

The engineering of potent prophylactic and therapeutic complexes has always required careful protein modification techniques with seamless capabilities. In this light, methods that favor unobstructed multivalent targeting and correct antigen presentations remain essential and very demanding. Sortase A (SrtA) transpeptidation has exhibited these attributes in various settings over the years. However, its applications for engineering avidity-inspired therapeutics and potent vaccines have yet to be significantly noticed, especially in this era where active targeting and multivalent nanomedications are in great demand. This review briefly presents the SrtA enzyme and its associated transpeptidation activity and describes interesting sortase-mediated protein engineering and chemistry approaches for achieving multivalent therapeutic and antigenic responses. The review further highlights advanced applications in targeted delivery systems, multivalent therapeutics, adoptive cellular therapy, and vaccine engineering. These innovations show the potential of sortase-mediated techniques in facilitating the development of simple plug-and-play nanomedicine technologies against recalcitrant diseases and pandemics such as cancer and viral infections.

Theoretical Studies of Leu-Pro-Arg-Asp-Ala Pentapeptide (LPRDA) Binding to Sortase A of Staphylococcus aureus.

Sortase A (SrtA) of Staphylococcus aureus is a well-defined molecular target to combat the virulence of these clinically important bacteria. However up to now no efficient drugs or even clinical candidates are known, hence the search for such drugs is still relevant and necessary. SrtA is a complex target, so many straight-forward techniques for modeling using the structure-based drug design (SBDD) fail to produce the results they used to bring for other, simpler, targets. In this work we conduct theoretical studies of the binding/activity of Leu-Pro-Arg-Asp-Ala (LPRDA) polypeptide, which was recently shown to possess antivirulence activity against S. aureus. Our investigation was aimed at establishing a framework for the estimation of the key interactions and subsequent modification of LPRDA, targeted at non-peptide molecules, with better drug-like properties than the original polypeptide. Firstly, the available PDB structures are critically analyzed and the criteria to evaluate the quality of the ligand-SrtA complex geometry are proposed. Secondly, the docking protocol was investigated to establish its applicability to the LPRDA-SrtA complex prediction. Thirdly, the molecular dynamics studies were carried out to refine the geometries and estimate the stability of the complexes, predicted by docking. The main finding is that the previously reported partially chaotic movement of the ß6/ß7 and ß7/ß8 loops of SrtA (being the intrinsically disordered parts related to the SrtA binding site) is exaggerated when SrtA is complexed with LPRDA, which in turn reveals all the signs of the flexible and structurally disordered molecule. As a result, a wealth of plausible LPRDA-SrtA complex conformations are hard to distinguish using simple modeling means, such as docking. The use of more elaborate modeling approaches may help to model the system reliably but at the cost of computational efficiency.

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.