Reversible modification of the histone H3 N-terminal tail is critical in regulating the chromatin structure, gene expression, and cell states, while its dysregulation contributes to disease pathogenesis. Understanding the crosstalk between H3 tail modifications in nucleosomes constitutes a central challenge in epigenetics. Here, we describe an engineered sortase transpeptidase, cW11, that displays highly favorable properties for introducing scarless H3 tails onto nucleosomes. This approach significantly accelerates the production of both symmetrically and asymmetrically modified nucleosomes. We demonstrate the utility of asymmetrically modified nucleosomes produced in this way in dissecting the impact of multiple modifications on eraser enzyme processing and molecular recognition by a reader protein. Moreover, we show that cW11 sortase is very effective at cutting and tagging histone H3 tails from endogenous histones, facilitating multiplex “cut-and-paste” middle-down proteomics with tandem mass tags. This cut-and-paste proteomics approach permits the quantitative analysis of histone H3 modification crosstalk after treatment with different histone deacetylase inhibitors. We propose that these chemoenzymatic tail isolation and modification strategies made possible with cW11 sortase will broadly power epigenetic discovery and therapeutic development.

Sortase ligation is a common method for chemical protein synthesis; however, the products often contain a reaction scar. Now a traceless sortase ligation method is reported in which cleavable sortase ...

Sortase-assembled pili contribute to virulence in many Gram-positive bacteria. In Enterococcus faeca

The electrostatic profile of sortase A pentamutant (SrtA-5M) is leveraged to improve SrtA-5M-mediated transpeptidations by incorporating short, charged peptidic modules into the substrates. The react....

Wild-type sortase A is an important virulence factor displaying a diverse array of proteins on the surface of bacteria and is engineered to allow protein ligation. Here, authors discover a previously unknown interaction between multiple classes of wild-type sortases and engineered sortase A and oligonucleotides. This property enables sortase-mediated oligonucleotide mammalian cell labeling, which can be applied in multiplexing samples for single-cell sequencing. Abstract Wild-type sortase A is an important virulence factor displaying a diverse array of proteins on the surface of bacteria. This protein display relies on the transpeptidase activity of sortase A, which is widely engineered to allow protein ligation and protein engineering based on the interaction between sortase A and peptides. Here an unknown interaction is found between sortase A from Staphylococcus aureus and nucleic acids, in which exogenously expressed engineered sortase A binds oligonucleotides in vitro and is independent of its canonical transpeptidase activity. When incubated with mammalian cells, engineered sortase A further mediates oligonucleotide labeling to the cell surface, where sortase A attaches itself and is part of the labeled moiety. The labeling reaction can also be mediated by many classes of wild-type sortases as well. Cell surface GAG appears involved in sortase-mediated oligonucleotide cell labeling, as demonstrated by CRISPR screening. This interaction property is utilized to develop a technique called CellID to facilitate sample multiplexing for scRNA-seq and shows the potential of using sortases to label cells with diverse oligonucleotides. Together, the binding between sortase A and nucleic acids opens a new avenue to understanding the virulence of wild-type sortases and exploring the application of sortases in biotechnology.

Nature - Ubiquitination of glycogen and metabolites in cells and tissues

In confronting the significant challenge posed by multidrug-resistant (MDR) pathogens, particularly methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), the development of innovative anti-infective...

Interactions between receptors and ligands on immune cells are visualized in vivo and in vitro using an enzyme-tagged ligand that, when cells interact, leaves behind a detectable label on the receptor...