Thioester‐Assisted Sortase‐A‐Mediated Ligation

Abstract

Sortase A (SrtA)-mediated ligation, a popular method for protein labeling and semi-synthesis, is limited by its reversibility and dependence on the LPxTG motif, where "x" is any amino acid. Here, we report that SrtA can mediate the efficient and irreversible ligation of a protein/peptide containing a Cterminal thioester with another protein/peptide bearing an N-terminal Gly, with broad tolerance for a wide variety of LPxT-derived sequences. This strategy, the thioester-assisted SrtA-mediated ligation, enabled the expedient preparation of proteins bearing various N-or C-terminal labels, including post-translationally modified proteins such as the Ser139-phosphorylated histone H2AX and Lys9-methylated histone H3, with less dependence on the LPxTG motif. Our study validates the chemical modification of substrates as an effective means of augmenting the synthetic capability of existing enzymatic methods.

The expedient, CAET-assisted synthesis of dual-monoubiquitinated histone H3 enables evaluation of its interaction with DNMT1

“…1,2 Our understanding of these processes necessitates the use of homogenously modied histones to assign functional consequences to site-specic ubiquitin marks. [3][4][5][6][7][8][9][10][11] In the past decade, site-selective conjugation chemistry using recombinant proteins as starting materials has been an attractive technology for making ubiquitinated histones, thereby facilitating the functional and mechanistic studies of histone ubiquitination in a chemically dened manner. 3,8,[12][13][14][15][16][17][18] For instance (Fig.…”

Chemical Synthesis of Post-Translationally Modified H2AX Reveals Redundancy in Interplay between Histone Phosphorylation, Ubiquitination, and Methylation on the Binding of 53BP1 with Nucleosomes

“…Different histone post-translational modifications (PTMs) (e.g., methylation, acetylation, phosphorylation, and ubiquitination) work together in a combinatorial fashion to alter the nucleosome structure or interact with the chromatin effector proteins in many chromatin-templated processes, including gene silencing, transcription, and DNA damage repair. , Although cell biology and genetic studies can reveal the functional contributions of different PTMs in various epigenetic events and whether they are synergistic or antagonistic, quantitative deciphering of the effect of each PTM pattern necessitates the use of chemical biology methods that enable their biochemical reconstitution in a chemically defined manner. − For instance, in a recent study on the regulatory mechanism of the histone methyltransferase Clr4 during heterochromatin formation, − an intein-based approach was used to prepare K14-ubiquitinated histone H3 (i.e., H3K14ub) to quantify the effect of ubiquitination on Clr4 activity . Meanwhile, to elucidate the activity and selectivity of different HDAC (histone deacetylase) complexes at the nucleosome level, a sortase-based approach was developed to make histones bearing site-specific modifications (e.g., H2BK11/12/20/46ac and H3K9/14/18/23/27ac). , Similarly, in our studies of the recruitment of p53-binding protein 1 (53BP1) in response to DNA double-strand breaks (DSBs), we used total chemical synthesis to make di-ubiquitinated histones and discovered that 53BP1 is a potential reader of both H2AK15 mono-ubiquitination and H2AK13 poly-ubiquitination. , In this context, we now report the synthesis of ubiquitinated and phosphorylated histone variant H2AX through an expedient semisynthetic strategy, integrating expressed protein hydrazinolysis and auxiliary-mediated protein ligation.…”

Chemically Synthetic d-Sortase Enables Enzymatic Ligation of d-Peptides

“…A notable constraint of synthetic d -sortase-mediated d -peptide ligation is reversible. Previous research demonstrates that engineering Sortase A with oxoester or thioester at the ligation site could enhance efficiency and provide a cost-effective approach for d -protein synthesis. − These findings imply that d -sortase ligation could potentially be engineered as a more efficient and cost-effective approach for d -protein synthesis. Overall, this study presents the first d -sortase ligation method, expanding the toolkit for generating d -proteins and D/L hybrid proteins with unique properties and potential applications in biology, biomedicine, and material science. − …”

“…Enzymatic protein ligation using Sortase A has been widely investigated for the production of homogeneously functional proteins such as antibody–drug conjugates. − Sortase-mediated ligation shows inherent reversibility, and a strong sequence and chirality preference for the conserved L-type “LPXTG” sequence, which results in poor ligation efficiency and restricted synthetic targets. The use of the engineered glycine amide structures such as an oxo-ester or thioester for an irreversible ligation can significantly improve the ligation efficiency. , Moreover, Sortase A has also been engineered or evolved to its substrate preference, thus extending its utility for protein ligation. − Despite these advances in improving sortase ligation, the high chirality preference of sortase ligation limits its use in d -protein synthesis. We envisioned that synthesizing a d -sortase could enable the d -sortase-mediated enzymatic synthesis of d -proteins.…”