Issue 2, 2025

The molecular features of non-peptidic nucleophilic substrates and acceptor proteins determine the efficiency of sortagging

Abstract

Sortase A-mediated ligation (SML) or “sortagging” has become a popular technology to selectively introduce structurally diverse protein modifications. Despite the great progress in the optimization of the reaction conditions and design of miscellaneous C- or N-terminal protein modification strategies, the reported yields of conjugates are highly variable. In this study, we have systematically investigated C-terminal protein sortagging efficiency using a combination of several rationally selected and modified acceptor proteins and a panel of incoming surrogate non-peptidic amine nucleophile substrates varying in the structural features of their amino linker parts and cargo molecules. Our data suggest that the sortagging efficiency is modulated by the combination of molecular features of the incoming nucleophilic substrate, including the ionization properties of the reactive amino group, structural recognition of the nucleophilic amino linker by the enzyme, as well as the molecular nature of the attached payload moiety. Previous reports have confirmed that the steric accessibility of the C-terminal SrtA recognition site in the acceptor protein is also the critical determinant of sortase reaction efficiency. We suggest a computational procedure for simplifying a priori predictions of sortagging outcomes through the structural assessment of the acceptor protein and introduction of a peptide linker, if deemed necessary.

Graphical abstract: The molecular features of non-peptidic nucleophilic substrates and acceptor proteins determine the efficiency of sortagging

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Article information

Article type
Paper
Submitted
09 Oct 2024
Accepted
19 Dec 2024
First published
26 Dec 2024
This article is Open Access
Creative Commons BY-NC license

RSC Chem. Biol., 2025,6, 295-306

The molecular features of non-peptidic nucleophilic substrates and acceptor proteins determine the efficiency of sortagging

T. Bondarchuk, E. Zhuravel, O. Shyshlyk, M. O. Debelyy, O. Pokholenko, D. Vaskiv, A. Pogribna, M. Kuznietsova, Y. Hrynyshyn, O. Nedialko, V. Brovarets and S. A. Zozulya, RSC Chem. Biol., 2025, 6, 295 DOI: 10.1039/D4CB00246F

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