A simple method for developing lysine targeted covalent protein reagents

Ronen Gabizon; Barr Tivon; Rambabu N. Reddi; Maxime C.M. van den Oetelaar; Hadar Amartely; Peter J. Cossar; Christian Ottmann; Nir London

doi:10.1038/s41467-023-42632-5

A simple method for developing lysine targeted covalent protein reagents

Ronen Gabizon, Barr Tivon, Rambabu N. Reddi, Maxime C.M. van den Oetelaar, Hadar Amartely, Peter J. Cossar, Christian Ottmann, Nir London^*

^*Corresponding author for this work

Department of Chemical and Structural Biology

Research output: Contribution to journal › Article › peer-review

7 Citations (Scopus)

Abstract

Peptide-based covalent probes can target shallow protein surfaces not typically addressable using small molecules, yet there is a need for versatile approaches to convert native peptide sequences into covalent binders that can target a broad range of residues. Here we report protein-based thio-methacrylate esters—electrophiles that can be installed easily on unprotected peptides and proteins via cysteine side chains, and react efficiently and selectively with cysteine and lysine side chains on the target. Methacrylate phosphopeptides derived from 14-3-3-binding proteins irreversibly label 14-3-3σ via either lysine or cysteine residues, depending on the position of the electrophile. Methacrylate peptides targeting a conserved lysine residue exhibit pan-isoform binding of 14-3-3 proteins both in lysates and in extracellular media. Finally, we apply this approach to develop protein-based covalent binders. A methacrylate-modified variant of the colicin E9 immunity protein irreversibly binds to the E9 DNAse, resulting in significantly higher thermal stability relative to the non-covalent complex. Our approach offers a simple and versatile route to convert peptides and proteins into potent covalent binders.

Original language	English
Article number	7933
Journal	Nature Communications
Volume	14
Issue number	1
DOIs	https://doi.org/10.1038/s41467-023-42632-5
Publication status	Published - Dec 2023

Bibliographical note

N.L. would like to acknowledge funding from the Israel Science Foundation (grant no. 2462/19), The Minerva Foundation, The Israel Cancer Research Fund, and the Moross Integrated Cancer Center. N.L. is also supported by the Honey and Dr. Barry Sherman Lab, Dr. Barry Sherman Institute for Medicinal Chemistry, Nelson P. Sirotsky, the Goldhirsh-Yellin Foundation and Celia Zwillenberg-Fridman. M.O. is supported by the NWO OCENW.KLEIN.300 10028177 project. The plasmids for expression and purification of E9 DNAse and immunity protein were a gift from the laboratory of Sarel Fleishman in the Weizmann Institute. We acknowledge DESY (Hamburg, Germany), a member of the Helmholtz Association HGF, for the provision of experimental facilities. Parts of this research were carried out at PETRA III, and we would like to thank Guillaume Pompidor for assistance in using beam P11. Beamtime was allocated for proposal I-20211300 EC.

All Science Journal Classification (ASJC) codes

General Chemistry
General Biochemistry,Genetics and Molecular Biology
General Physics and Astronomy

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10.1038/s41467-023-42632-5Licence: CC BY

Cite this

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title = "A simple method for developing lysine targeted covalent protein reagents",

abstract = "Peptide-based covalent probes can target shallow protein surfaces not typically addressable using small molecules, yet there is a need for versatile approaches to convert native peptide sequences into covalent binders that can target a broad range of residues. Here we report protein-based thio-methacrylate esters—electrophiles that can be installed easily on unprotected peptides and proteins via cysteine side chains, and react efficiently and selectively with cysteine and lysine side chains on the target. Methacrylate phosphopeptides derived from 14-3-3-binding proteins irreversibly label 14-3-3σ via either lysine or cysteine residues, depending on the position of the electrophile. Methacrylate peptides targeting a conserved lysine residue exhibit pan-isoform binding of 14-3-3 proteins both in lysates and in extracellular media. Finally, we apply this approach to develop protein-based covalent binders. A methacrylate-modified variant of the colicin E9 immunity protein irreversibly binds to the E9 DNAse, resulting in significantly higher thermal stability relative to the non-covalent complex. Our approach offers a simple and versatile route to convert peptides and proteins into potent covalent binders.",

author = "Ronen Gabizon and Barr Tivon and Reddi, {Rambabu N.} and {van den Oetelaar}, {Maxime C.M.} and Hadar Amartely and Cossar, {Peter J.} and Christian Ottmann and Nir London",

note = "N.L. would like to acknowledge funding from the Israel Science Foundation (grant no. 2462/19), The Minerva Foundation, The Israel Cancer Research Fund, and the Moross Integrated Cancer Center. N.L. is also supported by the Honey and Dr. Barry Sherman Lab, Dr. Barry Sherman Institute for Medicinal Chemistry, Nelson P. Sirotsky, the Goldhirsh-Yellin Foundation and Celia Zwillenberg-Fridman. M.O. is supported by the NWO OCENW.KLEIN.300 10028177 project. The plasmids for expression and purification of E9 DNAse and immunity protein were a gift from the laboratory of Sarel Fleishman in the Weizmann Institute. We acknowledge DESY (Hamburg, Germany), a member of the Helmholtz Association HGF, for the provision of experimental facilities. Parts of this research were carried out at PETRA III, and we would like to thank Guillaume Pompidor for assistance in using beam P11. Beamtime was allocated for proposal I-20211300 EC. ",

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AU - Ottmann, Christian

AU - London, Nir

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A simple method for developing lysine targeted covalent protein reagents

Abstract

Bibliographical note

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