Proteomics

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NeissLock: an inducible protein anhydride for covalent targeting of endogenous proteins


ABSTRACT: Effective and convenient covalent protein conjugation could benefit diverse biological systems, stabilizing key protein protein interactions which are otherwise reversible. Neisseria meningitidis contains a protein which undergoes autoproteolysis via an anhydride. Here, we harness this spontaneously generated electrophile for covalent targeting of unmodified endogenous proteins. In ‘NeissLock’, a binding protein genetically fused to the self-processing module (SPM) docks to its target protein. Upon triggering with calcium, the aspartic anhydride generated at the C-terminus of the binding protein allows nucleophilic attack by nearby residues on the target protein, so ligating the proteins. We established a computational tool to search the Protein Data Bank, assessing proximity of amines to C-termini. We validated and optimized the NeissLock concept using the Ornithine Decarboxylase/Antizyme complex. A range of nucleophiles on the target (α-amine or ε-amines) could rapidly react with the anhydride, but reaction was blocked if the partner protein did not dock. Surprisingly, the optimal pH for covalent ligation was 7.0. We then armed Transforming Growth Factor-α with SPM and established covalent targeting to Epidermal Growth Factor Receptor at the surface of living cells. NeissLock harnesses exceptional protein chemistry to allow covalent targeting of endogenous proteins under mild conditions with up to 80% yield, allowing new possibilities for molecular engineering.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Escherichia Coli

SUBMITTER: Shabaz Mohammed  

LAB HEAD: Mark Howarth

PROVIDER: PXD023073 | Pride | 2021-02-01

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
ODCK92R_OAZYSPM.raw Raw
ODC_OAZYSPM.raw Raw
TGFA.raw Raw
checksum.txt Txt
filtered_cross-linked_peptide_ODC_ODCK92R.csv Csv
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Publications

NeissLock provides an inducible protein anhydride for covalent targeting of endogenous proteins.

Scheu Arne H A AHA   Lim Sheryl Y T SYT   Metzner Felix J FJ   Mohammed Shabaz S   Howarth Mark M  

Nature communications 20210129 1


The Neisseria meningitidis protein FrpC contains a self-processing module (SPM) undergoing autoproteolysis via an aspartic anhydride. Herein, we establish NeissLock, using a binding protein genetically fused to SPM. Upon calcium triggering of SPM, the anhydride at the C-terminus of the binding protein allows nucleophilic attack by its target protein, ligating the complex. We establish a computational tool to search the Protein Data Bank, assessing proximity of amines to C-termini. We optimize Ne  ...[more]

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