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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

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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]

PMID: 33514717

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Project description:Fruits of transgenic tomato (Solanum lycopersicum) plants engineered with ripening-induced, yeast S-adenosylmethionine decarboxylase (ySAMdc) gene, accumulate the higher polyamines spermidine (Spd) and spermine (Spm) and demonstrate ameliorated phytonutrient content, juice quality, and prolonged vine life. Enhanced nitrogen-carbon interactions were revealed by comprehensive Nuclear Magnetic Resonance (NMR) spectroscopy-based metabolite profiling of the transgenics, suggesting that Spd and Spm are perceived as nitrogenous metabolites by the fruit cells (Mattoo et al., 2006). The recent work by our colleagues identified the effects of Spd/Spm accumulation on various functional classes of tomato genes affected during ripening by probing 1522 ESTs on a custom-made array (Srivastava et al., 2007). In this study we monitored alterations of genome-wide transcriptional patterns in pericarp of Spd/Spm-accumulating tomatoes by means of direct comparison with azygous controls using DNA-microarray technology. Consistent with the ySAMdc expression pattern, very minor transcriptional alterations were detected in mature green developmental stage. For both breaker and red stages, large mutual and unique gene sets displayed altered levels of transcript. Ontological term analysis of up- and down-regulated transcript groups revealed processes in cell metabolism that are regulated by increased levels of Spd/Spm in ripening tomato fruits. These processes mainly involve carbohydrate and amino acids metabolism and protein synthesis. Additionally, transcript levels of representative genes encoding structural enzymes for related biosynthetic pathways show strong relationship to specific metabolites that were identified as regulated in Spd/Spm-accumulating transgenics. Plants of both genotypes were grown in randomized blocks (3 blockes 8-10 plants each). At each of 3 developmental stages (mature green, breaker and pink-red) a single fruit was harvested from a randomly chosen plant of each genotype within each block. In each harvest different plants in each block were taken to represent each genotype. Following harvest, equal pericarp samples were cut from the equator region of each fruit. These samples were pooled according to genotype prior to RNA extraction, allowing equal representation of each block in the final sample. A total of 3 harvests taken from each genotype were eventually analyzed– each harvest representing a biological repeat.

Dataset Information

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

Publications

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

Similar Datasets

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