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Evolved, Selective Erasers of Distinct Lysine Acylations.

ABSTRACT: Lysine acylations, a family of diverse protein modifications varying in acyl-group length, charge, and saturation, are linked to many important physiological processes. Only a small set of substrate-promiscuous lysine acetyltransferases and deacetylases (KDACs) install and remove this vast variety of modifications. Engineered KDACs that remove only one type of acylation would help to dissect the different contributions of distinct acylations. We developed a bacterial selection system for the directed evolution of KDACs and identified variants up to 400 times more selective for butyryl-lysine compared to crotonyl-lysine. Structural analyses revealed that the enzyme adopts different conformational states depending on the type of acylation of the bound peptide. We used the butyryl-selective KDAC variant to shift the cellular acylation spectrum towards increased lysine crotonylation. These new enzymes will help in dissecting the roles of different lysine acylations in cell physiology.

SUBMITTER: Spinck M

PROVIDER: S-EPMC7317389 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Evolved, Selective Erasers of Distinct Lysine Acylations.

Spinck Martin M Neumann-Staubitz Petra P Ecke Maria M Gasper Raphael R Neumann Heinz H

Angewandte Chemie (International ed. in English) 20200424 27

Lysine acylations, a family of diverse protein modifications varying in acyl-group length, charge, and saturation, are linked to many important physiological processes. Only a small set of substrate-promiscuous lysine acetyltransferases and deacetylases (KDACs) install and remove this vast variety of modifications. Engineered KDACs that remove only one type of acylation would help to dissect the different contributions of distinct acylations. We developed a bacterial selection system for the dir ...[more]

PMID: 32187803

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Project description:Recent proteomic studies discovered histone lysines are modified by acylations beyond acetylation. These acylations derive from acyl-CoA metabolites, potentially linking metabolism to transcription. Bromodomains bind lysine acylation on histones and other nuclear proteins to influence transcription. However, the extent bromodomains bind non-acetyl acylations is largely unknown. Also unclear are the effects of neighboring post-translational modifications, especially within heavily modified histone tails. Using peptide arrays, binding assays, sucrose gradients, and computational methods, we quantified 10 distinct acylations for binding to the bromodomain and extraterminal domain (BET) family. Four of these acylations (hydroxyisobutyrylation, malonylation, glutarylation, and homocitrullination) had never been tested for bromodomain binding. We found N-terminal BET bromodomains bound acetylated and propionylated peptides, consistent with previous studies. Interestingly, all other acylations inhibited binding of the BET bromodomains to peptides and nucleosomes. To understand how context tunes bromodomain binding, effects of neighboring methylation, phosphorylation, and acylation within histone H4 tails were determined. Serine 1 phosphorylation inhibited binding of the BRD4 N-terminal bromodomain to polyacetylated H4 tails by >5-fold, whereas methylation had no effect. Furthermore, binding of BRDT and BRD4 N-terminal bromodomains to H4K5acetyl was enhanced 1.4-9.5-fold by any neighboring acylation of lysine 8, indicating a secondary H4K8acyl binding site that is more permissive of non-acetyl acylations than previously appreciated. In contrast, C-terminal BET bromodomains exhibited 9.9-13.5-fold weaker binding for polyacylated than for monoacylated H4 tails, indicating the C-terminal bromodomains do not cooperatively bind multiple acylations. These results suggest acyl-CoA levels tune or block recruitment of the BET bromodomains to histones, linking metabolism to bromodomain-mediated transcription.

Dataset Information

Evolved, Selective Erasers of Distinct Lysine Acylations.

Publications

Evolved, Selective Erasers of Distinct Lysine Acylations.

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