Electrostatic and steric effects underlie acetylation-induced changes in ubiquitin structure and function

Abstract

Covalent attachment of ubiquitin (Ub) to proteins is a highly versatile posttranslational modification. Moreover, Ub is not only a modifier but itself is modified by phosphorylation and lysine acetylation. However, the functional consequences of Ub acetylation are poorly understood. By generation and comprehensive characterization of all seven possible mono-acetylated Ub variants, we show that each acetylation site has a particular impact on Ub structure. This is reflected in selective usage of the acetylated variants by different E3 ligases and overlapping but distinct interactomes, linking different acetylated variants to different cellular pathways. Notably, not only electrostatic but also steric effects contribute to acetylation-induced changes in Ub structure and, thus, function. Finally, we provide evidence that p300 acts as a position-specific Ub acetyltransferase and HDAC6 as a general Ub deacetylase. Our findings provide intimate insights into the structural and functional consequences of Ub acetylation and highlight the general importance of Ub acetylation.

An expanded lexicon for the ubiquitin code

“…Using NMR, acetylation of ubiquitin at specific Lys residues was shown to enable ubiquitin to adopt distinct conformations that modulated its binding properties, leading to acetylation-site-specific ubiquitin interactomes. Acetylation at different Lys residues of ubiquitin also hampered the capacity of several E3s to undergo autoubiquitylation 158 .…”

Co‐translational Installation of Posttranslational Modifications by Non‐canonical Amino Acid Mutagenesis

“…However, a few examples showed that glutamine does not faithfully represent the property changes following the acetylation. [127,[135][136][137] By taking advantage of the high-fidelity and site-specificity of GCE-enabled incorporation, five lysine acetylation sites in the small GTP-binding protein Ran were studied using monoacetylated full-length proteins. [123] Results from the study revealed how the acetylation at a specific position in Ran impacts its interaction with the guanine exchange factor RCC1 and its essential functions, such as nucleotide hydrolysis, binding to import and export receptors.…”

“…Results from the study revealed how the acetylation at a specific position in Ran impacts its interaction with the guanine exchange factor RCC1 and its essential functions, such as nucleotide hydrolysis, binding to import and export receptors. The GCE‐enabled method was also adapted in the first comprehensive report on lysine acetylation in ubiquitin (Ub) [127] . As one major type of PTM, how ubiquitination is regulated through lysine acetylation of Ub was not well understood.…”

“…A well‐accepted approach was to use arginine or glutamine to mimic the lack or presence of an AcK residue at the site of interest, respectively. However, a few examples showed that glutamine does not faithfully represent the property changes following the acetylation [127,135–137] . By taking advantage of the high‐fidelity and site‐specificity of GCE‐enabled incorporation, five lysine acetylation sites in the small GTP‐binding protein Ran were studied using monoacetylated full‐length proteins [123] .…”

“…[119] Besides histone proteins, the GCE-enabled site-specific incorporation of AcK was broadly applied to investigate how lysine acetylation affects the functions of other structural, regulatory, and metabolic proteins. [120][121][122][123][124][125][126][127][128][129][130][131] Cyclophilin A (CypA) is a peptidyl-prolyl cis-trans isomerase (PPIase) with important implications in human health. [132] Its acetylation on lysine residues was detected in both intracellular and secreted forms.…”