Post-Translational Modifications of Protein Backbones: Unique Functions, Mechanisms, and Challenges

Abstract

Post-translational modifications (PTMs) dramatically enhance the capabilities of proteins. They introduce new functionalities and dynamically control protein activity by modulating intra- and intermolecular interactions. Traditionally, PTMs have been considered as reversible attachments to nucleophilic functional groups on amino acid side chains, whereas the polypeptide backbone is often thought to be inert. This paradigm is shifting as chemically and functionally diverse alterations of the protein backbone are discovered. Importantly, backbone PTMs can control protein structure and function just as side chain modifications do and operate through unique mechanisms to achieve these features. In this Perspective, I outline the various types of protein backbone modifications discovered so far and highlight their contributions to biology as well as the challenges in studying this versatile yet poorly characterized class of PTMs.

Figure 1

Examples of post-translational modifications of the polypeptide backbone (bbPTMs). This Perspective focuses on covalent modifications at C_α (blue dotted lines), the amide N (green), and C=O (orange) as well as backbone extensions (purple) on proteins (yellow shaded areas). Selected protein examples for the depicted modifications are listed below. Additional bbPTMs found in ribosomally synthesized and post-translationally modified peptides (RiPPs) are shaded with a blue background. MCR represents methyl-coenzyme M reductase.

Figure 2

Novel functions provided by backbone modifications. (a and b) Maturation of the GFP fluorophore via a backbone cyclization, dehydration, and oxidation [Protein Data Bank (PDB) entry 1EMA]. (c and d) Formation of the electrophilic cofactor 4-methylidene-5-imidazole-5-one (MIO) via backbone cyclization and dehydration steps (PDB entry 1GKJ).

Figure 3

Structural context of protein backbone modifications. (a) α-Hydroxyproline in the active site of Bacillus cereus peptidoglycan N-acetylglucosamine deacetylase (PDB entry 4L1G). (b) Stable succinimide residue in glutaminase from the hyperthermophilic archaeon Pyrococcus horikoshii (PDB entry 1WL8). (c) Isoaspartate-containing hairpin in MurA from Enterobacter cloacae (UDP-N-acetylglucosamine 1-carboxyvinyltransferase, PDB entry 1EJC). (d) bbPTMs discovered in the active site region of methyl-coenzyme M reductase from the methanogenic archaeon Methanothermobacter marburgensis (left, α-methylglutamine; center, thioglycine; right, dehydroaspartate; PDB entry 5A0Y).^, A potential n → π* interaction involving thioglycine is indicated by a dashed line.

Figure 4

Installation of bbPTMs by spontaneous and enzymatic pathways. (a) Spontaneous backbone rearrangements of Asn and Asp residues into isoAsp. IsoAsp formation and reversion may be catalyzed indirectly (red steps) by a glycosyltransferase (OGT) via a glycosyl aspartate intermediate and by protein isoaspartate methyltransferase (PIMT), respectively. (b) Proposed mechanism^, for thioamide formation in methyl-coenzyme M reductase via a kinase that targets the backbone amide, YcaO, and an auxiliary protein of unknown function, TfuA, which may be involved in substrate recognition or sulfur transfer.

Figure 5

Regulatory protein bbPTMs. (a) Switching of human protein tyrosine phosphatase 1B activity by reactive oxygen species (ROS) and glutathione (GSH). (b) Inactivation of host signaling pathways by the pathogenic phosphothreonine lyase OspF (red).

Figure 6

Conformational effects of protein backbone modifications. (a) Backbone modifications alter the positioning of side chains, exemplified by l-Asp and its derivatives. A green plate is drawn through the N–C_α–C(=O) plane for reference. In the case of isoAsp, the plate is drawn through N–C_α–C_β. Side chain rotatable bonds are indicated with dashed arrows. For the sake of clarity, only one of the backbone dihedral angles (red) is shown in each panel. (b) Ramachandran plots of native and selected modified amino acids indicating favorable conformations (shaded areas) and regions corresponding to α-helices, β-sheets, and selected β-turns. Indices in type I and II turns designate the i + 1 and i + 2 residues. (c) Differences in bond lengths between thioamide and amide groups.

Figure 7

Selected tools for studying the distribution and function of bbPTMs. (a) General proteomics approach for systematic characterization of the distribution of PTMs. (b) Strategies for the identification of isoAsp-containing proteins. (c) Preparation of site-specifically modified proteins via synthetic biology (left) and protein (semi)synthesis (right). aaRS represents aminoacyl-tRNA synthetase.