Site-Specific Introduction of an Acetyl-Lysine Mimic into Peptides and Proteins by Cysteine Alkylation

Abstract

Protein acetylation on Lys residues is recognized as a significant post-translational modification in cells, but it is often difficult to discern the direct structural and functional effects of individual acetylation events. Here we describe a new tool, methylthiocarbonyl-aziridine, to install acetylLys mimics site-specifically into peptides and proteins by alkylation of Cys residues. We demonstrate that the resultant thiocarbamate modification can be recognized by the Brdt bromodomain and site-specific anti-acetyl-Lys antibodies, is resistant to histone deacetylase cleavage, and can confer activation of the histone acetyltransferase Rtt109 by simulating autoacetylation. We also use this approach to obtain functional evidence that acetylation of CK2 protein kinase on Lys102 can stimulate its catalytic activity.Reversible acetylation of histones catalyzed by histone acetyltransferases (HATs) and histone deacetylases (HDACs) is recognized as a central mechanism of chromatin regulation. Beyond histones, Lys acetylation has been observed in more than 2500 mammalian proteins on more than 4000 sites and has been shown to govern many biological processes.1 Several approaches are available to explore the structural and functional consequences of protein acetylation. Traditional site-directed mutagenesis is used to replace Lys with Arg or Gln to provide indirect insights into Lys acetylation. Expressed protein ligation and unnatural amino acid mutagenesis via nonsense suppression can install acetylLys (AcK) at desired protein sites but have various technical limitations.2 pcole@jhmi.edu. Supporting Information Available. Detailed experimental procedures and Figures S1-S10. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public AccessAuthor Manuscript J Am Chem Soc. Author manuscript; available in PMC 2011 July 28. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptAs an analog of methyl-Lys, methyl-thiaLys can be introduced at targeted protein locations via a relatively simple strategy involving Cys alkylation with N-methyl-aminoethylbromide derivatives.3 To create an acetyl-thiaLys analog (Figure 1a), we attempted to use the corresponding acetamide reagent with Cys-containing peptide; however, no significant conversion was observed. We hypothesized that the reduced ability of the amide compounds to form three-membered ring intermediates and/or intramolecular imidate formation led to diminished activity of N-acetyl-aminoethylbromide. We then explored the reaction of Nacetyl-aziridine and a Cys-containing peptide (Figure 1b). The predominant product observed by mass spectrometry was M+42 suggesting that acetyl-aziridine treatment led to an undesired acetyl transfer by nucleophilic attack at the carbonyl rather than the aziridine methylene ( Figure S1).To reduce reactivity at the carbonyl, we explored methylthiocarbonyl-aziridine (MTCA) (Figure 1b).4 Cys alkylation with this reagent was proposed to provide methylthiocarbonylthiaLys (MTCTK), a thiocarbama...

Autoacetylation of the Histone Acetyltransferase Rtt109

“…Thus, the function of acetyl-lysine cannot be substituted with glutamine, a residue that conserves the neutral charge of the acetyl group. This is consistent with the observation that installation of a thiocarbamate analog of acetyl lysine at Lys-290 did not fully recover HAT activity (42). Moreover, introduction of alanine, arginine, glutamine, or tryptophan at residue Lys-290 into rtt109⌬ yeast cells did not rescue the DNA damage sensitivity phenotype (40).…”

“…Rtt109 Lys-290 mutants show decreased H3 acetylation in vitro and loss of Rtt109-dependent functions in vivo (40,41). However, installation of a thiocarbamate analog of acetyl lysine at position 290 exhibited only an ϳ4-fold increase in activity above a catalytically impaired K290C mutant (42). Thus, the functional role of Lys-290 acetylation remains unclear.…”

“…Moreover, previous studies reported that substituting Lys-290 to alanine results in decreased acetylation of Lys-56 on H3, while in vivo analyses demonstrated that mutation of this residue results in the loss of Rtt109-dependent functions. However, a recent study in which a thiocarbamate analog of acetyl lysine was installed at position 290 showed only ϳ4-fold activity increase above a catalytically impaired K290C mutant (42). Thus, the role of acetylated Lys-290 in histone acetylation remained unclear.…”

Conversion of Cysteine into Dehydroalanine Enables Access to Synthetic Histones Bearing Diverse Post‐Translational Modifications

“…[1] A unique Cys can therefore be positioned at desired sites of modification. Cys alkylation can then provide methylated lysine mimics [24] or a nonhydrolyzable thiocarbamate analogue of acetylated lysine [25] (Scheme 1 a). These aminoethylation protocols, which target protein nucleophiles, benefit from operational simplicity, thus enabling the study of lysine modifications in several contexts.…”

Regulation of S-Adenosylhomocysteine Hydrolase by Lysine Acetylation

“…Unnatural amino acid mutagenesis by nonsense suppression strategies (30,70), Cys derivatization (31,32,71), enzyme-catalyzed acetylation (56 -58, 72-74), and sortase-mediated semisynthesis (75) are viable strategies for site-specific introduction of acetyl-Lys or close mimics into proteins. Expressed protein ligation is especially attractive, however, when these modifications occur near the C terminus of proteins of interest (42, 55, 67-69, 76 -78).…”