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 acetyl-Lys 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 antiacetyl-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.

Figure 1

(a) Acetyl-Lys (AcK) and analog. (b) Strategies attempted to install acetyl-Lys analogs. (c) Competition assay of MTCA-modified H4 derived peptides by tetracetylated N-terminal H4 tail. Extracts from GFP-Brdt expressing cells were pre-incubated with peptides and then used in a pull-down assay with immobilized tetracetylated H4 tail or the same non-modified peptide (- lane). The GFP-Brdt retained on the beads was then revealed using an anti-GFP antibody. C* corresponds to MTCTK. (d) Western blots with site-specific anti-AcK9 Ab on MTCA-treated mutant H3.

Figure 2

(a) HAT activity of Rtt109 K290C and RTT109 K290R HAT activity before and after MTCA treatment. (autoradiograph below). (b) Kinase activity of CK2α (carrying C147A and C220A mutations) containing either Cys102 or Lys102 after MTCA treatment.