Engineering Methyllysine Writers and Readers for Allele-Specific Regulation of Protein-Protein Interactions

Abstract

Protein-protein interactions mediated by methyllysine are ubiquitous in biological systems. Specific perturbation of such interactions has remained a challenging endeavor. Herein, we describe an allele-specific strategy toward an engineered protein-protein interface orthogonal to the human proteome. We develop a methyltransferase (writer) variant that installs aryllysine moiety on histones that can only be recognized by an engineered chromodomain (reader). We establish biochemical integrity of the engineered interface, provide structural evidence for orthogonality and validate its applicability to identify transcriptional regulators. Our approach provides an unprecedented strategy for specific manipulation of the methyllysine interactome.

Figure 1.

Strategy for allele-specific protein–protein interactions. Methyllysine writer (KMT) is first engineered to introduce “bumped” lysine and disrupt interaction with wild-type readers. Binding is restored using “hole-modified” reader, allowing conditional manipulation of methyllysine pathways.

Figure 2.

Engineering of G9a and CBX1. (A) G9a-Y1154A mutant transfers alkenyl groups from the corresponding SAM analogues to TAMRA-labeled H3K9 peptide. (B) MALDI-MS showing benzylation of the substrate peptide by G9a-Y1154G mutant using benzyl-SAM. (C) Heat-map diagram showing dissociation constants (K_d) for the binding of modified H3K9 peptides by wild-type CBX1 and “hole-modified” mutants.

Figure 3.

Characterization of the histone-reader pairs. (A, B) Isothermal titration calorimetric (ITC) measurements for binding between wild-type CBX1 and H3K9me3 (A), and Y26F/F50G-CBX1 and H3K9bn (B). (C, D) View of the aromatic cage from crystal structures of wild-type CBX1 chromodomain bound to H3K9me3 peptide (C) and “hole-modified” CBX1 (Y26F/F50G) bound to H3K9bn (D).

Figure 4.

Generality in methyllysine writer-reader engineering. (A, B) Sequence alignments of KMTs (A) and methyllysine readers (B) showing Y1154 of G9a and F50 in CBX1 are highly conserved. (C) Substrate benzylation by Suv39H2-Y372G mutant on full-length histone H3. (D) Binding of wild-type CBX3 toward H3K9me3 (K_d = 12.2 ± 0.2 μM) and H3K9bn (K_d > 1 mM). (E) Binding of CBX3-F44G mutant toward H3K9me3 (K_d > 1 mM) and H3K9bn (K_d = 49 ± 2.4 μM).

Figure 5.

Allele-specific interactions demonstrated in cell extracts. (A) Interaction between “hole-modified” CBX1 and “bumped” histone in the presence of endogenous methyllysine readers (step 1). The engineered complex binds to transcriptional regulators (step 2). (B) Western blot (indicated antibody) of nuclear extracts pulled-down using biotin-attached H3K9me3 or H3K9bn peptide. CBX1 mutant carries HA tag. (C) Western blot (anti-HA antibody) of nuclear extracts pulled-down using biotin-attached H3K4me3, H3K27me3, H3K36me3 or H3K9bn peptide. (D) Western blot (anti-TIF1β antibody) of nuclear extracts pulled-down using biotin-attached H3K9me3 or H3K9bn or unmodified peptide.