Probing the Binding Mechanism of Dimethyllysine Reader Proteins

Student Classification

Junior

Faculty Mentor

Scott Harrison, Ph.D.

Department

Department of Biology

Document Type

Poster

Publication Date

Fall 2019

Disciplines

Biology

Abstract

Post-translational modifications (PTMs), such as methylation, are epigenetic mechanisms that regulate gene expression. Histones are known hotspots for PTMs. Lethal(3)malignant brain tumor-like protein 1 (L3MBTL1) and p53-binding protein 1 (53BP1) are “reader” proteins that recognize and bind dimethylated lysine (Kme2) on histone tails. Reader proteins bind to Kme2 using an “aromatic cage” that consists of multiple aromatic residues. In Kme2-reader proteins, this cage also includes an acidic residue such as an aspartic acid (D). Previous studies have been conducted to understand the contributions of individual residues to binding. Here, the aspartic acid was studied to determine its contribution to binding. Using site-directed mutagenesis and recombinant protein expression, we made mutants of both L3MBTL1 and 53BP1, changing the aspartic acid to alanine(A) and asparagine (N), with the 53BP1 aspartic acid to asparagine being a previously uncharacterized mutation. Using solid-phase peptide synthesis, we synthesized a Kme2 peptide substrate based on the histone 4 lysine 20 binding site. Isothermal titration calorimetry was used to measure the binding affinity of wild-type and mutant proteins with Kme2 peptide. It is expected that these mutations in 53BP1 and L3MBTL1 will decrease the binding affinity to H4K20me2 peptide, consistent with previous studies and indicating that this aspartic acid residue in the aromatic cage is important for binding in Kme2 reader proteins.

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