Deciphering the Role of the Ser-Phosphorylation Pattern on the DNA-Binding Activity of Max Transcription Factor Using Chemical Protein Synthesis.

Abstract

The chemical synthesis of site-specifically modified transcription factors (TFs) is a powerful method to investigate how post-translational modifications (PTMs) influence TF-DNA interactions and impact gene expression. Among these TFs, Max plays a pivotal role in controlling the expression of 15% of the genome. The activity of Max is regulated by PTMs; Ser-phosphorylation at the N-terminus is considered one of the key regulatory mechanisms. However, the molecular impact of the phosphorylation pattern on the DNA binding and sequence specificities remains elusive. Here, we developed a practical synthetic strategy to prepare homogeneous full-length Max for the first time, to explore the impact of Max phosphorylation. We prepared a focused library of eight Max variants, with distinct modification patterns, including mono-phosphorylated, and doubly-phosphorylated analogs at Ser2/ Ser11 as well as fluorescently-labeled variants via native chemical ligation. Through comprehensive DNA binding analyses, we discovered that the phosphorylation position plays a crucial role in Max's DNA binding activity. Furthermore, in-vitro high-throughput analysis using DNA microarrays revealed that the N-terminus phosphorylation pattern does not interfere with Max's DNA sequence specificity. Our work provides insights into the regulatory role of Max's phosphorylation on the DNA interactions and sequence specificity, shedding light on how PTMs influence TF function.