Mechanistic insights into the phosphorylation-regulated a disordered protein interaction module

The TFIIS N-terminal domain (TND) is a crucial protein scaffold that selectively recognizes disordered ligands, known as TND-interacting motifs (TIMs). Understanding the specific mechanisms of TND-TIM interactions is essential for deciphering the transcription machinery. Here, we investigated the conformational ensembles of the TND-TIM interaction module using molecular dynamics simulations. The study revealed that the experimental structures of TND-TIM complexes, including P75-PogZ and P75-IWS1, maintained stable conformations during microsecond-long simulations, even when the linked proteins between TND and TIM were removed or when TIM was phosphorylated. Conversely, both P75-ASK and HRP2-IWS1, prepared based on the structure of P75-IWS1, are unstable in simulations; for example, the helix-1 of TIMs shifts from their initial binding site on TND. However, phosphorylation enhances TND-TIM interactions and rapidly stabilizes the complex structure. A general rule for phosphorylation regulation of TND-TIM interactions is identified: the phosphoryl group of TIM forms hydrogen bonds with the positively charged side chains of TND residues, promoting dynamic correlation between TND and the Ser-containing acidic linker of TIM, and enhancing residue-residue interactions among helix-1 and FXGF motif of TIM with TND. These phosphorylation-induced changes resulted in a higher affinity between TND and TIM. Our study provides insights into the phosphorylation-regulated TND-TIM interaction module at an atomic level, facilitating a deeper understanding of the molecular mechanisms of protein interactome assembly in transcription machinery.