# Exploring the CNOT1(800–999) HEAT Domain and Its Interactions with Tristetraprolin (TTP) as Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry

## TL;DR

This study uses mass spectrometry to explore how mutations in a protein domain affect its interaction with another protein involved in mRNA decay.

## Contribution

The study applies HDX MS to analyze HEAT repeat domains and reveals subtle structural changes caused by point mutations in CNOT1.

## Key findings

- Point mutations in CNOT1(800–999) affect interactions with TTP due to loss of critical residues.
- HDX MS detects minor structural changes from mutations that are often overlooked in interaction studies.

## Abstract

CNOT1, a key scaffold in the CCR4-NOT complex, plays a critical role in mRNA decay, particularly in the regulation of inflammatory responses through its interaction with tristetraprolin. A fragment of the middle part of CNOT1 (residues 800–999) is an example of an α-helical HEAT-like repeat domain. The HEAT motif is an evolutionarily conserved motif present in scaffolding and transport proteins across a wide range of organisms. Using hydrogen/deuterium exchange mass spectrometry (HDX MS), a method that has not been widely explored in the context of HEAT repeats, we analysed the structural dynamics of wild-type CNOT1(800–999) and its two double point mutants (E893A/Y900A, E893Q/Y900H) to find the individual contributions of these CNOT1 residues to the molecular recognition of tristetraprolin (TTP). Our results show that the differences in the interactions of CNOT1(800–999) variants with the TTP peptide fragment are due to the absence of the critical residues resulting from point mutations and not due to the perturbation of the protein structure. Nevertheless, the HDX MS was able to detect slight local changes in structural dynamics induced by protein point mutations, which are usually neglected in studies of intermolecular interactions.