# Congenital heart disease missense mutations in the TBX5 DNA-binding domain alter thermal stability and DNA-binding affinity

## TL;DR

This study explores how specific mutations in the TBX5 protein affect heart development by altering its stability and DNA-binding ability.

## Contribution

The study provides new biophysical and biochemical insights into how TBX5 missense mutations contribute to congenital heart disease.

## Key findings

- Mutants I54T and M74V showed decreased thermal stability, while I101F and R113K showed increased stability.
- All five missense mutations reduced DNA-binding affinity to TBX5 binding sites in regulatory elements of Nppa and Camta1 genes.
- Structural modeling predicted altered conformations and stability due to amino acid residue interactions.

## Abstract

Missense mutations can alter the biochemical properties of proteins, including stability, structure, and function, potentially contributing to the development of multiple human diseases. Mutations in TBX5, a transcription factor necessary for heart development, are among the causes of congenital heart diseases. However, further research on biophysical and biochemical mechanisms is needed to understand how missense mutations in transcription factors alter their function in regulating gene expression. In this work, we applied in vitro and in silico approaches to understand how 5 missense mutations in the TBX5 T-box DNA-binding domain (I54T, M74V, I101F, R113K, and R237W) impact protein structure, thermal stability, and DNA-binding affinity to known TBX5 cognate binding sites. Differential scanning fluorimetry showed that mutants I54T and M74V had decreased thermal stability, mutants I101F and R113K had increased stability, and R237W had no significant effect on stability. Additionally, DNA-binding affinity decreased for all 5 missense mutants when evaluated in vitro for known TBX5 genomic binding sites within regulatory elements of Nppa and Camta1 genes. Structural modeling of the TBX5 predicted altered protein conformations and stability due to the loss or gain of amino acid residue interactions. Together, our findings provide biophysical and biochemical mechanisms that can be further explored to establish causality between TBX5 missense mutations and the development of congenital heart diseases.

Graphical Abstract