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

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 (TF) necessary for heart development, are among the causes of congenital heart diseases (CHD). However, further research on biophysical and biochemical mechanisms is needed to understand how missense mutations in TFs alter their function in regulating gene expression. In this work, we applied in vitro and in silico approaches to understand how five 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 decreased thermal stability, whereas I101F and R113K had increased stability. Additionally, DNA-binding affinity decreased for all five 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 T-box domain predicted altered protein conformation 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 CHDs. Article SummaryMutations in genes crucial for heart development, such as TBX5, a cardiac transcription factor (TF), have been shown to cause congenital heart diseases. However, the functional consequences of missense mutations in cardiac TF have not been fully explored. In this work, we performed biochemical evaluations and found significant changes in thermal stability and DNA-binding of five TBX5 missense mutants (I54T, M74V, I101F, R113K, and R237W). Additionally, we generated in silico predictions of protein structure, stability, and pathogenicity for the five missense mutants. Our approach is scalable to other TF mutants crucial for organ development to further our understanding of congenital disease etiology. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/654568v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@188cb2corg.highwire.dtl.DTLVardef@1d2e08corg.highwire.dtl.DTLVardef@19424fcorg.highwire.dtl.DTLVardef@1083f0d_HPS_FORMAT_FIGEXP M_FIG C_FIG