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Binding affinity of TWIST2 wild type and mutant proteins and their potential role in the Barber‐Say Syndrome

Barber Say Syndrome (BSS) is a rare autosomal dominant disease, classified as an ectodermal dysplasia, affecting the eyelids and skin as well as exhibiting dysmorphic facial features. BSS exhibits variable phenotypes. Molecular analysis of patients with BSS identified mutations in the TWIST2 gene, involving amino acid substitutions of Glutamate 75 to Glutamine (E75Q) or Alanine (E75A).TWIST2 is a transcription factor of the bHLH superfamily, belonging to the class II group. Glutamate 75 is a highly conserved amino acid in the basic region of bHLH proteins, which is responsible for nucleotide binding in both class I and II groups. Both the I and II bHLH classes are known for their specificity towards canonical E‐box (CANNTG) DNA response elements. The E75Q and E75A mutations have been suggested to alter the DNA‐binding activity of TWIST2, leading to both dominant‐negative and gain‐of‐function effects. In this study, we expressed His‐Tagged TWIST2, and the E75Q and E75A mutant proteins, in E. coli plysBL21 cells. Subsequently, proteins were purified by affinity chromatography and yields between 1.2 to 1.7 mg/L of pure recombinant protein were obtained from 2 liter cultures. Protein affinities towards known E‐boxes in the human TNFa, MCK1 and POSTN genes were measured using Biolayer Interferometry technology. TWIST2 wild type equilibrium constants were in the range of 0.003573 to 0.7929 uM. The equilibrium constants of the E75Q and E75A mutants were three to six order of magnitude higher when compared to the wild type protein. Therefore, the TWIST2 wild type protein showed higher binding affinities towards double‐stranded oligonucleotides containing E‐boxes, while the pathological variants found in BSS patients showed lower affinities to the same oligonucleotides. These results suggest that basic region variants of the TWIST2 glutamate 75 may cause gene dysregulation by their reduced capacity to bind their cognate DNA response elements.