The impact of R53C mutation on the three-dimensional structure, stability, and DNA-binding properties of the human Hesx-1 homeodomain.

Septo-optic dysplasia (SOD) is loosely defined as any combination of optic nerve hypoplasia, pituitary gland hypoplasia, and midline abnormalities of the brain. Recent studies have shown that this rare disease has its origin in key mutations in Hesx-1, a protein that plays a critical role in normal development of the forebrain, eyes, and other anterior structures during embryogenesis. R53C mutation in the Hesx-1 homeodomain has recently been identified in some patients with SOD. However, no detailed description of the effect of this mutation on the protein structure, stability, and function has been reported so far. The impact of R53C substitution on Hesx-1 homeodomain structure, stability, and DNA-binding properties was analyzed by using a combination of NMR spectroscopy, molecular modeling and circular dichroism experiments. Although R53C mutation has very slight effects on protein structure, it has a profound impact on homeodomain stability. We show that intramolecular disulfide formation can be easily accomplished in the mutated homeodomain, which suggests that such bond formation could take place in vivo. This modification has a key effect on the homeodomain structure and stability, mainly through its effect on helix I/helix III packing. Finally, CD titrations allowed us to establish the energy cost of the R53C substitution with respect to Hesx-1-homeodomain-DNA complex stability. Detailed structural models are provided for the wild-type human Hesx-1 homeodomain and the R53C mutant, which is associated with SOD in humans. The reported effect of R53C mutation on protein stability and DNA-binding properties, together with the significant structural perturbations induced by disulfide formation in the mutated polypeptide might explain the loss of activity of the mutant in vivo.