Investigating the mechanistic difference between the D46 and H46 of the hedgehog hint domain in hedgehog autoprocessing

Abstract

The overexpression of the Hedgehog signaling pathway is involved in the development of many cancers. The pathway is activated by the autoprocessing of the Hedgehog precursor, without the need of any accessory protein. Understanding the mechanism of autoprocessing could potentially contribute to the development of novel anti-cancer therapy. The aspartic acid at position 46 (D46) of the Drosophila melanogaster Hedgehog precursor Hint domain plays a vital role in autoprocessing, and a catalytic mechanism was proposed. On the other hand, other organisms such as Caenorhabditis elegans have a naturally occurring histidine at position 46 (H46). Interestingly, the H46 does not follow the previous proposed mechanism of the Drosophila melanogaster Hedgehog. In this study, the wild type D46 Hint domain of Hedgehog precursor was overexpressed and purified. HSQC and HMQC NMR experiments were conducted to verify the structural properties of D46. pKa value of D46H was determined as 5.79 by pH titration based on HMQC. The HSQC and HMQC of the wild type D46 were compared with the previously determined structural data of a D46H mutant.