Effect of pH on the stability of hemochromatosis factor E: a combined spectroscopic and molecular dynamics simulation-based study

Abstract

Hereditary hemochromatosis is an iron overburden condition, which is mainly governed by hereditary hemochromatosis factor E (HFE), a member of major histocompatibility complex class I. To understand the effect of pH on the structure and stability of HFE, we have cloned, expressed, and purified the HFE in the bacterial system and performed circular dichroism, fluorescence, and absorbance measurements at a wide pH range (pH 3.0–11.0). We found that HFE remains stable in the pH range 7.5–11.0 and gets completely acid denatured at low pH values. In this work, we also analyzed the contribution of salt bridges to the stability of HFE. We further performed molecular dynamics simulations for 80 ns at different pH values. An excellent agreement was observed between results from biophysical and MD simulation studies. At lower pH, HFE undergoes denaturation and may be driven toward a degradation pathway, such as ubiquitination. Hence, HFE is not available to bind again with transferrin receptor1 to negatively regulate iron homeostasis. Further we postulated that, might be low pH of cancerous cells helps them to meet their high iron requirement.

Keywords:

• iron-homeostasis
• hemochromatosis factor E
• hereditary hemochromatosis
• molecular dynamics simulations
• pH-induced denaturation

Acknowledgements

PK thanks the Indian Council of Medical Research, India, for the award of a fellowship. MIH and FA thank the Department of Science and Technology, India, and the Indian Council of Medical Research for financial support. Suggestions from Dr Abdul Waheed and Robert E. Fleming, St. Louis, University are highly acknowledged. We sincerely thank Tracy L. Baird for editorial assistance. The Authors thank the Harvard UniversityPlasmID Repository for providing the HFE gene. The FIST support of the Department of Science and Technology, India, is highly acknowledged.