Unravelling the unfolding pathway of human Fas-activated serine/threonine kinase induced by urea

Abstract

Fas-activated serine/threonine kinase (FASTK) is a mitochondria-associated nuclear protein that inhibits Fas- and UV-induced apoptosis. This protein is generally activated during Fas-mediated apoptosis by phosphorylating a nuclear RNA-binding protein T-cell intracellular antigen-1 and thus considered as a modulator of apoptosis. In the present study, we have examined the equilibrium unfolding and conformational stability of the kinase domain of FASTK (FASTK_353-444). The kinase domain of FASTK_353-444 was cloned, expressed, and purified. The folding ↔ unfolding transitions of urea-induced denaturation was monitored with the help of circular dichroism, intrinsic fluorescence, and UV absorption spectroscopies. Analysis of transition curves obtained from different probes revealed a coincidence of denaturation curves, suggesting that folding/unfolding of FASTK follows a two-state process with the midpoint (C_m) value at 3.50 ± 0.1 M. Urea-induced denaturation curves were further analyzed to estimate change in the Gibbs free energy in the absence of urea (ΔG_D⁰) associated with the equilibrium of denaturation. To get atomistic insights into the urea-induced denaturation of FASTK, we performed an all-atom molecular dynamics simulation for 100 ns. A close agreement was noticed between experimental and computational studies. This study will help to understand the unfolding mechanism and structural stability of the kinase domain of FASTK.

Communicated by Ramaswamy H. Sarma

Keywords:

• Fas-activated serine/threonine kinase
• kinase domain
• protein folding and dynamics
• urea-induced denaturation
• Gibbs free energy
• molecular dynamics simulation

Acknowledgements

The authors, acknowledge King Abdulaziz University, Jeddah, Saudi Arabia, for technical and financial support (KEP-57-130-38) under Deanship of Scientific Research (DSR) scheme. MIH thanks to the Department of Science and Technology and Indian Council of Medical Research for financial support. FA is thankful to the Indian National Science Academy for the award of Senior Scientist Position. SS is grateful to the University Grants Commission, Government of India, New Delhi for the award of DS Kothari Postdoctoral fellowship (BSR/BL/13-14/0483). PK is thankful to the Department of Biotechnology, New Delhi for the award of DBT-Research Associate. Authors thank Harvard University-PlasmID providing facility for providing the FASTK gene. PG thanks DST for the award of National-Post doctoral fellowship (File no. PDF/2017/001084).

Disclosure statement

The authors have no conflict of interest with the current work.

Additional information

Funding

This project was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, Saudi Arabia, under grant no. (KEP-57-130-38).