Elucidation of binding mechanism of stigmasterol with human serum albumin: a biophysical and molecular dynamics simulation approach

Abstract

In the present study, we have analyzed the interaction of a phytochemical, stigmasterol (Stig), with human serum albumin (HSA) under physiological conditions using fluorescence quenching, circular dichroism and molecular modeling methods. Cytotoxic studies with Stig in mouse macrophages (RAW 246.7) and HeLa cell lines showed anti-inflammatory and anti-cancer properties. Further, the intrinsic fluorescence of HSA was quenched by Stig, which was considered a static quenching mechanism. The site-specific marker experiments revealed that Stig binds to the IIIA subdomain of HSA with a binding constant of K_Stig=1.8 ± 0.03 × 10⁵ M⁻¹ and free energy of −7.26 ± 0.031 Kcal/mol. The secondary structure of HSA was partially unfolded after binding of Stig, which indicates an alteration in the microenvironment of the protein binding site. Molecular docking experiments found that Stig binds strongly with HSA at the IIIA domain of the hydrophobic pocket with one hydrogen bond. The rigidity of the protein-Stig complex and free energies were analyzed by molecular dynamic simulation (MDS) for 100 ns, where the HSA-Stig was stabilized after 40 ns. MDS studies revealed that HSA does not significantly change the secondary structure when it binds with Stig, which is in agreement with the circular dichroism data. Overall, the results obtained gave qualitative and quantitative insight into the binding interaction between HSA and Stig, which is essential in understanding the latter as a therapeutic molecule.

Communicated by Ramaswamy H. Sarma

Keywords:

• Protein-ligand interaction
• conformation
• molecular docking
• molecular dynamic simulations

Acknowledgments

This work was supported by the Science and Engineering Research Board (SB/EMEQ-064/2014 dated 14-07-2015), India, DST-FIST, UGC-SAP and UPE-2 University of Hyderabad. In addition, we thank BIF, University of Hyderabad, for its Bioinformatics facilities. The authors are grateful to Steven G. Doll, Thomas Jefferson University, USA, for the critical reading of this manuscript.

Disclosure statement

No potential conflict of interest was reported by the authors.