Molecular dynamics study of in silico mutations in the auto-inhibitory loop of human endothelial nitric oxide synthase FMN sub-domain

Journal of Molecular Modeling volume 27, Article number: 63 (2021) Cite this article

Abstract

Structural flexibility of the peptide linker connecting two domains is essential for the functioning of multi-domain complex. Nitric oxide synthase (NOS) isoforms contain the oxygenase and the reductase domains connected by calmodulin binding linker (CBL) region. Additionally, the endothelial NOS (eNOS) isoform contain an auto-inhibitory loop (AI) in the FMN reductase sub-domain which represses the inter-domain electron transfer process. Binding of Ca2+-Calmodulin complex on the CBL region relieves the AI loop repression and facilitates electron transfer from FMN in the reductase domain to the heme in the oxygenase domain. Few experimental studies have reported that in vitro mutation of Serine-615 (S615D) and Serine-633 (S633D) in the FMN reductase sub-domain to aspartic acid increased NO production and increased Ca2+ sensitivity. To understand the role of AI loop in eNOS repression and activation in serine mutants (S615D and S633D), we modelled the FMN reductase sub-domain of human eNOS protein with and without the CBL region. Molecular dynamics simulations performed indicated that the mutant protein AI loop structure was stabilized by salt bridge formed between D615 and R602. It was also found that mutation increased the flexibility of C-terminal residues of eNOS CBL region. The hinge-like movement of the AI loop allowed rotation of the FMN sub-domain clockwise which may favour electron-transfer in the mutant protein. This study provides insight on mutation (S615D and S633D) induced changes in AI loop and increased flexibility of CBL region which may lead to the protein activation and may also facilitate Calmodulin binding at physiological Ca2+ concentration.

Mutation of amino acid residues contribute to structural changes at molecular level leading to alteration in protein dynamics and its function. Serine-615 and Serine-633 in the auto-inhibitory loop of human eNOS reductase model was mutated to aspartic acid in silico and molecular dynamics simulations of the protein showed that steric hindrance due to mutation altered the auto-inhibitory loop rearrangement and the FMN sub-domain movement favouring electron transfer.

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Acknowledgements

We acknowledge AU-KBC Research Centre, MIT Campus, Anna University, Chennai and BTIS facility, Centre for Biotechnology, Anna University, Chennai for providing us with space and facility for the research work. PG would like to thank the Department of Biotechnology and Department of Science and Technology, Government of India for their support. DP is thankful to the Council of Scientific and Industrial Research, Government of India for their financial support through CSIR-NET fellowship grant 09/468(0505)/2017-EMR-I. We would like to thank Dr.Kothai and Dr. Suvro Chatterjee, Anna University for their valuable inputs and suggestions.

Funding

This work was funded by Council of Scientific and Industrial Research, Government of India, New Delhi through CSIR-NET fellowship grant 09/468(0505)/2017-EMR-I.

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  1. Centre for Biotechnology, Anna University, Chennai, Tamil Nadu, 600 025, India

    D. Preethi, Sharmila Anishetty & P. Gautam

  2. AU-KBC Research Centre, Anna University, Chennai, Tamil Nadu, 600 044, India

    P. Gautam

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  1. D. Preethi
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Preethi, D., Anishetty, S. & Gautam, P. Molecular dynamics study of in silico mutations in the auto-inhibitory loop of human endothelial nitric oxide synthase FMN sub-domain. J Mol Model 27, 63 (2021). https://doi.org/10.1007/s00894-020-04643-7

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Keywords

  • Endothelial nitric oxide synthase
  • Homology Modelling
  • Molecular dynamic simulation
  • S615D and S633D
  • Calmodulin binding linker region