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BioTechnologia
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3/2024
vol. 105
 
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abstract:
RESEARCH PAPERS

Investigation of the new substitution glycine to alanine within the Kringle-2 domain of reteplase: a molecular dynamics study

Kaveh Haji-Allahverdipoor
1
,
Habib Eslami
2
,
Koosha Rokhzadi
1
,
Mokhtar Jalali Javaran
3
,
Sajad Rashidi Monfared
3
,
Mohamad Bagher Khademerfan
1

  1. Cellular and Molecular Research Center, Research Institute for Health Development, Kurdistan University of Medical Sciences, Sanandaj, Iran
  2. Department of Pharmacology and Toxicology, Faculty of Pharmacy, Molecular Medicine Research Center, Hormozgan Health Institute, Hormozgan University of Medicinal Sciences, Bandar Abbas, Iran
  3. Department of Plant Biotechnology, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran
BioTechnologia vol. 105 (3) ∙ pp. 201–213 ∙ 2024
DOI: https://doi.org/10.5114/bta.2024.141801
Online publish date: 2024/09/30
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Background.
Recombinant plasminogen activator (r-PA) consists of the Kringle-2 and protease domains of human tissue-type plasminogen. It is used clinically to treat coronary artery thrombosis and acute myocardial infarction. However, the expression and production of reteplase (r-PA) are limited due to its susceptibility to proteolysis during manufacturing processes. Therefore, efforts have been made to address this limitation.

Materials and methods.
To enhance the conformational stability of r-PA and increase its resistance to proteolysis, we used Gly → Ala substitutions in the Kringle-2 domain through in silico. We created an in silico mutant collection with eight structures, incorporating four designated mutations (R103S, G39A, G53A, and G55A). Using MODELLER software and homology modeling, we developed three-dimensional structures for two Kringle-2 and tissue plasminogen activator protease domains, including the wild noncleavable form (R103S) and mutants with all four designated mutations. We assessed protein stability using a dynamic cross-correlation matrix by extracting global properties such as Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) from trajectory files.

Results.
The findings revealed that a single glycine–alanine substitution (G39A) enhanced the conformational stability of r-PA, as evidenced by improvements in RMSD, RMSF, radius of gyration, surface accessibility, hydrogen bond formation, eigenvector projection, and density analysis.

Conclusions.
The conformational stability of r-PA conferred by glycine replacement with alanine may decrease the propensity for proteolysis in protease – rich environments across various recombinant systems and potentially enhance its production and expression levels.

keywords:

reteplase, stability, mutations, molecular dynamic simulations, homology modeling


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