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Understanding the Structural and Functional Intricacies That Govern the Dynamic Regulation of Pyruvate Kinase Muscle Isoform 2(PKM2)
CC BY 4.0 · Indian J Med Paediatr Oncol 2024; 45(S 01): S1-S16
DOI: DOI: 10.1055/s-0044-1788238
*Corresponding author: (e-mail: kbose@actrec.gov.in).
Abstract
Background: The lesser active dimeric PKM2 (pyruvate kinase muscle isoform 2), which exists in dimer–tetramer equilibrium, is overexpressed in cancers. Both metabolic and nonmetabolic PKM2 functions (interacts with JMJD5—a positive tumorigenesis regulator) contribute to tumorigenesis. We aim to design PKM2 variants that will shift the equilibrium toward tetramer and negate JMJD5 interaction.
Materials and Methods: PKM2 mutations were identified through structure-guided design and generated using site-directed mutagenesis. PKM2_WT (wildtype), its nontumorigenic tetrameric isoform PKM1, and JMJD5 constructs with N-terminal his6 and GST-tags, respectively, were bacterially expressed and affinity purified. PKM proteins were biophysically characterized. JMJD5–PKM2 interacting surface(s) was also identified using in silico and biochemical tools.
Results: Critical residues at the dimer–tetramer interface of PKM2 were identified using PDBSum analysis (PDB ID: 1T5A). Two of them were mutated (PKM2_DM) followed by comparative biophysical characterization with PKM2_WT and PKM1. Circular-dichroism analyses depicted minimal conformational changes due to the mutation and overall structural integrity is maintained in each protein. Fluorescence emission studies showed a slight red shift in PKM2_DM compared to PKM2_WT. Thermal-denaturation studies showed a slight increase in PKM2_DM’s stability. In silico scanning of PKM2–JMJD5 interaction surface identified crucial interaction patches, later validated for disruption of interaction in vitro by alanine mutation and pull-down assays.
Conclusion: Our study shows that PKM2_DM is well-folded and has higher stability compared to PKM2_WT, which hints toward a shift in the dimer–tetramer equilibrium toward the latter, which needs further validation using size-exclusion chromatography. The JMJD5–PKM2 interaction region was identified in silico, further in-vitro validation for disruption of interaction is underway.
Publication History
Article published online:
08 July 2024
© 2024. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/)
Thieme Medical and Scientific Publishers Pvt. Ltd.
A-12, 2nd Floor, Sector 2, Noida-201301 UP, India
*Corresponding author: (e-mail: kbose@actrec.gov.in).
Abstract
Background: The lesser active dimeric PKM2 (pyruvate kinase muscle isoform 2), which exists in dimer–tetramer equilibrium, is overexpressed in cancers. Both metabolic and nonmetabolic PKM2 functions (interacts with JMJD5—a positive tumorigenesis regulator) contribute to tumorigenesis. We aim to design PKM2 variants that will shift the equilibrium toward tetramer and negate JMJD5 interaction.
Materials and Methods: PKM2 mutations were identified through structure-guided design and generated using site-directed mutagenesis. PKM2_WT (wildtype), its nontumorigenic tetrameric isoform PKM1, and JMJD5 constructs with N-terminal his6 and GST-tags, respectively, were bacterially expressed and affinity purified. PKM proteins were biophysically characterized. JMJD5–PKM2 interacting surface(s) was also identified using in silico and biochemical tools.
Results: Critical residues at the dimer–tetramer interface of PKM2 were identified using PDBSum analysis (PDB ID: 1T5A). Two of them were mutated (PKM2_DM) followed by comparative biophysical characterization with PKM2_WT and PKM1. Circular-dichroism analyses depicted minimal conformational changes due to the mutation and overall structural integrity is maintained in each protein. Fluorescence emission studies showed a slight red shift in PKM2_DM compared to PKM2_WT. Thermal-denaturation studies showed a slight increase in PKM2_DM’s stability. In silico scanning of PKM2–JMJD5 interaction surface identified crucial interaction patches, later validated for disruption of interaction in vitro by alanine mutation and pull-down assays.
Conclusion: Our study shows that PKM2_DM is well-folded and has higher stability compared to PKM2_WT, which hints toward a shift in the dimer–tetramer equilibrium toward the latter, which needs further validation using size-exclusion chromatography. The JMJD5–PKM2 interaction region was identified in silico, further in-vitro validation for disruption of interaction is underway.
No conflict of interest has been declared by the author(s).
Publication History
Article published online:
08 July 2024
© 2024. The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. (https://creativecommons.org/licenses/by/4.0/)
Thieme Medical and Scientific Publishers Pvt. Ltd.
A-12, 2nd Floor, Sector 2, Noida-201301 UP, India