NMR-Chemical-Shift-Driven Protocol Reveals the Cofactor-Bound, Complete Structure of Dynamic Intermediates of the Catalytic Cycle of Oncogenic KRAS G12C Protein and the Significance of the Mg2+ Ion
Abstract
:1. Introduction
2. Results
2.1. Ab Initio Structure Models of the Mg2+-Bound KRAS-G12C/GDP by Chemical-Shift-Rosetta
2.2. Chemical-Shift-Driven MD Refinement—Reinsertion of the Nonprotein Components in the Case of the KRAS-G12C/GDP-Mg2+ System
2.3. Comparison of the CS-Rosetta Ensembles of KRAS-G12C/GDP-Mg2+ and the Mg2+-Free KRAS-G12C/GDP System
2.4. Comparison of the csdMD Refined Ensembles of KRAS-G12C/GDP-Mg2+ and the Mg2+-Free KRAS-G12C/GDP Systems
2.5. Structure Determination of the Mg2+-Free and GDP-Bound KRAS-G12C by ARTINA
3. Discussion
4. Materials and Methods
4.1. Protein Expression and Purification
4.2. NMR Spectroscopy
4.3. CS-Rosetta Structure Determination
4.4. csdMD Model Refinement
4.5. ARTINA Calculations
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
BMRB | Biological Magnetic Resonance Data Bank |
csdMD | Chemical-shift-driven MD |
CS-Rosetta | Chemical-Shift-Rosetta |
χ1 | Chi-1 dihedral (or torsion) angle |
GDP | Guanosine diphosphate |
GEF | Guanosine exchange factor |
GTP | Guanosine triphosphate |
MD | Molecular dynamics |
NMR | Nuclear magnetic resonance |
NOE | Nuclear Overhauser effect |
ω | Omega dihedral (or torsion) angle |
ϕ | Phi dihedral (or torsion) angle |
PEF | Potential energy function |
PDB | Protein Data Bank |
ψ | Psi dihedral (or torsion) angle |
RMSD | Root-mean-square deviation |
RMSF | Root-mean-square fluctuation |
SOS | Son of Sevenless |
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Gadanecz, M.; Fazekas, Z.; Pálfy, G.; Karancsiné Menyhárd, D.; Perczel, A. NMR-Chemical-Shift-Driven Protocol Reveals the Cofactor-Bound, Complete Structure of Dynamic Intermediates of the Catalytic Cycle of Oncogenic KRAS G12C Protein and the Significance of the Mg2+ Ion. Int. J. Mol. Sci. 2023, 24, 12101. https://doi.org/10.3390/ijms241512101
Gadanecz M, Fazekas Z, Pálfy G, Karancsiné Menyhárd D, Perczel A. NMR-Chemical-Shift-Driven Protocol Reveals the Cofactor-Bound, Complete Structure of Dynamic Intermediates of the Catalytic Cycle of Oncogenic KRAS G12C Protein and the Significance of the Mg2+ Ion. International Journal of Molecular Sciences. 2023; 24(15):12101. https://doi.org/10.3390/ijms241512101
Chicago/Turabian StyleGadanecz, Márton, Zsolt Fazekas, Gyula Pálfy, Dóra Karancsiné Menyhárd, and András Perczel. 2023. "NMR-Chemical-Shift-Driven Protocol Reveals the Cofactor-Bound, Complete Structure of Dynamic Intermediates of the Catalytic Cycle of Oncogenic KRAS G12C Protein and the Significance of the Mg2+ Ion" International Journal of Molecular Sciences 24, no. 15: 12101. https://doi.org/10.3390/ijms241512101