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 Mg<sup>2+</sup> Ion
In this work, catalytically significant states of the oncogenic G12C variant of KRAS, those of Mg<sup>2+</sup>-free and Mg<sup>2+</sup>-bound GDP-loaded forms, have been determined using CS-Rosetta software and NMR-data-driven molecular dynamics simulations. There are several...
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-07-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/24/15/12101 |
| _version_ | 1797586710053257216 |
|---|---|
| author | Márton Gadanecz Zsolt Fazekas Gyula Pálfy Dóra Karancsiné Menyhárd András Perczel |
| author_facet | Márton Gadanecz Zsolt Fazekas Gyula Pálfy Dóra Karancsiné Menyhárd András Perczel |
| author_sort | Márton Gadanecz |
| collection | DOAJ |
| description | In this work, catalytically significant states of the oncogenic G12C variant of KRAS, those of Mg<sup>2+</sup>-free and Mg<sup>2+</sup>-bound GDP-loaded forms, have been determined using CS-Rosetta software and NMR-data-driven molecular dynamics simulations. There are several Mg<sup>2+</sup>-bound G12C KRAS/GDP structures deposited in the Protein Data Bank (PDB), so this system was used as a reference, while the structure of the Mg<sup>2+</sup>-free but GDP-bound state of the RAS cycle has not been determined previously. Due to the high flexibility of the Switch-I and Switch-II regions, which also happen to be the catalytically most significant segments, only chemical shift information could be collected for the most important regions of both systems. CS-Rosetta was used to derive an “NMR ensemble” based on the measured chemical shifts, which, however, did not contain the nonprotein components of the complex. We developed a torsional restraint set for backbone torsions based on the CS-Rosetta ensembles for MD simulations, overriding the force-field-based parametrization in the presence of the reinserted cofactors. This protocol (csdMD) resulted in complete models for both systems that also retained the structural features and heterogeneity defined by the measured chemical shifts and allowed a detailed comparison of the Mg<sup>2+</sup>-bound and Mg<sup>2+</sup>-free states of G12C KRAS/GDP. |
| first_indexed | 2024-03-11T00:26:57Z |
| format | Article |
| id | doaj.art-fa6e1a18e3f5422b93bd04c9d40180cd |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T00:26:57Z |
| publishDate | 2023-07-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-fa6e1a18e3f5422b93bd04c9d40180cd2023-11-18T23:00:25ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-07-0124151210110.3390/ijms241512101NMR-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 Mg<sup>2+</sup> IonMárton Gadanecz0Zsolt Fazekas1Gyula Pálfy2Dóra Karancsiné Menyhárd3András Perczel4Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Pázmány Péter stny. 1/A, H-1117 Budapest, HungaryLaboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Pázmány Péter stny. 1/A, H-1117 Budapest, HungaryLaboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Pázmány Péter stny. 1/A, H-1117 Budapest, HungaryLaboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Pázmány Péter stny. 1/A, H-1117 Budapest, HungaryLaboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Pázmány Péter stny. 1/A, H-1117 Budapest, HungaryIn this work, catalytically significant states of the oncogenic G12C variant of KRAS, those of Mg<sup>2+</sup>-free and Mg<sup>2+</sup>-bound GDP-loaded forms, have been determined using CS-Rosetta software and NMR-data-driven molecular dynamics simulations. There are several Mg<sup>2+</sup>-bound G12C KRAS/GDP structures deposited in the Protein Data Bank (PDB), so this system was used as a reference, while the structure of the Mg<sup>2+</sup>-free but GDP-bound state of the RAS cycle has not been determined previously. Due to the high flexibility of the Switch-I and Switch-II regions, which also happen to be the catalytically most significant segments, only chemical shift information could be collected for the most important regions of both systems. CS-Rosetta was used to derive an “NMR ensemble” based on the measured chemical shifts, which, however, did not contain the nonprotein components of the complex. We developed a torsional restraint set for backbone torsions based on the CS-Rosetta ensembles for MD simulations, overriding the force-field-based parametrization in the presence of the reinserted cofactors. This protocol (csdMD) resulted in complete models for both systems that also retained the structural features and heterogeneity defined by the measured chemical shifts and allowed a detailed comparison of the Mg<sup>2+</sup>-bound and Mg<sup>2+</sup>-free states of G12C KRAS/GDP.https://www.mdpi.com/1422-0067/24/15/12101protein structure determinationprotein dynamicsNMR spectroscopymolecular dynamics simulationKRASligand binding |
| spellingShingle | Márton Gadanecz Zsolt Fazekas Gyula Pálfy Dóra Karancsiné Menyhárd András Perczel 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 Mg<sup>2+</sup> Ion International Journal of Molecular Sciences protein structure determination protein dynamics NMR spectroscopy molecular dynamics simulation KRAS ligand binding |
| title | 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 Mg<sup>2+</sup> Ion |
| title_full | 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 Mg<sup>2+</sup> Ion |
| title_fullStr | 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 Mg<sup>2+</sup> Ion |
| title_full_unstemmed | 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 Mg<sup>2+</sup> Ion |
| title_short | 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 Mg<sup>2+</sup> Ion |
| title_sort | 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 mg sup 2 sup ion |
| topic | protein structure determination protein dynamics NMR spectroscopy molecular dynamics simulation KRAS ligand binding |
| url | https://www.mdpi.com/1422-0067/24/15/12101 |
| work_keys_str_mv | AT martongadanecz nmrchemicalshiftdrivenprotocolrevealsthecofactorboundcompletestructureofdynamicintermediatesofthecatalyticcycleofoncogenickrasg12cproteinandthesignificanceofthemgsup2supion AT zsoltfazekas nmrchemicalshiftdrivenprotocolrevealsthecofactorboundcompletestructureofdynamicintermediatesofthecatalyticcycleofoncogenickrasg12cproteinandthesignificanceofthemgsup2supion AT gyulapalfy nmrchemicalshiftdrivenprotocolrevealsthecofactorboundcompletestructureofdynamicintermediatesofthecatalyticcycleofoncogenickrasg12cproteinandthesignificanceofthemgsup2supion AT dorakarancsinemenyhard nmrchemicalshiftdrivenprotocolrevealsthecofactorboundcompletestructureofdynamicintermediatesofthecatalyticcycleofoncogenickrasg12cproteinandthesignificanceofthemgsup2supion AT andrasperczel nmrchemicalshiftdrivenprotocolrevealsthecofactorboundcompletestructureofdynamicintermediatesofthecatalyticcycleofoncogenickrasg12cproteinandthesignificanceofthemgsup2supion |