Cryo-EM Structures of CRAF2/14-3-32 and CRAF2/14-3-32/MEK12 Complexes
Dirk Dedden1, Julius Nitsche1, Elisabeth V Schneider1
1Proteros biostructures GmbH, Bunsenstraße 7a, D-82152 Planegg-Martinsried, Germany.
Abstract:
RAF protein kinases are essential effectors in the MAPK pathway and are important cancer drug targets. Structural understanding of RAF activation is so far based on cryo-electron microscopy (cryo-EM) and X-ray structures of BRAF in different conformational states as inactive or active complexes with KRAS, 14-3-3 and MEK1. In this study, we have solved the first cryo-EM structures of CRAF2/14-3-32 at 3.4 Å resolution and CRAF2/14-3-32/MEK12 at 4.2 Å resolution using CRAF kinase domain expressed as constitutively active Y340D/Y341D mutant in insect cells. The overall architecture of our CRAF2/14-3-32 and CRAF2/14-3-32/MEK12 cryo-EM structures is highly similar to corresponding BRAF structures in complex with 14-3-3 or 14-3-3/MEK1 and represent the activated dimeric RAF conformation. Our CRAF cryo-EM structures provide additional insights into structural understanding of the activated CRAF2/14-3-32/MEK12 complex.
Insights
Structural insights into CRAF activation were revealed using cryo-electron microscopy (cryo-EM). These findings advance understanding of the mitogen-activated protein kinase (MAPK) pathway and RAF kinase drug targets.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- RAF protein kinases are crucial components of the MAPK pathway.
- RAF kinases are significant targets for cancer drug development.
- Existing structural data primarily focuses on BRAF, with limited information on CRAF activation states.
Purpose of the Study:
- To determine the first cryo-electron microscopy (cryo-EM) structures of CRAF in complex with 14-3-3 and MEK1.
- To elucidate the structural basis of CRAF activation and dimerization.
- To provide insights into the activated dimeric conformation of CRAF.
Main Methods:
- Utilized cryo-electron microscopy (cryo-EM) to solve protein structures.
- Expressed constitutively active CRAF kinase domain (Y340D/Y341D mutant) in insect cells.
- Determined structures of CRAF dimer/14-3-3 dimer and CRAF dimer/14-3-3 dimer/MEK1 dimer complexes.
Main Results:
- Achieved 3.4 Å resolution for the CRAF dimer/14-3-3 dimer structure.
- Achieved 4.2 Å resolution for the CRAF dimer/14-3-3 dimer/MEK1 dimer structure.
- Observed an overall architecture highly similar to activated BRAF structures, indicating a conserved dimeric conformation.
Conclusions:
- The study presents the first cryo-EM structures of activated CRAF complexes.
- The findings reveal conserved structural features between activated CRAF and BRAF dimers.
- These structures offer valuable insights into the molecular mechanisms of CRAF activation within the MAPK pathway.
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