Cryo-EM Structures of CRAF₂/14-3-3₂ and CRAF₂/14-3-3₂/MEK1₂ Complexes

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.