Reconstitution and characterization of BRAF in complex with 14-3-3 and KRAS4B on nanodiscs

Area of Science:

• Biochemistry
• Cell Signaling
• Molecular Biology

Background:

• RAF kinases are central to the RAS-RAS-MAPK pathway, regulating cell growth and implicated in cancer.
• RAF activation involves RAS recruitment to the membrane, promoting a conformational change from an inactive monomer to an active dimer.
• The precise mechanisms of RAF activation, especially the roles of RAS and the membrane, remain incompletely understood.

Purpose of the Study:

• To elucidate the mechanistic details of RAF activation by reconstituting a functional signaling complex.
• To investigate the influence of RAS GTPase and membrane properties on RAF conformational changes and kinase activity.
• To provide a biophysical perspective on the regulation of RAF-RAS-MAPK signaling.

Main Methods:

• Reconstitution of a BRAF-14-3-3-KRAS4B complex on a nanodisc bilayer system.
• GTP-dependency verification of complex assembly.
• Biolayer interferometry (BLI) to assess binding affinities and the impact of lipid composition and KRAS4B density.
• Dissection of kinase activity using the reconstituted system.

Main Results:

• A GTP-dependent active complex of dimeric BRAF, 14-3-3 dimer, and KRAS4B was successfully reconstituted.
• Membrane lipid composition (DOPS) and higher KRAS4B density enhanced BRAF:14-3-3 binding to RAS-nanodiscs.
• KRAS4B and nanodiscs individually did not stimulate BRAF activity, but the RAS-nanodisc complex activated both monomeric and dimeric BRAF states.

Conclusions:

• The reconstituted cell-free system provides a holistic biophysical approach to study RAF-RAS-MAPK signaling complex regulation.
• RAS GTPase and membrane interactions are critical for mediating RAF conformational rearrangement and kinase activation.
• This work deepens the understanding of RAF activation mechanisms at the cell membrane, relevant for cancer research.