Anionic Lipids Impact RAS-Binding Site Accessibility and Membrane Binding Affinity of CRAF RBD-CRD.

Area of Science:

• Molecular biology
• Cell signaling
• Biophysics

Background:

• CRAF activation depends on binding to GTP-bound RAS at the cell membrane.
• CRAF has a RAS-binding domain (RBD) and a cysteine-rich domain (CRD) linked together.
• The interplay between RBD and CRD in membrane association and RAS interaction is not fully understood.

Purpose of the Study:

• To investigate the dynamics of CRAF's RBD when tethered to CRD anchored to a model membrane.
• To understand how CRD influences RBD's accessibility to RAS.
• To explore the synergistic effects of RBD and CRD on membrane binding.

Main Methods:

• Coarse-grained and all-atom molecular dynamics simulations of a CRAF RBD-CRD construct.
• Surface plasmon resonance (SPR) measurements using liposomes.
• Mutational analysis of key residues in the RBD.

Main Results:

• The RBD exhibits dynamic positioning near the membrane surface.
• Membrane-bound RBD's RAS-binding interface is largely inaccessible.
• Positively charged residues in RBD are crucial for membrane association, confirmed by SPR.
• RBD presence locally enriches anionic lipids, enhancing overall membrane affinity.
• RBD-CRD shows stronger liposome partitioning than CRD alone.

Conclusions:

• CRAF's RBD and CRD exhibit synergistic effects on membrane dynamics.
• CRD facilitates RBD proximity to the membrane, affecting RAS accessibility.
• RBD enhances membrane affinity through local lipid enrichment.
• These findings impact understanding of RAS-CRAF interactions at the membrane.