Effector Binding Sequentially Alters KRAS Dimerization on the Membrane: New Insights Into RAS-Mediated RAF Activation

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• RAS proteins are key regulators of cell proliferation and differentiation.
• RAS-RAF complex formation at the plasma membrane is implicated in ~25% of human cancers.
• The precise mechanism of RAS-RAF complex assembly and subsequent RAF activation remains poorly understood.

Purpose of the Study:

• To determine the structure of the hetero-tetrameric KRAS-RAF1 complex.
• To elucidate the mechanism by which RAF1 binding alters KRAS dimerization and activates RAF signaling.
• To provide insights into the allosteric regulation of KRAS.

Main Methods:

• Utilized nanodisc platforms to stabilize membrane-associated protein complexes.
• Employed paramagnetic relaxation enhancement (PRE) analyses to determine complex structure.
• Performed interface-specific mutagenesis to validate binding interfaces and functional effects.

Main Results:

• Demonstrated that RAF1 binding differentially alters KRAS dimerization modes on membranes.
• Showed that RAS-binding domain (RBD) binding allosterically induces distinct KRAS dimer interfaces.
• Revealed that the cysteine-rich domain (CRD) cooperatively stabilizes a novel KRAS dimer configuration with RAF1.
• Identified a sequential alteration of KRAS dimerization upon RAF binding, leading to CRD interaction and RAF release.

Conclusions:

• RAF1 binding sequentially modulates KRAS dimerization, providing a mechanism for RAF activation.
• A configurational transition of the KRAS dimer creates an interaction site for the RAF1 CRD, releasing the autoinhibited RAF complex.
• The employed nanodisc and PRE methods are broadly applicable to studying membrane protein complexes.