Oncogenic KRAS G12D mutation promotes dimerization through a second, phosphatidylserine-dependent interface: a model...

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• KRAS protein forms dimers and nanoclusters on cell membranes, driving MAPK signaling and proliferation.
• The KRAS G12D mutation, common in cancer, impairs GTP hydrolysis and enhances nanoclustering.
• Studying dynamic KRAS membrane assemblies is challenging.

Purpose of the Study:

• To elucidate the structural basis of KRAS G12D mutant self-association on membranes.
• To investigate the role of specific lipid interactions in KRAS nanoclustering.
• To characterize the mechanism of action for small molecule inhibitors targeting KRAS assembly.

Main Methods:

• Refined NMR spectroscopy using nanodiscs to study membrane-associated KRAS.
• Paramagnetic relaxation enhancement (PRE) titrations and interface mutagenesis.
• Utilized engineered mutants and PRE probes to map KRAS interfaces and interactions.

Main Results:

• Identified a novel asymmetric 'α-β' dimerization interface for KRAS G12D, dependent on phosphatidylserine lipids.
• Proposed a model where 'α-α' dimerization facilitates the formation of 'α-β' interfaces, initiating nanoclustering.
• Demonstrated that BI-2852 binds a β-β interface, stabilizing an inhibitory dimer configuration.

Conclusions:

• KRAS self-association involves a dynamic equilibrium sensitive to mutations and inhibitors.
• The G12D mutation alters KRAS assembly through distinct interfaces, promoting oncogenic signaling.
• Developed methods applicable to studying other transient membrane protein interactions.