Revealing KRas4b topology on the membrane surface

Abstract

KRas4b is a membrane-bound regulatory protein belonging to the family of small GTPases that function as a molecular switch, facilitating signal transduction from activated membrane receptors to intracellular pathways controlling cell growth and proliferation. Oncogenic mutations locking KRas4b in the active GTP state are responsible for nearly 85% of all Ras-driven cancers. Understanding the membrane-bound state of KRas4b is crucial for designing new therapeutic approaches targeting oncogenic KRas-driven signaling pathways. Extensive research demonstrates the significant involvement of the membrane bilayer in Ras-effector interactions, with anionic lipids playing a critical role in determining protein conformations The preferred topology of KRas4b for interacting with signaling partners has been a long-time question. Computational studies suggest a membrane-proximal conformation, while other biophysical methods like neutron reflectivity propose a membrane-distal conformation. To address these gaps, we employed FRET measurements to investigate the conformation of KRas4b. Using fully post-translationally modified KRas4b, we designed a Nanodisc based FRET assay to study KRas4b-membrane interactions. We suggest an extended conformation of KRas4b relative to the membrane surface. Measurement of FRET donor - acceptor distances reveal that a negatively charged membrane surface weakly favors closer association with the membrane surface. Our findings provide insights into the role of anionic lipids in determining the dynamic conformations of KRas4b and shed light on the predominant conformation of its topology on lipid headgroups.

Keywords: Cancer signaling; KRas4b; Lipid specificity; Membrane topology; Nanodisc.

Figure 1.

Calculated dependence of average FRET efficiency between donor and acceptor (dashed curve, open circles) as a function of KRas4b height above the membrane bilayer, (see methods). The experimentally measured FRET efficiencies, indicated as red squares, obtained through FRET-lifetime measurements (Table1). This graph then allows correlating the FRET efficiency with an average height above the membrane surface of Nanodiscs.

Figure 2.

Dependence of KRas4b height above the membrane bilayer as a function of the formal charge on the Nanodisc. Indicated are the calculated heights for the extended and membrane local conformations as determined from molecular dynamics simulations (see text).