Membrane interactions of the globular domain and the hypervariable region of KRAS4b define its unique diffusion behavior

Abstract

The RAS proteins are GTP-dependent switches that regulate signaling pathways and are frequently mutated in cancer. RAS proteins concentrate in the plasma membrane via lipid-tethers and hypervariable region side-chain interactions in distinct nano-domains. However, little is known about RAS membrane dynamics and the details of RAS activation of downstream signaling. Here, we characterize RAS in live human and mouse cells using single-molecule-tracking methods and estimate RAS mobility parameters. KRAS4b exhibits confined mobility with three diffusive states distinct from the other RAS isoforms (KRAS4a, NRAS, and HRAS); and although most of the amino acid differences between RAS isoforms lie within the hypervariable region, the additional confinement of KRAS4b is largely determined by the protein’s globular domain. To understand the altered mobility of an oncogenic KRAS4b, we used complementary experimental and molecular dynamics simulation approaches to reveal a detailed mechanism.

Crystal Structure Reveals the Full Ras–Raf Interface and Advances Mechanistic Understanding of Raf Activation

“…The allosteric connections linking residues D113 at the Galectin-binding pocket of each Raf-RBD molecule in the dimer, which also includes residue D117 [13] (Tables S5-S7), suggest a model where Ras-Raf-RBD_CRD dimers couple with Galectin dimers to form a higher-order macromolecular platform involved in signal amplification and kinetic proofreading [8] (Figure 3e), similar to that described for the LAT/Grb2/SOS system [58]. This model is consistent with the functional importance of Galectins 1 and 3 for signaling through the MAPK pathway [15,16,59,60] and with results from single molecule tracking experiments correlating immobile species of Ras observed in live cells to active signaling through the Ras-Raf-MEK-ERK pathway [61][62][63].…”

Recent Updates on the Significance of KRAS Mutations in Colorectal Cancer Biology

“…G domain sequences are highly homologous, while the hypervariable regions (HVR) differ across the RAS isoforms [ 3 ]. The G domain is responsible for catalytic activity and effector binding, while the function of the HVR is mainly binding to the plasma membrane [ 4 ]. The HVR region, which is responsible for plasma membrane (PM) association, undergoes a series of posttranslational modifications (PTMs) required for it to bind the membrane.…”

RAS Nanoclusters: Dynamic Signaling Platforms Amenable to Therapeutic Intervention

“…Recently, work using improved imaging techniques and more photostable fluorescent tags indicates that KRAS membrane dynamics can be described by a three-state diffusion model ( Figure 4 A,B) [ 20 , 21 ]. In this model, KRAS4b diffusion in the plasma membrane is described by fast (~1 μm 2 /s), intermediate (~0.2 μm 2 /s) and slow (~0.05 μm 2 /s) diffusion states.…”

“…Transitions from the fast to the slow state always occur via the intermediate state, perhaps suggesting a distinct assembly process. Interestingly, this diffusion pattern was unique for full-length KRAS4b, among other RAS isoforms [ 20 ]. One model to describe this behavior suggests that freely diffusing KRAS samples the plasma membrane with the polybasic region of the HVR, engaging with negatively charged lipids that cluster and facilitate the formation of nanodomains.…”

“…One model to describe this behavior suggests that freely diffusing KRAS samples the plasma membrane with the polybasic region of the HVR, engaging with negatively charged lipids that cluster and facilitate the formation of nanodomains. Upon activation, KRAS4b interacts with effectors and lipids to form confined transient signaling complexes, represented by the slow diffusion state [ 20 ].…”