KRAS Prenylation Is Required for Bivalent Binding with Calmodulin in a Nucleotide-Independent Manner

Abstract

Deregulation of KRAS4b signaling pathway has been implicated in 30% of all cancers. Membrane localization of KRAS4b is an essential step for the initiation of the downstream signaling cascades that guide various cellular mechanisms. KRAS4b plasma membrane (PM) binding is mediated by the insertion of a prenylated moiety that is attached to the terminal carboxy-methylated cysteine, in addition to electrostatic interactions of its positively charged hypervariable region with anionic lipids. Calmodulin (CaM) has been suggested to selectively bind KRAS4b to act as a negative regulator of the RAS/mitogen-activated protein kinase (MAPK) signaling pathway by displacing KRAS4b from the membrane. However, the mechanism by which CaM can recognize and displace KRAS4b from the membrane is not well understood. In this study, we employed biophysical and structural techniques to characterize this mechanism in detail. We show that KRAS4b prenylation is required for binding to CaM and that the hydrophobic pockets of CaM can accommodate the prenylated region of KRAS4b, which might represent a novel CaM-binding motif. Remarkably, prenylated KRAS4b forms a 2:1 stoichiometric complex with CaM in a nucleotide-independent manner. The interaction between prenylated KRAS4b and CaM is enthalpically driven, and electrostatic interactions also contribute to the formation of the complex. The prenylated KRAS4b terminal KSKTKC-farnesylation and carboxy-methylation is sufficient for binding and defines the minimal CaM-binding motif. This is the same region implicated in membrane and phosphodiesterase6- δ binding. Finally, we provide a structure-based docking model by which CaM binds to prenylated KRAS4b. Our data provide new insights into the KRAS4b-CaM interaction and suggest a possible mechanism whereby CaM can regulate KRAS4b membrane localization.

A Non-Canonical Calmodulin Target Motif Comprising a Polybasic Region and Lipidated Terminal Residue Regulates Localization

“…Remarkably, farnesyl cysteine methyl ester (FCME), representing the farnesylated C-terminal residue of KRAS4b, and farnesol alone, an alcohol derivative of the farnesyl moiety, induced a similar pattern of extensive Ca 2+ -dependent chemical shift perturbations in 15 N CaM without causing line broadening [55]. Consistently, addition of a farnesylated polybasic 6-mer peptide from the KRAS4b C-terminus produced nearly identical changes in the CaM spectrum [54].…”

“…Structural data show that the KRAS4b-CaM interaction is principally mediated through sequestration of the KRAS4b farnesyl group, likely further stabilized by electrostatic contacts between the polybasic KRAS4b HVR and the acidic CaM surface [55]. Ca 2+ -CaM therefore sequesters the hydrophobic KRAS4b membrane localization moiety, and KRAS4b association with Ca 2+ -CaM was reported to be~10-fold tighter than with the membrane [54,56]. Consistently, surface plasmon resonance (SPR) experiments with a lipid bilayer membrane captured on the biosensor chip showed that Ca 2+ -CaM extracted KRAS4b from this bilayer in a nucleotide-independent manner [46], and the transfer of prenylated KRAS4b between vesicles was accelerated by Ca 2+ -CaM [57].…”

“…The KRAS4b C-terminus has some propensity to form a helix [49], however, it lacks the hydrophobic anchor residues of the canonical CaM targets. Most, but not all studies have reported that CaM binding to KRAS4b requires farnesylation [43,44,46,[50][51][52][53][54][55], and there are discrepancies in the literature about whether or not the GTPase domain or the nucleotide to which it is bound (GTP versus GDP) plays a role in the interaction.…”

“…Not surprisingly, earlier studies focused on probing the direct interaction between CaM and the GTPase-domain of KRAS and proposed this interaction is GTP-dependent [43,45,50,51]. On the other hand, other researchers reported negative evidence for this interaction and concluded that KRAS G-domain does not bind CaM [44,46,51,53,54]. Recent in vitro biophysical and structural studies of the KRAS4b-CaM interaction [54,55] improved our understanding of this controversial interaction and revealed the detail of the CaM interaction with KRAS4b, which involves the farnesylated tail for binding to CaM.…”

Biochemical and structural analysis reveals the Neurofibromin (NF1) protein forms a high-affinity dimer

“…Protein production E. coli proteins were expressed as described in (21). For insect cell expression, baculoviruses were generated by transfection of bacmid DNA into Sf9 cells as previously described (22). Titered viruses were used at an MOI of 3 to infect Tni-FNL cells (23) which were grown at 21 °C for 72 hours prior to harvest.…”