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.

Figure 1

A schematic representation of KRAS4b and CaM domains.

Figure 2

GDP- and GNP-bound KRAS4b-FMe binds to CaM in a nucleotide-independent manner. (A) Shown are the sedimentation velocity and normalized absorbance c(s) profiles for solutions containing 42 μM CaM (green), 8 μM GDP-bound KRAS4b-FMe (red), and 10 μM GNP-bound KRAS4b-FMe (blue). C(s) distributions for mixtures of 20 μM KRAS4b-FMe and 40 μM CaM as well as 14 μM KRAS4b-FMe and 6 μM CaM are shown as long dash and short dash plots, respectively. Red plots are data for GDP-bound KRAS4b-FMe, and blue plots are for GNP-bound KRAS4b-FMe. (B and C) Shown are SPR binding kinetics of 10–0.2 μM GDP- and GNP-bound KRAS4b-FMe to avi-CaM. Data were fit and yielded K_d values of 0.4 and 0.5 μM, respectively. (D) Fits of the steady-state binding isotherms derived from the SPR data are shown.

Figure 3

KRAS4b-FMe forms a 2:1 complex with CaM. (A) Shown are the absorbance (blue) and interference (red) S_w isotherms for the titration of CaM into 3 μM GDP-bound KRAS4b-FMe (left panel) and 6 μM GDP-bound KRAS4b-FMe (center panel) as well as the titration of GDP-bound KRAS4b-FMe into 6 μM CaM (right panel). Experimental data (circles) were fitted globally to a two-site equilibrium model with two nonsymmetric sites and microscopic K as described in the text to obtain the isotherms shown (solid lines). (B) MALS analysis was performed on GDP-bound KRAS4b-FMe-CaM complex eluted from an SEC column. The absolute molar mass versus elution time displays a monodisperse peak at 57 kDa.

Figure 4

KRAS4b-FMe forms a 1:1 complex with CaM-N and CaM-C. (A) Shown are the absorbance (blue) and interference (red) S_w isotherms for the titration of CaM-C into 3 μM GDP-bound KRAS4b-FMe. Experimental data (circles) were fitted to an A + B = AB heteroassociation model to obtain the best-fit isotherms shown (solid lines). (B) Shown are the absorbance (blue) and interference (red) S_w isotherms for the titration of CaM-N into 3 μM GDP-bound KRAS4b-FMe. Experimental data (circles) were fitted to an A + B = AB heteroassociation model to obtain the best-fit isotherms shown (solid lines). Sedimentation velocity c(s) profiles for these isotherms are shown in Supporting Materials and Methods. (C) Shown are SPR binding kinetics of 10–0.2 μM GDP and GNP-bound KRAS4b-FMe to avi-CaM-C. Data were fit using a one-site model and yielded K_d values of ∼0.6 μM for both GDP- and GNP-bound KRAS4b-FMe to CaM-C. (D) Shown are SPR binding kinetics of 10–0.2 μM GDP- and GNP-bound KRAS4b-FMe to avi-CaM-N. Data were fit using a one-site model and yielded K_d values of ∼2 μM for both GDP- and GNP-bound KRAS4b-FMe to CaM-N. (E) Fits of the steady-state binding isotherms derived from the SPR data are shown.

Figure 5

Electrostatic interactions contribute to KRAS4b-FMe/CaM binding. (A) Steady-state binding of GDP-bound KRAS4b-FMe to avi-CaM at 50, 150, and 500 mM NaCl is shown. (B) Data were fit using a one-site model and yielded K_d values of 0.1, 0.4, and 6 μM, respectively.

Figure 6

CaM disrupts KRAS4b G-domain-HVR interaction. (A) 2D ¹H-¹⁵N TROSY-HSQC spectra were obtained from GDP-bound ¹⁵N-labeled KRAS4b-FMe (70 μM) in the free (black) and CaM-bound (magenta) states. Amide signals that exhibited chemical shift changes in the G-domain of KRAS4b (blue) and HVR (green) are highlighted. (B) Selected region of 2D ¹H-¹⁵N TROSY-HSQC spectra was obtained of a GDP-bound ¹⁵N-labeled KRAS4b-FMe in the free (black) overlaid with CaM-bound (magenta) states and free GDP-bound ¹⁵N-labeled KRAS 2-169 (blue).

Figure 7

KSKTKC-FMe is the minimal CaM-binding domain. (A) ITC data obtained for GDP-bound KRAS4b-FMe binding to CaM. (B) ITC data obtained for GDP-bound KRAS4b-FMe binding to CaM-C. (C) ITC data obtained for GDP-bound KRAS4b-FMe binding to CaM-N. ITC data obtained for (D) GDP-bound KRAS4b-farnesyl, (E) GDP-bound KRAS4b 2-180 (AAAAC)-farnesyl, (F) HVR-FMe, (G) KSKTKC-FMe, (H) KSKTKC-GMe, (I) and KSTKTC-PMe binding to CaM. Fit data yielded K_d values of 0.3, 0.5, 4, 2, 10, 0.3, 0.4, 3, and 30 μM, respectively.

Figure 8

KRAS4b-FMe induces major changes in CaM. (A) 2D ¹H-¹⁵N TROSY-HSQC spectra obtained of a ¹⁵N-labeled CaM (200 μM) in the free (black) and KSKTKC-FMe bound (red states) are shown. The amide signals that exhibited significant chemical shift changes correspond to residues throughout the N- and C-terminal lobes as well as the central linker of CaM. (B) Shown is a histogram of normalized ¹H-¹⁵N chemical shift changes versus residue number calculated from the HSQC spectra for CaM upon the addition of KSKTKC-FMe. (C) Shown is the surface representation of CaM structure (Protein Data Bank (PDB): 3CLN) highlighting residues that exhibited substantial (>0.1 ppm) chemical shift changes. Residues that were significantly perturbed in CaM-N (blue), central linker (orange), and CaM-C (magenta) regions have been highlighted.

Figure 9

CaM displaces membrane-bound GDP- and GNP-bound KRAS4b-FMe. (A) Left: shown are the sedimentation velocity absorbance c(s) profiles for 42 μM CaM (gray), 9 μM GDP-bound KRAS4b-FMe (green), 4.5 μM MSP1D1 70:30 POPC/POPS nanodisc (black), and mixtures of 2.5 μM nanodisc and 10 μM GDP-bound KRAS4b-FMe without (blue) and with 11 μM CaM (red). Shown are the sedimentation velocity absorbance c(s) profiles for mixtures of 10 μM nanodisc and 40 μM GDP-bound KRAS4b-FMe without (purple) and with 44 μM CaM (red). (B) Shown is a normalized SPR sensogram of single injection of GDP- and GNP-bound KRAS4b-FMe (20 μM) onto immobilized MSP1D1: 50:50 POPC/POPS nanodisc followed by injection of buffer (orange), apo-CaM (magenta), CaM (black) onto GDP-bound nanodisc, and CaM (blue) onto GNP-bound nanodisc. Notice that CaM can displace KRAS4b-FMe from the membrane, whereas buffer and apo-CaM do not.

Figure 10

Proposed structural docking model of CaM-KRAS4b interaction. (A) Cartoon showing two methylated and farnesylated KRAS4b subunits (PDB: 5TAR), color-coded magenta and cyan, modeled into two hydrophobic pockets of the pseudosymmetrical N- and C-terminal domains of calmodulin structure, color-coded green (PDB: 2MGU). The last five residues of KRAS4b (181–185) critical for binding to calmodulin and the hydrophobic residues in the hydrophobic pockets of calmodulin are shown in blue and yellow sticks, respectively. The farnesyl group covalently linked to the last residue of KRAS4b is also shown in sticks, having the same color as its respective KRAS protein subunit. (B) A surface representation of the structural model shown in (A) with the last residue of KRAS4b (185) and the farnesyl group displayed as sticks is given.