Research ArticleBiochemistry

Calmodulin disrupts plasma membrane localization of farnesylated KRAS4b by sequestering its lipid moiety

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Science Signaling  31 Mar 2020:
Vol. 13, Issue 625, eaaz0344
DOI: 10.1126/scisignal.aaz0344
  • Fig. 1 CaM interacts strongly with farnesylated KRAS4b and farnesyl compounds.

    (A) Schematic of farnesylated KRAS4b purified from High Five insect cells engineered to enhance farnesylation capacity. The aaX (VIM) is cleaved off after farnesylation at Cys185, and the exposed terminal cysteine is carboxymethylated. The polybasic region of the HVR is highlighted in blue. (B) 1H-15N HSQC of uniformly 15N-labeled CaM alone (red) and in the presence of fourfold excess KRAS4b-FMe-GDP (blue). The presence of KRAS4b-FMe induces CSPs and extensive signal broadening. Unbroadened signals that experience perturbations are highlighted (arrows) and used as probes for the interaction. (C) Chemical structures of tested farnesyl-derived compounds. (D) 1H-15N HSQC of uniformly 15N-labeled Ca2+-bound CaM alone (red) and in the presence of 11-fold excess E,E-farnesol (blue). Probe peaks from (B) are marked with arrows in the larger, full spectrum. Regions of the larger spectrum were extracted for a closer view in the boxes marked 1 to 5. All spectra were collected in NMR buffer containing 50 mM Hepes (pH 7.3), 100 mM NaCl, 10 mM Ca2+, and 1 mM TCEP at 25°C and 600 MHz for eight scans. ppm, parts per million.

  • Fig. 2 Crystal structure of CaM in complex with FCME.

    (A) Schematic diagram of the CaM-KRAS4b-FMe interaction based on the NMR data presented in Fig. 1; the hydrophobic core of CaM sequesters the farnesyl moiety of KRAS4b-FMe. (B to E) X-ray crystallography structure of Ca2+-CaM bound to FCME, resolved to 1.8 Å (PDB: 6OS4). The cartoon diagram of the CaM-FCME structure (B) shows a compact CaM conformation with significant interlobe contacts. FCME is buried in the CaM C-lobe hydrophobic pocket with the aminoacyl (C185) group exiting through a gap between α-helices 1, 6, and 7. The surface electrostatic representation of CaM (C) shows the channel formed by α-helices 1, 6, and 7 through which the cysteinyl moiety of FCME exits [rotated 45° relative to (B)] and the highly acidic surface accessible to an adjacent polybasic HVR. Electrostatics were modeled with the APBS PyMol plugin. Panel (D) shows a zoomed perspective of FCME in the CaM C-lobe hydrophobic pocket [rotated 90° relative to (B), into the interlobe plane]. Ten residues involved in hydrophobic contacts with the ligand are depicted as sticks and labeled. Panel (E) shows a close-up surface representation of FCME in the C-lobe hydrophobic pocket [oriented as in (D)] with interacting residues colored gray. (F) A ligand interaction map highlighting the hydrophobic interactions between FCME and the CaM C-lobe pocket.

  • Fig. 3 Alternative isotopic labeling schemes of KRAS4b-FMe show CaM binding is mutually exclusive with membrane association.

    (A to E) Fully processed KRAS4b-FMe, specifically labeled with 15N-lysine in insect cells, provides 5 NMR probes in the HVR and 11 probes in the G-domain. The chosen window highlights the lysine residues in the HVR (in italics). Samples contain 20 μM acetyl glycine (AcGly) as an internal standard. Shown are 1H-15N HSQC spectra for (A) KRAS4b-FMe alone; (B) KRAS4b-FMe after addition of NDs composed of 80% DOPC and 20% DOPS (fivefold excess KRAS4b-FMe); (C) KRAS4b-FMe in the presence of NDs and Ca2+-CaM (1:1 KRAS4b-FMe molar ratio); (D) KRAS4b-FMe and Ca2+-CaM in the absence of NDs, overlaid with (C), in the presence of NDs; and (E) KRAS4b-FMe and Ca2+-CaM overlaid with (A), KRAS4B-FMe alone. Data are representative of two experiments each. (F) Model of the predicted interactions between KRAS4b-FMe, membranes (NDs), and CaM in the presence and absence of Ca2+ using paramagnetic relaxation enhancement (PRE). Without Ca2+, KRAS4b-FMe is associated with the membrane through HVR electrostatics and farnesyl insertion and experiences significant peak broadening by membrane embedded PRE-active Gd3+. After addition of Ca2+ and activation of CaM, KRAS4b-FMe is extracted from the membrane by CaM and is thus unaffected by PRE-broadening at the membrane. (G) Experimentally derived probe peaks for PRE interactions (Ile24, Val45, Ile55, and Leu113) within KRAS4b-FMe, specifically methyl-labeled with 13C-Thr, Ile, Leu, Val, and Met (TMILV) in an insect cell expression system, in the presence or absence of 5 mM Ca2. A PRE reading of 1 means complete loss of signal, and a reading of 0 signifies no change in peak intensity relative to pre-Ca2+. Spectra were normalized by 0.6 mM sodium trimethylsilylpropanesulfonate (DSS) internal standard peak intensities. Error bars represent spectral noise. Data are representative of two experiments.

  • Fig. 4 CaMeRAS FRET construct demonstrates Ca2+- and farnesyl-dependent FRET signal change in vitro.

    (A) Schematic of chimeric construct expressed in HEK293 and HeLa cells and bimodal use for CaM-KRAS4b binding model validation. When transiently expressed in mammalian cells, the chimeric construct is natively processed at the C-terminal CaaX box, allowing tethering of the construct to the PM. Upon Ca2+ influx, Ca2+-CaM extracts KRAS4b from the membrane, visible with fluorescence imaging, and the structural rearrangement of the chimera leads to detectable FRET. (B) FRET signal (SFYP2/mTq2 emission ratio) changes during in vitro Ca2+ titration into purified CaMeRASWT (black) CaMeRAS bearing a mutation that prevents farnesylation (CaMeRAS185A:FLAG-mTq2-CaM-SYFP2-KRAS4b-C185Astop, orange) or mutations that block Ca2+ binding (CaMeRAS1234Q:FLAG-mTq2-CaM[E31Q, E67Q, E104Q, E140Q]-SYFP2-KRAS4b, red). Data are presented as means ± SEM of three independent measurements. FRET signal was calculated by using the peak intensities of mTq2 (at 472 nm) and SYFP2 (at 522 nm) in emission spectra as shown in fig. S12. Data were fit with sigmoidal dose-response equation on Prism8 software (GraphPad). (C) In vitro Ca2+-dependent CaMeRAS FRET changes in the presence of 0.5 mM Ca2+ and after addition of 2.5 mM EDTA. CaMeRASWT, CaMeRAS185A, and CaMeRAS1234Q are described in (B). Data are presented as means ± SEM of three independent measurements.

  • Fig. 5 Tracking cellular localization of CaMeRAS constructs in HeLa cells following histamine stimulation.

    (A) Schematic of CaMeRAS constructs transiently expressed (CaMeRASWT, CaMeRAS185A, and CaMeRAS1234Q), with a red “X” indicating where the mutants are deficient. The farnesyl moiety is represented by the zig-zag line. (B to D) Live-cell imaging tracking the cellular localization (mTq2) of constructs before (B) and 60 s after (C) 100 μM histamine stimulation. Panel (D) shows representative mTq2 intensity distributions at the indicated cross sections [yellow lines in (B)] over time, from 0 s [as shown in (B)] to 160 s; the 120-s time point is as shown in (C); histamine was added at 60 s (yellow arrowheads) after starting imaging. Scale bars, 10 μm. Data are representative of five experiments, each analyzing five to six cells.

  • Fig. 6 CaMeRAS demonstrates reversible Ca2+-/farnesyl-dependent internalization and FRET change in live cells.

    (A) Cellular localization (mTq2) of CaMeRAS constructs in HeLa cells before and after 100 μM histamine stimulation. BAPTA-AM (10 μM) treatment was added to HeLa cells expressing CaMeRASWT to examine reversibility. Regions of interest on the PM or in the cytosol were drawn manually as shown with white outlines. Data are representative of five experiments, each analyzing 5 to 10 cells. (B and C) In the cells described in (A), cytoplasmic Ca2+ concentration [assessed as normalized Calbryte 630 intensity tracking; (B)] and cytoplasmic and PM localization of CaMeRAS constructs (C) were traced over time after histamine stimulation and, for CaMeRASWT cells, BAPTA-AM treatment (red trace). In these traces, the bold lines denote the mean, and the shadows represent the SEM from five independent cells pooled from a single representative experiment. (D) Histograms of FRET readouts (ratio of SYFP2/mTq2 emissions) of the localization of CaMeRAS constructs [as aligned under (A)] at the PM and cytoplasm before (Cyt-Pre) and after (Cyt-Post) histamine stimulation (and for the WT construct, BAPTA-AM; “BAPTA”) in the marked regions of interest (see fig. S14). Data fitting of the normal distributions was performed using Gaussian functions, and data were pooled from 15 to 20 cells across three independent experiments.

  • Table 1 Crystallographic statistics reported for deposited CaM-FCME complex structure (PDB: 6OS4).

    This crystal was grown at room temperature in 0.2 mM sodium acetate, 0.1 mM sodium cacodylate (pH 6.7), and 28% PEG 8000, with 1.25 mM CaM and 5 mM FCME, using the sitting drop-vapor diffusion method. The protein crystal was diffracted on the Canadian Macromolecular Crystallography Facility 08ID-1 Beamline at the Canadian Light Source at two wavelengths to collect native (n = 1) and anomalous scattering (n = 1) datasets, which were processed using HKL2000 and refined with Phenix. Values in parentheses are for the highest resolution shell. a, b, c, unit cell measurements; α, β, γ, unit cell angles; RMS, root mean square.

    6OS4Native
    FP2, SigFP2
    Anomalous
    I(+), SigI(+), I(−), SigI(−)
    Wavelength1 Å1.77 Å
    Resolution range34.78–1.81
    (2.01–1.81)
    34.78–2.05
    (2.123–2.05)
    Space groupP 61 2 2P 61 2 2
    Unit cell: a, b, c
    α, β, γ
    40.379, 40.379, 338.137
    90, 90, 120
    40.379, 40.379, 338.137
    90, 90, 120
    Total reflections16289*19319*
    Unique reflections11386 (1091)11372 (1075)
    Multiplicity
    Completeness (%)99.91 (99.63)99.84 (98.71)
    Mean I/sigma(I)28*19.2*
    Wilson B factor35.1542.8
    Reflections used in refinement11380 (1087)11370 (1075)
    Reflections used for R-free1139 (109)1138 (108)
    R-work0.2024 (0.1934)0.1912 (0.2200)
    R-free0.2418 (0.2195)0.2253 (0.2586)
    Number of nonhydrogen atoms11941194
      Macromolecules11071107
      Ligands2727
      Solvent6060
    Protein residues144144
    RMS (bonds)0.0060.006
    RMS (angles)0.750.75
    Ramachandran favored (%)98.5998.59
    Ramachandran allowed (%)1.411.41
    Ramachandran outliers (%)00
    Rotamer outliers (%)1.831.83
    Clash score0.930.93
    Average B factor47.6747.67
      Macromolecules47.1247.12
      Ligands78.1378.13
      Solvent44.2144.21

    *Results from Phenix Xtriage.

    Supplementary Materials

    • stke.sciencemag.org/cgi/content/full/13/625/eaaz0344/DC1

      Fig. S1. RAS isoform-specific HVRs.

      Fig. S2. There is no major interaction between unfarnesylated KRAS4b (1–185) and CaM, independent of bound nucleotide.

      Fig. S3. A new class of noncanonical CaM binding targets: Singly lipidated polybasic peptides.

      Fig. S4. Chemical shift analysis of CaM residues upon farnesol binding.

      Fig. S5. Farnesol and CaM do not interact in the absence of Ca2+.

      Fig. S6. Farnesyl compound titrations into CaM.

      Fig. S7. KRAS4b-FMe and farnesol perturbations of 15N-CaM share a characteristic pattern.

      Fig. S8. Structure comparison of CaM conformations bound to FCME and CAP23/NAP22 polybasic myristoylated peptide.

      Fig. S9. Lipid orientation in CaM:FCME complex structure.

      Fig. S10. Solution NMR PRE validation of CaM-FCME crystal structure.

      Fig. S11. Membrane-based PRE probe for KRAS4b-FMe interaction with CaM in solution.

      Fig. S12. KRAS4b retains function in the CaMeRAS chimera.

      Fig. S13. Calcium-dependent change in emission spectra of CaMeRAS chimera FRET protein.

      Fig. S14. Tracking cellular localization of CaMeRASWT in HeLa cells after ionomycin treatment.

      Fig. S15. Cellular image segmentation for FRET histogram analysis by Gaussian modeling.

      Fig. S16. Fluorescence lifetime of mTq2 of CaMeRASWT is reduced by histamine stimulation in HeLa cells.

      Fig. S17. ATP-evoked Ca2+ signal induces CaMeRASWT internalization and FRET change in HEK293 cells.

      Fig. S18. The nucleotide and amino acid sequences of CaMeRASWT chimeric FRET construct.

      Table S1. Peak values of FRET histograms and the numbers of the ROIs analyzed.

      Table S2. P values from Student’s t tests comparing Gaussian curve models of FRET histogram data.

      Table S3. Primer sequences used for plasmid construction.

      Movie S1. Co-imaging CaMeRASWT and calcium response to histamine stimulation in HeLa cells.

      Movie S2. Slow motion highlight: Co-imaging CaMeRASWT and calcium response to histamine stimulation in HeLa cells.

      Movie S3. Coimaging CaMeRASWT and calcium response to histamine stimulation in HeLa cells and calcium chelation.

      Movie S4. Coimaging CaMeRAS185A and calcium response to histamine stimulation in HeLa cells.

      Movie S5. Coimaging CaMeRAS1234Q and calcium response to histamine stimulation in HeLa cells.

      Data file S1. CaM-farnesol binding CSP by residue.

      Data file S2. CaM-FCME PRE.

      Data file S3. KRAS4b-ND PRE.

      Data file S4. In vitro CaMeRAS Ca2+ titration.

      Data file S5. Relative fluorophore intensity in vitro as a function of Ca2+ titration for CaMeRAS.

      Data file S6. In vivo FRET from CaMeRAS contructs in response to histamine stimulation and Ca2+ influx.

    • The PDF file includes:

      • Fig. S1. RAS isoform-specific HVRs.
      • Fig. S2. There is no major interaction between unfarnesylated KRAS4b (1–185) and CaM, independent of bound nucleotide.
      • Fig. S3. A new class of noncanonical CaM binding targets: Singly lipidated polybasic peptides.
      • Fig. S4. Chemical shift analysis of CaM residues upon farnesol binding.
      • Fig. S5. Farnesol and CaM do not interact in the absence of Ca2+.
      • Fig. S6. Farnesyl compound titrations into CaM.
      • Fig. S7. KRAS4b-FMe and farnesol perturbations of 15N-CaM share a characteristic pattern.
      • Fig. S8. Structure comparison of CaM conformations bound to FCME and CAP23/NAP22 polybasic myristoylated peptide.
      • Fig. S9. Lipid orientation in CaM:FCME complex structure.
      • Fig. S10. Solution NMR PRE validation of CaM-FCME crystal structure.
      • Fig. S11. Membrane-based PRE probe for KRAS4b-FMe interaction with CaM in solution.
      • Fig. S12. KRAS4b retains function in the CaMeRAS chimera.
      • Fig. S13. Calcium-dependent change in emission spectra of CaMeRAS chimera FRET protein.
      • Fig. S14. Tracking cellular localization of CaMeRASWT in HeLa cells after ionomycin treatment.
      • Fig. S15. Cellular image segmentation for FRET histogram analysis by Gaussian modeling.
      • Fig. S16. Fluorescence lifetime of mTq2 of CaMeRASWT is reduced by histamine stimulation in HeLa cells.
      • Fig. S17. ATP-evoked Ca2+ signal induces CaMeRASWT internalization and FRET change in HEK293 cells.
      • Fig. S18. The nucleotide and amino acid sequences of CaMeRASWT chimeric FRET construct.
      • Table S1. Peak values of FRET histograms and the numbers of the ROIs analyzed.
      • Table S2. P values from Student’s t tests comparing Gaussian curve models of FRET histogram data.
      • Table S3. Primer sequences used for plasmid construction.
      • Legends for movies S1 to S5
      • Legends for data files S1 to S6

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Movie S1 (.avi format). Co-imaging CaMeRASWT and calcium response to histamine stimulation in HeLa cells.
      • Movie S2 (.avi format). Slow motion highlight: Co-imaging CaMeRASWT and calcium response to histamine stimulation in HeLa cells.
      • Movie S3 (.avi format). Coimaging CaMeRASWT and calcium response to histamine stimulation in HeLa cells and calcium chelation.
      • Movie S4 (.avi format). Coimaging CaMeRAS185A and calcium response to histamine stimulation in HeLa cells.
      • Movie S5 (.avi format). Coimaging CaMeRAS1234Q and calcium response to histamine stimulation in HeLa cells.
      • Data file S1 (Microsoft Excel format). CaM-farnesol binding CSP by residue.
      • Data file S2 (Microsoft Excel format). CaM-FCME PRE.
      • Data file S3 (Microsoft Excel format). KRAS4b-ND PRE.
      • Data file S4 (Microsoft Excel format). In vitro CaMeRAS Ca2+ titration.
      • Data file S5 (Microsoft Excel format). Relative fluorophore intensity in vitro as a function of Ca2+ titration for CaMeRAS.
      • Data file S6 (.zip file). In vivo FRET from CaMeRAS contructs in response to histamine stimulation and Ca2+ influx.

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