1. Computational Modeling Reveals that Signaling Lipids Modulate the Orientation of K- Ras4A at the Membrane Reflecting Protein Topology 
Zhen-Lu Li, Matthias Buck 
Structure 
Volume 25, Issue 4, Pages e2 (April 2017)
DOI: /j.str
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2. Structure 2017 25, 679-689.e2DOI: (10.1016/j.str.2017.02.007)
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3. Figure 1 Sequence and Structure of K-Ras
(A) Sequence of human K-Ras4A and K-Ras4B. Basic residues are in blue, acidic residues in red, polar residues in green, and non-polar residues in black. Note that only four residues differ in the CD while the HVR is considerably different.
(B) Structural model of K-Ras4A. Residues 1–86 (lobe 1) are colored pink and residues 87–166 (lobe 2) cyan. HVR (residues 167–186) is in gray.
(C) Initial simulation system. CD (residues 1–166) is in surface representation and HVR in trace representation, except that the FAR group is highlighted by green beads. The lipid membrane is denoted by gray lines.
See also Table S1.
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4. Figure 2 Binding of K-Ras4A to the Membrane
Row 1: time evolution of the distance in the z direction between center of mass of the K-Ras4A CD and the center of mass of the membrane (see also Movies S1 and S2). Rows 2–5: contact map of residues of the CD with the membrane. Residues within 0.3 nm of membrane lipids are treated as contact residues.
(A–D) Results for the POPC (A), POPC/POPS (B), and POPC/PIP2 (C and D) systems.
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5. Figure 3
Role of HVR
(A) Average distance and standard deviations in the z direction between the center of mass of residues in the HVR and that of membrane. The average distance and deviations are given for the simulations by each kind of model membrane.
(B) The contact probability (percent time of contact at <0.3 nm) of HVR with the CD averaged over all the simulations. The inserted snapshot (final structure from the PI1 system) highlights the interaction residues.
See also Figures S1 and S2.
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6. Figure 4 Multiple Orientation of K-Ras4A
Contour maps of the relative orientation of K-Ras4A with respect to the membrane for the (A) POPC, (B) POPC/POPS, and (C) POPC/PIP2 systems. D1 is the distance between the vertical center of membrane and that of lobe 1. θ is the cross angle between the normal direction of the membrane and a directional vector of K-Ras4A CD as explained in STAR Methods and Figure S3. The color scale from gray to red denotes the relative population of K-Ras4A in different orientation states (D1, θ). The probability is calculated by dividing the number of each state by the total number of sampling frames and then scaled to 1 for the maximally populated state at each membrane. See also Figure S3.
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7. Figure 5 Typical Orientations of K-Ras4A Relative to Membranes
Lobe 1 domain is colored pink, lobe 2 and HVR cyan, POPC gray, and POPS and PIP2 green. The main secondary structure elements in contact with the membrane are labeled. GTP nucleotide (shown as surface representation in purple) and the two switch regions are typically located at least 1.0 nm away from the membrane surface.
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8. Figure 6 Charge of Lipid Membrane and K-Ras4A
(A) Distribution of charges due to placement of phosphate in the lipid headgroups at the surface of the three model membranes in a 100 ns simulation (without protein).
(B) Net distribution of charge of lipid head-phosphate groups in the POPC/POPS system or POPC/PIP2 system by subtracting the distribution of charges in the POPC system.
(C) Radial distribution function of ions (sodium and chlorine ion) relative to the center of mass of the head group of a POPC, POPS, or PIP2 lipid in the membrane-only simulations.
(D and E) Protein residues that are in frequent contact with the membrane for the POPC/POPS (D) and POPC/PIP2 (E) systems. The frequency is calculated by taking the time average of contacts seen in all the coordinate frames for each kind of membrane system.
(F and G) The density of POPS (F) and PIP2 (G) from the center of the K-Ras4A CD (density scaled to 1 for maximum). The CD is centered at (5.0 nm, 5.0 nm), and the direction vector connecting the center of CD and HVR is aligned in the direction of the x axis.
(H) Charged lipid molecules gather within 2.5 nm of the center of the Ras CD (which itself has a radius of gyration of about 1.54 nm) in the POPC/POPS or POPC/PIP2 systems.
See also Figure S4.
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9. Figure 7
Snapshot of Anionic Lipid Interacting with Nearby Charged Protein Residues
Coordinate frames for PS1 (A) and PS2 (B) in O1-like orientation; and for PI1 (C), PI3 (D), and PI4 (E) in O3-like orientation. Na+ ions that were found to mediate the interaction of negative residues with anionic lipids in (B) and (D) are shown as yellow beads. See also Table S2.
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10. Figure 8
Electrostatic Interaction and Protein Topology in Regulating K-Ras Membrane Orientation
(A) Arrangement of surface secondary structure elements of K-Ras4A. The center of mass of β1-β3, α2-α5 are projected on the membrane surface, respectively, with direction vector Vy (see Figure S3) perpendicular to the membrane surface. Each two adjacent elements constitute a surface with a certain orientation. Charged residues exposed to the membrane in each kind of orientation (contact probability larger than 10%) are denoted. Residues are shown as balls for Cα and Cβ atoms, indicating the main chain and the side chain. Negatively charged residues are denoted in red. Positively charged residues are denoted in blue or orange. The blue residues, but not the orange residues, are found to directly interact with anionic lipids.
(B) Competitive interaction of the anionic membranes interacting with positive and negative residues. O1 has more negatively charged residues exposed to the membrane, while PIP2 preferred O3 orientation has more negative charges above the membrane surface. Rather than a rotation the protein is flipped, using the opposite surface to contact the membrane.
(C) Solvent-accessible surface buried between protein and lipid bilayers (on the side of the protein) for different orientations. Parts of the simulations corresponding to the specific orientation states are used to calculate mean values and the standard deviations.
See also Figure S5 and Table S3.
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