Influence of the lipid anchor motif of N-ras on the interaction with lipid membranes: a surface plasmon resonance study

Abstract

Ras GTPases play a crucial role in signal transduction cascades involved in cell differentiation and proliferation, and membrane binding is essential for their proper function. To determine the influence of the nature of the lipid anchor motif and the difference between the active (GTP) and inactive (GDP) forms of N-Ras on partitioning and localization in the lipid membrane, five different N-Ras constructs with different lipid anchors and nucleotide loading (Far/Far (GDP), HD/Far (GDP), HD/HD (GDP), Far (GDP), and HD/Far (GppNHp)) were synthesized. Using the surface plasmon resonance technique, we were able to follow the insertion and dissociation process of the lipidated proteins into and out of model membranes consisting of pure liquid-ordered (l(o)) or liquid-disordered (l(d)) phase and a heterogeneous two-phase mixture, i.e., a raft mixture with l(o) + l(d) phase coexistence. In addition, we examined the influence of negatively charged headgroups and stored curvature elastic stress on the binding properties of the lipidated N-Ras proteins. In most cases, significant differences were found for the various anchor motifs. In general, N-Ras proteins insert preferentially into a fluidlike, rather than a rigid, ordered lipid bilayer environment. Electrostatic interactions with lipid headgroups or stored curvature elastic stress of the membrane seem to have no drastic effect on the binding and dissociation processes of the lipidated proteins. The monofarnesylated N-Ras exhibits generally the highest association rate and fastest dissociation process in fluidlike membranes. Double lipidation, especially including farnesylation, of the protein leads to drastically reduced initial binding rates but strong final association. The change in the nucleotide loading of the natural N-Ras HD/Far induces a slightly different binding and dissociation kinetics, as well as stability of association, and seems to influence the tendency to segregate laterally in the membrane plane. The GDP-bound inactive form of N-Ras with an HD/Far anchor shows stronger membrane association, which might be due to a more pronounced tendency to self-assemble in the membrane matrix than is seen with the active GTP-bound form.

Figure 1

Schematic of the semisynthetic N-Ras with the different anchor systems and nucleotides investigated (SH, unmodified; HD, hexadecyl; Far, Farnesyl). If no nucleotide is mentioned, the protein is in the inactive, GDP-bound state.

Figure 2

Sensorgrams of 1 μM N-Ras Far (blue), Far/Far (red), HD/HD (turquoise), HD/Far (GDP) (pink), HD/Far (GTP) (green), and nonlipidated N-Ras (olive) in the presence of a DOPC lipid bilayer. Respective fits to a two-step reaction model (Eqs. 2–4) are shown as dotted black lines. Sensorgrams were recorded at a frequency of 10 Hz.

Figure 3

(A) k_on,1 and (B) k_off,1 values of N-Ras Far (GDP), HD/Far (GDP), HD/Far (GTP), HD/HD (GDP), and Far/Far (GDP) binding to a DOPC (red), DPPC/Chol (7:3) (green), DOPC/DPPC/Chol (1:2:1) (blue), DOPC/DOPG (7:3) (turquoise), or DOPC/DOPE (7:3) (pink) lipid bilayer. Values were derived from the SPR data by applying a two-step model fit. The error bars represent the standard deviation from at least three (up to six) independently conducted experiments.

Figure 4

(A) k_on,2 and (B) k_off,2 values of N-Ras Far (GDP), HD/Far (GDP), HD/Far (GTP), HD/HD (GDP), and Far/Far (GDP) binding to a DOPC (red), DPPC/Chol (7:3) (green), DOPC/DPPC/Chol (1:2:1) (blue), DOPC/DOPG (7:3) (turquoise), or DOPC/DOPE (7:3) (pink) lipid bilayer. Values were derived from the SPR data by applying a two-step model fit. The error bars represent the standard deviation from at least three (up to six) independently conducted experiments.

Figure 5

(A) Initial slope and (B) averaged dissociation constant of N-Ras Far (GDP), HD/Far (GDP), HD/Far (GTP), HD/HD (GDP), and Far/Far (GDP) binding to a DOPC (red), DPPC/Chol (7:3) (green), DOPC/DPPC/Chol (1:2:1) (blue), DOPC/DOPG (7:3) (turquoise), or DOPC/DOPE (7:3) (pink) lipid bilayer. (C) Corresponding relative amount of quasi-irreversibly bound protein. The error bars represent the standard deviation from at least three (up to six) independently conducted experiments.