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. 2008 Sep 24;130(38):12624-5.
doi: 10.1021/ja805110q. Epub 2008 Aug 30.

Similar membrane affinity of mono- and Di-S-acylated ras membrane anchors: a new twist in the role of protein lipidation

Alemayehu A Gorfe  1 , J Andrew McCammon
Affiliations

Similar membrane affinity of mono- and Di-S-acylated ras membrane anchors: a new twist in the role of protein lipidation

Alemayehu A Gorfe et al. J Am Chem Soc. .

Abstract

The functionally required membrane attachment of Ras is achieved through an invariant isoprenylation of a C-terminal Cys, supplemented by further lipid modification of adjacent Cys residues by one (N-ras) or two (H-ras) palmitoyls. However, whether the triply lipidated membrane anchor of H-ras has a higher membrane affinity than its doubly lipidated counterpart, or whether the affinity contribution of the two palmitates and the farnesyl is additive, was not known. To address this issue, we carried out potential of mean force (PMF or free energy profile) calculations on a hexadecylated but nonpalmitoylated anchor (Cys186-HD), hexadecylated and monopalmitoylated anchors (Cys181-monopalmitate and Cys184-monopalmitate), and a nonlipid-modified anchor. We found that the overall insertion free energy follows the trend Cys181/Cys184-bipalmitate (wild type) approximately Cys181-monopalmitate > Cys184-monopalmitate >> nonpalmitoylated anchor. Consistent with suggestions from recent cell biological experiments, the computed PMFs, coupled with structural analysis, demonstrate that membrane affinity of the Ras anchor depends on both the hydrophobicity of the palmitate and the prenyl groups and the spacing between them. The data further suggest that while Cys181-palmitate and Cys186-farnesyl together provide sufficient hydrophobic force for tight membrane binding, the palmitoyl at Cys184 is likely designed to serve another, presumably functional, role.

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Figures

Figure 1
Figure 1
Insertion free energy and membrane interaction of Ras. (A) Potential of mean force (PMF) for the insertion of wild-type and mutant H-ras anchors into a DMPC bilayer. The computed PMF is plotted against the average distance of backbone C and O atoms’ center-of-geometry from the bilayer center (r). (B) Evolution of anchor−DMPC nonpolar contacts. Contacts are defined as the number of selected Ras heavy atoms within 4 Å of DMPC tail carbon atoms. Contacts by Ras lipid tail (heavy lines), Met182 side chain (medium thick lines), and Lys side chain (thin lines) are shown.
Figure 2
Figure 2
Radial distribution functions (g(r)) for backbone nitrogen atoms and DMPC phosphorus atoms. Snapshots representing the distribution of anchor atoms at the approximate location of the free energy minimum are provided as insets. Color code: carbon, green; nitrogen, blue; oxygen, red; sulfur, yellow.
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