Extended Data Figure 9. Proposed reaction cycle for ICMT.

Major steps along the reaction coordinate (I–V). From the AdoHcy-bound state (I), a hinged displacement of the M6–M7 connector loop (II) allows release of AdoHcy and exchange for AdoMet from the cytosol (III). The C-terminal prenyl group of the substrate is located within the ER membrane prior to methylation, as depicted for k-Ras4b in the figure, and approaches the enzyme by two-dimensional diffusion within the membrane. The prenylcysteine reaches the active site by passing between the M5 and M8 helices (coloured magenta and blue in the figure, respectively). In the ternary complex (IV), substrate-enzyme contacts are limited to interactions with the prenyl group and the C-terminal carboxylate, giving rise to specificity for these elements within the context of a wide range of protein substrates. A cytosolic cleft above the M5–M8 crevice that leads to the active site accommodates the polar C-terminal peptide of protein substrates (e.g. the polybasic region of k-Ras4b, coloured blue in the figure). The inhibitory monobody occupies this region. Catalysis proceeds from the ternary complex, and the product, made more lipophilic by methylation and neutralization of a negative charge, is able to diffuse into the ER membrane (V). ICMT is show as a cartoon with helices drawn as cylinders and the AdoHcy/AdoMet cofactors depicted as cyan sticks. A black sphere indicates the methyl group of AdoMet. Prenylated k-Ras4b (based upon PDB ID 5TAR⁴²) is depicted as a molecular surface and coloured according to electrostatic potential (red, negative; blue, positive) with its prenyl group shown as orange spheres. The ER membrane is depicted in gray and curved in the vicinity of the active site to suggest that the enzyme might modulate the local distribution of lipid molecules to facilitate substrate access.