Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT

Abstract

The maturation of RAS GTPases and approximately 200 other cellular CAAX proteins involves three enzymatic steps: addition of a farnesyl or geranylgeranyl prenyl lipid to the cysteine (C) in the C-terminal CAAX motif, proteolytic cleavage of the AAX residues and methylation of the exposed prenylcysteine residue at its terminal carboxylate1. This final step is catalysed by isoprenylcysteine carboxyl methyltransferase (ICMT), a eukaryote-specific integral membrane enzyme that resides in the endoplasmic reticulum2. ICMT is the only cellular enzyme that is known to methylate prenylcysteine substrates; methylation is important for the biological functions of these substrates, such as the membrane localization and subsequent activity of RAS1, prelamin A3 and RAB4. Inhibition of ICMT has potential for combating progeria3 and cancer5,6,7,8. Here we present an X-ray structure of ICMT, in complex with its cofactor, an ordered lipid molecule and a monobody inhibitor, at 2.3 Å resolution. The active site spans cytosolic and membrane-exposed regions, indicating distinct entry routes for the cytosolic methyl donor, S-adenosyl-l-methionine, and for prenylcysteine substrates, which are associated with the endoplasmic reticulum membrane. The structure suggests how ICMT overcomes the topographical challenge and unfavourable energetics of bringing two reactants that have different cellular localizations together in a membrane environment—a relatively uncharacterized but defining feature of many integral membrane enzymes.

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Figure 1: Architecture of ICMT.
Figure 2: The active site.
Figure 3: Lipid-binding cavity and monobody complex.
Figure 4: Substrate access and transition-state models.

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Acknowledgements

We thank D. Julius, C. D. Lima, M. Luo, N. P. Pavletich, R. K. Hite, S. Shuman and members of the Long laboratory for discussions. Beamlines 24-ID and 23-ID at the Advanced Photon Source are supported by NIH grants ACB-12002, AGM-12006, P41 GM103403 and S10 RR029205, under DOE contract DE-AC02-06CH11357. This work was supported, in part, by an American Heart Association Pre-doctoral Fellowship (M.M.D.), a Burroughs Wellcome Career Award (S.B.L), the Geoffrey Beene Cancer Research Center at MSKCC (S.B.L.), a core-facilities support grant to MSKCC (P30 CA008748) and NIH grant U54-GM087519 (S.K.).

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M.M.D. and L.P. cloned, expressed and purified proteins. A.K. and S.K. developed monobodies. M.M.D. performed all other experiments. M.M.D. and S.B.L. designed experiments, determined structures, analysed results and prepared the manuscript with contributions from all authors.

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Correspondence to Stephen B. Long.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks M. Bergö, P. Casey and O. Nureki for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 The structure identifies functions for key residues.

ICMT is displayed as a progression from primary sequence alignment, to the effects of scanning mutagenesis (bar graphs), to observed secondary structure (α-helices depicted as ribbons), to the assigned role of amino acids. Results from scanning mutagenesis experiments12 are plotted above the sequence alignment as a bar graph showing the reduction of specific activity in comparison to wild-type ICMT, with 0% representing wild-type activity and 100% reflecting no detectable activity (indicated by a horizontal dashed line). The inset key denotes the colouring of the bar graph according to the functional role of the amino acid inferred from the atomic structure. Labelled brackets above the secondary structure denote the general function of the indicated regions of the primary sequence. In the bar graph: magenta, amino acids that contact AdoHcy; red, amino acids that line the lipid-binding cavity; blue, arginine residues that are proposed to form hydrogen bonds with the carboxylate of the prenylcysteine substrate, and the residues that position them; grey, residues proposed to form hydrogen bonds to the methyl of AdoHcy in the transition state; hydrogen bonds made with backbone atoms are indicated by parentheses surrounding the amino acid label. The mutagenesis data are derived from experiments using A. gambiae ICMT12, and are normalized for expression level. Except where noted, the mutations were alanine substitutions. In some cases, leucine substitutions (L) were made (for example, when the wild-type amino acid was a glycine or alanine). The data represent triplicate measurements for each mutation and the mean s.d. is 11%. Gaps in the bar graph indicate amino acid positions that were not analysed by mutagenesis. Based on size-exclusion chromatography that was performed for each of the mutants, only the E141A mutation was found to be notably destabilizing (asterisk). A few mutations increased the activity relative to wild-type; these are shown as exhibiting 0% reduction in activity. The amino acid sequences included in the alignment are: T. castaneum ICMT (beetle ICMT), human (Hs), A. gambiae (Ag), Saccharomyces cerevisiae (Sc), and Arabidopsis thaliana (At) (UniProt accession numbers: D6WJ77, O60725, Q7PXA7, P32584 and Q93W54, respectively). The alignment is coloured according to the ClustalW convention.

Extended Data Figure 2 Enzymatic activity and monobody inhibition of beetle ICMT.

a, Schematic of the ICMT reaction. The shaded region represents the endoplasmic reticulum membrane; R, the protein portion of the substrate. In the minimal substrate AGGC, R is an acetyl group. For BFC, R is a biotin group. be, Activity of beetle ICMT, purified in DMNG detergent (b, c) or in cell lysate (d, e), shown as a plot of the formation of BFC-[3H]methyl ester as a function of BFC concentration (b, d) or AdoMet concentration (c, e). For assays using HEK293 cells (d, e), ICMT was expressed as a fusion protein with GFP in order to quantify the amount of enzyme in the cell lysate (Methods). f, Kinetic parameters determined from the curves in be. Best-fit values (calculated in GraphPad Prism 7) are reported with the standard error of the fit. We observed a degree of substrate inhibition at higher concentrations of BFC (Ki ~40 μM) when using ICMT in detergent (b, f), which may be due to the dispersive nature of BFC when it exceeds the detergent concentration (10 μM). g, Dose–response curve showing the inhibitory effects of the monobody on the activity of purified beetle ICMT in detergent. h, Comparison of the effects of the monobody on the activities of beetle, human, and A. gambiae ICMT in HEK293 cell lysates. GFP–ICMT fusion proteins were used for the three orthologues and the concentration of each enzyme was determined using the fluorescence of GFP (Methods). The monobody inhibited beetle ICMT with an IC50 of ~7 μM in this assay, whereas no detectable inhibition of human or A. gambiae ICMT was observed. Individual data points are shown on the graphs (be, g, h).

Extended Data Figure 3 Electron density maps and crystal lattice.

a, Stereo representation of the electron density for the ICMT–monobody complex (blue mesh, 2FoFc, countoured at 1.1σ, calculated from 35 to 2.3 Å resolution) drawn around the atomic model (stick representation). The monobody is coloured magenta; ICMT is tan. b, Electron density for the lipid in the active site from a composite simulated-annealing omit-electron density map (2FoFc, contoured at 1σ and calculated from 35 to 2.3 Å resolution). The density would accommodate a geranylgeranyl lipid (orange sticks), as shown here. c, Electron density (blue mesh; 2FoFc map contoured at 1σ) for ordered monoolein lipids (magenta sticks) around ICMT. Monoolein lipids surround the transmembrane region of ICMT and collectively resemble a bilayer with typical thickness (arrows). The positioning of monoolein lipids in the vicinity of the M4–M5 connector (yellow) is consistent with the hypothesis that the enzyme would cause a slight depression in the membrane in this region, as illustrated in Extended Data Fig. 9. d, Crystal lattice in the ICMT–monobody complex, with ICMT coloured and the monobody in grey. e, A composite omit-electron-density map (2FoFc, contoured at 1σ and calculated from 35 to 4.0 Å resolution) for the X-ray structure of ICMT without the monobody. The composite omit maps, which reduce model bias, were obtained using Phenix36.

Extended Data Figure 4 Interactions with the M5–M6 connector, interactions with active site arginine residues and cross-section of the active site.

a, Interactions between the M5–M6 and M7–M8 connectors. The side chains of Glu141 (on M5) and Lys153 (on the M5–M6 connector) form hydrogen bonds (dashed lines) that cap the C-terminal end of M7 and the N-terminal end of M8, respectively. Portions of the amino acids involved are drawn as sticks. Parentheses indicate hydrogen bonds made with backbone atoms. Grey, carbon; blue, nitrogen; red, oxygen. b, Hydrogen-bonding network involving Arg173 and Arg246. Bonds (dashed lines) are made with surrounding amino acids (labelled sticks) and two water molecules (red spheres) in the active site. c, Cutaway view of the molecular surface of ICMT (grey), showing labelled regions of the active site. The view is approximately orthogonal to that shown in Fig. 2a. AdoHcy is drawn as sticks; approximate boundaries of the membrane are indicated by horizontal bars.

Extended Data Figure 5 The M4–M5 connector.

a, Close up view of the M4–M5 connector with side chains depicted as sticks. ICMT is predominately grey, with the M4–M5 connector coloured yellow. Amino acids that interact with Phe123 (Ile64, Leu71 and Phe107) are coloured grey. b, The N-terminal end of the M5 helix is capped by Ser128. The side chain of Ser128 is shown in stick representation, with the hydrogen bond to the backbone nitrogen atom of Tyr131 indicated by a dotted line.

Extended Data Figure 6 The M1–M2 portion of ICMT is an integral part of the enzyme.

a, The GXXXG packing motif between helices M1 and M3 of ICMT. Residues on M1 and M3 that make contacts between these helices are drawn as sticks and coloured magenta. Because the M1 and M2 helices are not present in S. cerevisiae ICMT, we hypothesize that the GXXXG motif in the first transmembrane helix of yeast ICMT (equivalent to M3 of beetle ICMT) may cause dimerization of the yeast enzyme through packing of these helices40, whereas ICMT enzymes that contain M1 and M2, which include human and beetle ICMT, are monomeric. The location of a PreScission protease cleavage site (PS site) that was introduced at Asn58, in the connection between the M2 and M3 helices, for experiments outlined in this figure, is indicated. b, Anti-His western blot showing that this cleavage site can be cleaved by PreScission protease. In this experiment, ICMT was expressed in HEK293 cells with an N-terminal His–GFP tag (His–GFP–ICMT), with or without the cleavage site. The addition of PreScission protease to detergent-solubilized sample 4 (His–GFP–ICMT with the cleavage site), but not control samples 1–3, results in a cleavage product consisting of His–GFP followed by amino acids 1–58 of ICMT (His–GFP–ICMT 1–58), which is detected by anti-His western blot. This confirms that the loop connecting the M2 and M3 helices is cut by the protease. Samples 1–3 are control experiments, as indicated. For gel source data, see Supplementary Fig. 1. c, FSEC profiles of the samples evaluated by western blot in b and numbered accordingly. Elution volumes for the void, His–GFP–ICMT and free GFP are indicated on the plot for sample 1. The cleavage of the M2–M3 loop by PreScission protease does not alter the elution profile (sample 4) in comparison to the other samples, which indicates that the cleaved portion (His–GFP–ICMT 1–58) is associated with the remainder of the enzyme via non-covalent interactions. No replicates of these experiments were performed.

Extended Data Figure 7 Comparison of ICMT and MaMTase.

a, Sequence alignment between beetle ICMT, human ICMT, and MaMTase (referred to as Ma-ICMT in ref. 16). In the alignment, red colouring indicates identical residues and pink indicates similar residues. The secondary structures of ICMT and MaMTase (PDB ID: 4A2N) are shown above and below the alignment, respectively. The region of highest sequence conservation corresponds to the cofactor-binding domain (bracketed); elsewhere there is low sequence similarity. Asterisks indicate amino acids of ICMT that contact AdoHcy; surrounding parentheses, where present, indicate that hydrogen bonds are made with backbone atoms. bd, Superposition of the structures of beetle ICMT and MaMTase, shown from three vantage points in cartoon representations. ICMT is coloured according to the colouring of its secondary structure in a; MaMTase is coloured grey.

Extended Data Figure 8 Comparison of the active sites of ICMT, MaMTase and MaSR1.

a, Two orientations of the overall structure of ICMT are shown with the active site depicted as a molecular surface. The portion that binds AdoHcy is coloured cyan and is partially transparent to reveal a stick representation of the cofactor. The cavity that would be exposed to the membrane and forms the presumed binding site of the prenylcysteine moiety of the substrate is coloured orange. An arrow marks a pathway by which the prenyl group of the substrate could reach the active site via the membrane. Horizontal lines denote the approximate boundaries of the endoplasmic reticulum membrane. b, Overall structure of MaMTase (PDB ID: 4A2N) shown in two orientations, obtained by superposing with ICMT. A cavity, denoting the active site, is drawn as a molecular surface. The portion in which AdoHcy binds is coloured cyan; the remainder is dark grey and is likely to represent the binding site of its biological substrates, which are unknown. Structural elements are coloured as for ICMT in a. The cavity is exposed to the membrane on the opposite side of the enzyme relative to ICMT. Instead of a crevice between the magenta- and dark-blue-coloured helices like ICMT (H2 and H5 of MaMTase, which roughly correspond to M5 and M8 of ICMT, respectively), MaMTase has a crevice between H1 (light blue) and H2 (magenta), suggesting that its substrates access it from the ‘right’ (arrow) rather than from the ‘left’. In ICMT, this route is blocked by the M4–M5 linker. The dimensions of the cavity in MaMTase and its exposure to the membrane suggest that the substrates of MaMTase are hydrophobic molecules that are smaller than a farnesyl or geranylgeranyl prenyl group. c, Overall structure of the prokaryotic integral membrane sterol reductase MaSR1 (PDB ID: 4QUV)18. The orientation is based on superposition of the cofactor-binding domain with ICMT, with corresponding colouring. The carbon atoms of a bound NADPH cofactor are coloured cyan. A crevice between the magenta and dark blue-coloured helices may serve as access for lipophilic sterol substrates18 (arrow).

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 endoplasmic reticulum membrane before methylation, as depicted for KRAS4B in the figure, and approaches the enzyme by 2D diffusion within the membrane. The prenylcysteine reaches the active site by passing between the M5 and M8 helices (coloured magenta and blue, 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 (for example, the polybasic region of KRAS4B, coloured blue). 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 membrane (V). ICMT is shown as a cartoon with helices drawn as cylinders and the AdoHcy or AdoMet cofactors depicted as cyan sticks. A black sphere indicates the methyl group of AdoMet. Prenylated KRAS4B (based on PDB ID: 5TAR41) 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 endoplasmic reticulum membrane is depicted in grey 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.

Extended Data Table 1 Data collection, phasing and refinement statistics

Supplementary information

Supplementary Figure 1

The uncropped scan of the Western blot in Extended Data Figure 6b. The boxed regions are displayed in Extended Data Figure 6b. The band at ~ 28 kDa corresponds to PreScission protease, which contains a His-tag. Lanes that are not boxed are not relevant to this experiment. (PDF 144 kb)

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Diver, M., Pedi, L., Koide, A. et al. Atomic structure of the eukaryotic intramembrane RAS methyltransferase ICMT. Nature 553, 526–529 (2018). https://doi.org/10.1038/nature25439

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