Figure 2: VCP-KMT interacts with and methylates VCP in vitro.
From: Lysine methylation of VCP by a member of a novel human protein methyltransferase family
(a) Structure of hexameric VCP. Three of the protomers have been individually colour-labeled and darker colour indicates the selected interaction domain (SID) involved in VCP-KMT interaction. The structure was rendered using symmetry expansion on a published VCP structure (pdb 1oz4)8. (b) Domain structure of VCP showing the N-terminal, D1 ATPase and D2 ATPase domains, and indicating the SID and VCPΔD2. (c) VCP-KMT methylates VCP and VCPΔD2. Ten microgram of substrate were incubated with varying amounts of VCP-KMT at pH 7.5. Data are represented as means±s.d., n=3. (d) VCP-KMT disrupts the hexamer and forms a complex with VCPΔD2. Hexameric VCP or VCPΔD2 were incubated with a twofold molar excess of VCP-KMT, followed by SEC. Left, Elution profiles (absorbance at 280 nm) of protein samples and size standards. Right, Coomassie blue-stained SDS–PAGE of indicated fractions. (e) Methylation assay using purified hexameric VCP and VCPΔD2. Ten microgram of protein before SEC, hexameric protein purified as shown in (d), or aggregated protein eluting in the void volume were incubated with 100 pmol VCP-KMT. Error bars indicate range between duplicate samples. (f) MS/MS sequencing of Arg-C generated peptide VCPΔD2(288–322)-Kme3 ([M+1H]+=4,007.124 Da) showing trimethylation of Lys315. The corresponding spectrum is shown in Supplementary Fig. S2a. (g) Mutational analysis of VCPΔD2 methylation. VCPΔD2 variants with point mutations in the region surrounding Lys315 were incubated with 100 pmol VCP-KMT. Data are represented as means±s.e.m., n=4. Four variants (#) showed changed assembly and/or aggregation, see Supplementary Fig. S4. (h) Methylation status of VCPΔD2 incubated with varying amounts of VCP-KMT. The methylation status of Lys315 in peptide VCPΔD2(288–322) was quantified by MS. See also Supplementary Fig. S2c.
