Fig. 1

TRAF7 mutations disrupt protein structure and interactions. (A) Protein structure of TRAF7; developmental variants V142M, V442M, and c.1998+2T>G and the locations of previously reported (16) meningioma-associated mutations (asterisks; 93% map to the WD40 domains), including those analyzed in this study. (B) Predicted structure of TRAF7. The RING finger (green), coiled-coil (blue), and putative 7-WD40-repeat-containing putative ligand-binding (red) domains are indicated. The mutations (gray circles) are primarily localized to one face of the latter. (C) Representation of the WD40-domain molecular surface, exhibiting a hydrophobic patch (pink, reflecting concentration of white [small hydrophobic] and magenta [aromatic] residues) surrounding the pore of the β-propeller surface. Yellow: cysteine; pale green: proline; green: glycine; cyan: polar; blue: positively charged residues; red: negatively charged residues. (D) The N (red) to S (blue) mutation at position 520 changes hydrogen bonding (yellow dashes) between the β-strands of the preceding blade of the WD40 β-propeller domain. (E) Substitution of Y residues 563, 603, and 621 (red) with charged and polar (blue) residues results in loss of hydrophobic interaction (purple – light pink dashes). (F) The W (red) to R (blue) substitution at position 400 abrogates hydrophobic interactions with several residues involving multiple β-propeller units. (G) WT and meningioma-associated mutant forms of TRAF7 can form homo- and hetero-dimers. (H) Mutant TRAF7 disrupts the interaction with endogenous MEKK3. Coimmunoprecipitation analysis in HEK293 cells. (I) Low concentrations of mutant TRAF7 (C388Y) are sufficient to disrupt the interaction of WT TRAF7 with MEKK3. Plasmids expressing C388Y (1, 2, 3, or 4 μg) and WT (4 μg) TRAF7 were cotransfected in HEK293 cells followed by immunoprecipitation for WT-TRAF7. (J and K) TRAF7 mutants display reduced ubiquitination in the absence (J) or presence (K) of exogenous MEKK3. (L) Surgically resected TRAF7-mutant meningiomas highly express TRAF7 (genotypes of tumors shown: MUN290: NF2; Yale 132: SMO W535L; MUN105: TRAF7 R641C/AKT1 E49K; MUN201: TRAF7 L580del/AKT1 E49K; Yale 25: TRAF7 G536S/KLF4 K409Q; MUN191: TRAF7 K615E/KLF4 K409Q). (Scale bar, 50 μm.) Confocal images captured under identical settings.