(A) Tumor derived mutants (red sticks) are shown in the context of the overall structure of the FERM domain of human Merlin (colored ribbons; PDB 1ISN). The α-helical coiled-coil domain from the crystal structure of the closed form of Spodoptera frugiperda Moesin (thin grey αcarbon trace; PDB 2I1K), which is highly homologous to human Merlin, is shown for reference. The coiled-coil domain was positioned by superimposing the FERM domains from the structures of Merlin and Moesin, which have a root mean square deviation for α-carbon atoms of 1.3 Å and share 62% sequence identity.
(B) Close-up view of tumor-derived mutations (red sticks) located in subdomain A (green ribbon) in the crystal structure of human Merlin. Residues in contact with mutated residues are drawn as sticks and colored according to atom type (carbon, yellow; sulfur, green).
(C) Subdomain B represented as in (B), with oxygens as magenta sticks. Red sphere, G197.
(D) Meso-33 cells were transiently transfected with empty vector or vectors encoding wild type Merlin or indicated mutants. Asynchronized cells growing in 10% FCS were subjected to BrdU incorporation assay. The graph indicates the percentage (± SEM) of BrdU-positive cells. Pictures show representative fields of cells transfected with indicated vectors and stained with anti-BrdU (red) and DAPI (blue).
(E) Cos7 cells were transfected with FH-tagged versions of wild type Merlin or indicated mutants. Flag- or control-immunoprecipitates (M2 and C, respectively) and total lysates were immunoblotted as indicated.
See also Supplemental Figure S5.