Figure 3. Heterohexameric ring assembly.
(A) Heterohexameric model of alternating LAP1 (light blue) and TorsinA (green hues), based on the hexameric ring of ClpC-D2 domains (3PXI; Wang et al., 2011). The C-terminal domain of Torsin is colored bright green. An ATP molecule (red) is modeled into the TorsinA nucleotide binding pocket. The conserved arginine finger in LAP1 is in blue. The disulfide bridge within LAP1 is in yellow. (B) Schematic diagram of (A), same coloring scheme. (C) A montage of representative particle classes of the TorsinA(E171Q):LULL1 complex obtained from negatively stained particles are shown. The particles are consistent with a toroidal hexameric conformation as shown in (A and B). The particle diameter is approximately 120 Å (double arrow).
Figure 3—figure supplement 1. Analytical gel filtration of TorsinA(E171Q):LAP1 and TorsinA(E171Q):LULL1 complexes.
Elution profiles of TorsinA(E171Q):LAP1 (red) and TorsinA(E171Q):LULL1 (red dashes) are compared with uncomplexed LAP1 (blue) and LULL1 (blue dashes), respectively. The experiment was performed on a Superdex S200 HR10/300 column. Elution volumes of standard globular protein markers are displayed. SDS-PAGE analysis of peak fractions is shown. The fractions are indicated below the chromatogram. The TorsinA:LAP1 and TorsinA:LULL1 protein complexes elute at positions consistent with a heterodimer. SDS-PAGE analysis indicates a 1:1 stoichiometry for the complexes.
Figure 3—figure supplement 2. Negative-stain micrographs.
Protein samples were negatively stained with uranyl acetate and analyzed by electron microscopy. All samples were measured at closely comparable concentrations. Scale bars represent 100 nm. While ring-like structures are visible in the TorsinA(E171Q):LULL1 and TorsinA(E171Q):LAP1 samples (A and B), no discernible objects are seen on the micrographs of uncomplexed LAP1 and LULL1 (C and D). This indicates that LAP1 and LULL1 do not form homohexameric rings in the absence of TorsinA.