Figure 1. Crystal structure of human LAP1.
(A) Crystal structure of the luminal domain of LAP1 from Homo sapiens. The helices are numbered according to the nomenclature used for AAA+ proteins (Erzberger and Berger, 2006). A disulfide bridge (yellow) attaches the C terminus to helix α1. (B) Same as (A), but rotated around the x-axis by 50°. (C) Crystal structure of the AMPPCP-bound ClpB-D2 domain from Thermus thermophilus (4LJ6; Zeymer et al., 2014) in the same orientation as LAP1 in (A) revealing the striking topological similarity. In comparison to ClpB, LAP1 does not bind a nucleotide and has no C-terminal domain.
Figure 1—figure supplement 1. Phylogenetic analysis of LAP1 homologs.
Sequence alignment of phylogenetically diverse sequences of the luminal domain of LAP1 and LULL1. Residues are color-coded from dark blue (most conserved) to white (not conserved). Secondary structure elements of human LAP1 are shown above the sequences. The conserved disulfide bond is indicated with a yellow line. The strictly conserved arginine (‘R-finger’) is marked by a green circle.
Figure 1—figure supplement 2. Representative 2Fo-Fc electron density of the final model.
Stereo view of the binding site of VHH-BS1 (gray) with LAP1 (blue). Contacting residues between LAP1 and the variable loops of VHH-BS1 (red) are shown as sticks and are labeled. Waters, with direct contact to both binding partners, are in green. The 2Fo-Fc map is contoured at 1σ.
Figure 1—figure supplement 3. VHH-BS1 competes with TorsinA for LAP1 binding.
(A) Overall structure of the LAP1-VHH-BS1 complex. LAP1 is in light blue, VHH-BS1 in gray. The variable loops, responsible for high affinity interaction with LAP1, are colored in red. The binding site on LAP1 is centered around arginine 563 (blue sticks). (B) Model of LAP1 (light blue) interacting with TorsinA (green) in a putative heterohexameric ring assembly in the same orientations as in (A). TorsinA-bound ATP molecule is in red. Note the overlap between the VHH-BS1 and the putative TorsinA binding site on LAP1. (C and D) In vitro precipitation assay shows that VHH-BS1 competes with TorsinA(E171Q) for LAP1 binding. Preformed TorsinA(E171Q)-LAP1 complex was incubated for several hours with VHH-BS1 (C) or a control VHH (D). The mixtures were centrifuged at the indicated times to separate soluble from insoluble protein. The samples were analyzed by SDS-PAGE. Due to the competition with VHH-BS1, TorsinA(E171Q) is released from LAP1. Since free TorsinA(E171Q) is insoluble, the process of the reaction can be monitored.