Fig. 4: Bortezomib induces a sixfold symmetrization of the LONP1 protease domains and promotes activation of the proteolytic active site.
From: Structures of the human LONP1 protease reveal regulatory steps involved in protease activation
a Two external axial views of the bortezomib- and substrate-bound human LONP1Bz atomic model (left) is shown next to a cutaway lateral view of LONP1, using the same representation and coloring as in Fig. 1. Bortezomib (blue) bound within the protease active sites are shown using a sphere representation, with one bortezomib molecule denoted by a dashed box. b A twelve-residue polyalanine chain is modeled into the substrate density within the central channel of the LONP1Bz structure, shown as in Fig. 2. The pore loop 1 Y565 residues from subunits ATP1-4 maintain intercalating, zipper-like interactions with substrate; however, Y565 from ADP1 in LONP1Bz is positioned further from substrate as compared to our substrate-bound structure (see Fig. 2). c When the subunits are positioned side-by-side, the protease domains are aligned along a single plane. d Bortezomib covalently attached to S855 in ATP1 is shown using a ribbon representation within a mesh representation of the cryo-EM map, where the loop containing the catalytic serine S855 is observed extended toward the neighboring protease domain and interacting with conserved residues V809, P854, and E882 from the neighboring protease domain, shown in blue. e, f Graphs showing rates of FITC-casein degradation (e) or ATPase rate (f) for LONP1 harboring alanine mutations in the conserved residues V809, P854, and E882 involved in stabilizing the catalytic loop in the active conformation of the protease active site. Data are presented as bar graphs showing mean values with error bars showing standard deviation from three independent replicates. ****p < 0.0001 relative to wild type calculated by one-sided ANOVA. g A single protease domain is shown as a worm representation from the substrate-bound LONP1 structure in the absence (left) and presence (right) of bortezomib. Regions showing a decrease in D2O exchange measured by HDX-MS upon bortezomib binding are colored red in the inactive state (signifying greater exchange) and blue in the bortezomib-bound state (signifying decreased exchange).
