Loqs dsRBD3 dimerizes via a conserved interface. (A) Structure model of a dimer of native Loqs-dsRBD3 molecules. The side chain of residue L426 is shown as yellow sticks and I461 as red sticks. (B) Crosslinking assay of purified His-dmDcr1 and HA-Loqs-3 (aa 368–463). Concentrations of DSS-crosslinker are indicated on top. Both proteins are detected by immunoblot—Loqs by an anti-HA-antibody (green) and dmDcr1 by a specific antibody (red). Positions of monomeric and dimeric Loqs-3 are indicated on the right. (C) Size-exclusion chromatography of Loqs-dsRBD2 (green), Loqs-3 (black), Loqs-3 L426R (orange), Loqs-3 K460E (red), and Loqs-3 KI460/61EA (blue). Migration of a protein standard is shown on top. (D, top) Pulldown assay of His-dmDcr1 with GST-Loqs-3 and mutants as indicated. Bound dmDcr1 is detected by immunoblotting using a specific antibody; bait proteins are visualized by Coomassie staining. (Bottom) HA-tagged wt Loqs (lanes 2,4) or HA-Loqs L426R (lanes 1,3) were cotransfected with myc-dmDcr1 into HEK 293 cells. Myc-dmDcr1 was immunoprecipitated using anti-myc antibodies and the immunoprecipitates were analyzed by Western blotting using anti-dmDcr1 (upper panel) or -HA antibodies (lower panel). (E) Sequence alignment of Loqs-dsRBD3 with functional homologs of the TRBP and PACT protein families from different organisms.
