Fig. 1: Three-dimensional model of UNC-89 PK2, and CRISPR/Cas9 generation of a nematode expressing PK2 with an inactivating KtoA mutation.
a Cartoon representation (yellow) of the 3D model generated by AlphaFold68. Catalytically relevant segments are color-coded as in (c). As a transparent ribbon trace, the crystal structure of the active, canonical kinase DAPK1 in complex with the ATP-analog AMPPNP and Mg2+ and in the catalytically committed state is shown (PDB code 3F5U). b Close-up of the active site of UNC-89-PK2. Color code is as in (a). Catalytically relevant residues described in the main text are displayed and annotated. c Alignment of catalytically relevant sequence motifs in UNC-89-PK2 with those of representative DMT kinases: UNC-89-PK2 (PK2), human death-associated-protein kinase (DAPK1), twitchin kinase from C. elegans (TwcK), human myosin light-chain kinase (MLCK). d On top is a schematic representation of domains within the largest isoform of UNC-89. Ig domains, purple; Fn3 domains, green; KSPs, a region with tandem repeats and 44 KSPs; other domains as indicated. Note the two protein kinase domains, PK1 and PK2, at the C-terminus. Below is representation of part of the exon/intron organization of part of the unc-89 gene, with the location of the PK2 coding region. Bottom right: confirmation of the CRISPR/Cas9 generated aag to gcc sequence change in unc-89(sf22) which specifies a K7816A mutation in PK2. Bottom, left: western blot comparing the levels of UNC-89 giant isoforms in wild type vs. unc-89(sf22). The giant isoforms of UNC-89 are ~800 kDa. The asterisk indicates a protein of nonspecific reactivity detected by the anti-UNC-89 antibodies46.
