Structural Insights into Separase Architecture and Substrate Recognition through Computational Modelling of Caspase-Like and Death Domains
Fig 5
The central region of separases may be similar to death domains and harbours a conserved WWxxRxxLD motif.
(A) The region N-terminal to the catalytically active caspase-like domain (black) is made up of six α-helices (grey) and may be structurally similar to death domains. A novel WWxxRxxLD-motif was found in the second helix of this domain whose function remains to be elucidated. Helices are numbered and indicated as grey bars, and their boundaries in C. elegans separase annotated. The region encompassing three β-strands is shown in light grey. Catalytic residues are marked with white bars. (B) Three-dimensional model of the proposed death domain in separase from C. elegans using the prodomain of human procaspase-9 as template. The six-helix bundle is shown in cartoon view with amino acids belonging to the proposed the WR motif shown as sticks (orange). A surface-exposed cysteine, C866 is indicated in magenta. Figure prepared with PyMol. (C) Surface representation and electrostatics of the proposed CARD domain show a large electropositive patch where the WR motif is located. Left: front view, same as view in (B), Right: view from back of molecule via vertical rotation by 180°. Figure prepared with PyMol. (D) Sequence alignment of the novel WR motif shows their high conservation within the central region of separase proteins. Sequences from mammals (Homo sapiens, Mus musculus), Caenorhabditis elegans, insects (Spodoptera frugiperda, Drosophila melanogaster, Drosophila virilis, Drosophila willistoni), fungi (Schizosaccharomyces pombe, Saccharomyces cerevisiae, Exophiala dermatitidis, Blumeria graminis), microsporidia (Encephalitozoon hellem, Encephalitozoon intestinalis, Encephalitozoon romaleae), protozoa (Giardia lamblia, Giardia intestinalis, Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, Perkinsus marinus, Cryptosporidium muris, Cryptosporidium hominis, Cryptosporidium parvum), plants (Arabidopsis thaliana, Medicago truncatula, Ricinus communis) green algae (Ostreococcus tauri, Chlamydomonas reinhardtii, Volvox carteri) and the diatom Phaeodactylum tricornutum were aligned. Highlighted residues have 80% or more sequence identity (white letters on black background), 60–80% sequence identity (grey), or 40–60% (light grey). ‘Conservation’ indicates the degree of conservation of physico-chemical properties in each column of the alignment and is represented by numbers from 0 to 10. (E) Weblogo representation of the second predicted helix of the proposed CARD domain. The overall height of the stack indicates the sequence conservation at that position, while the height of symbols within the stack indicates the relative frequency of each amino acid at that position.