Structural Insights into Separase Architecture and Substrate Recognition through Computational Modelling of Caspase-Like and Death Domains.

Separases are large proteins that mediate sister chromatid disjunction in all eukaryotes. They belong to clan CD of cysteine peptidases and contain a well-conserved C-terminal catalytic protease domain similar to caspases and gingipains. However, unlike other well-characterized groups of clan CD peptidases, there are no high-resolution structures of separases and the details of their regulation and substrate recognition are poorly understood. Here we undertook an in-depth bioinformatical analysis of separases from different species with respect to their similarity in amino acid sequence and protein fold in comparison to caspases, MALT-1 proteins (mucosa-associated lymphoidtissue lymphoma translocation protein 1) and gingipain-R. A comparative model of the single C-terminal caspase-like domain in separase from C. elegans suggests similar binding modes of substrate peptides between these protein subfamilies, and enables differences in substrate specificity of separase proteins to be rationalised. We also modelled a newly identified putative death domain, located N-terminal to the caspase-like domain. The surface features of this domain identify potential sites of protein-protein interactions. Notably, we identified a novel conserved region with the consensus sequence WWxxRxxLD predicted to be exposed on the surface of the death domain, which we termed the WR motif. We envisage that findings from our study will guide structural and functional studies of this important protein family

Comparison of the structures of human caspase 3, human MALT-1 and gingipain-R.

<p>(A) Overlaid structures of human caspase 3 (cyan, PDB code 3EDQ), human MALT-1 (green, PDB code 3UO8) and gingipain-R (magenta, PDB code 1CVR) show a similar overall fold consisting of a central six-stranded β -sheet flanked by α-helices. In gingipain-R, a helix connects the first four with the last two β-strands and replaces the inter-subunit linker present in caspases. In caspases the first four β -strands belong to the larger subunit p20 and the remaining two β-strands to the smaller subunit p10. The C-terminal Ig domain of MALT-1, subdomain A and the C-terminal IgSF domain of gingipain-R were omitted for clarity. (B) Comparison of the substrate binding pockets reveal a very similar binding mode of peptide inhibitors ace-LDESD-cho (for caspase 3), z-VRPR-fmk (MALT-1) and FFR-cmk (gingipain-R). Peptide inhibitors (magenta) and catalytic residues His and Cys (green) are shown as sticks. Residues mainly responsible for the recognition of the respective peptide P1 are shown as cyan sticks: Arg64 (for caspase 3), Asp163 (for gingipain-R) and Asp365 (for MALT-1). Figures were prepared with PyMol.</p

The central region of separases may be similar to death domains and harbours a conserved WWxxRxxLD motif.

<p>(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 <i>C</i>. <i>elegans</i> 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 <i>C</i>. <i>elegans</i> 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 (<i>Homo sapiens</i>, <i>Mus musculus</i>), <i>Caenorhabditis elegans</i>, insects (<i>Spodoptera frugiperda</i>, <i>Drosophila melanogaster</i>, <i>Drosophila virilis</i>, <i>Drosophila willistoni</i>), fungi (<i>Schizosaccharomyces pombe</i>, <i>Saccharomyces cerevisiae</i>, <i>Exophiala dermatitidis</i>, <i>Blumeria graminis</i>), microsporidia (<i>Encephalitozoon hellem</i>, <i>Encephalitozoon intestinalis</i>, <i>Encephalitozoon romaleae</i>), protozoa (<i>Giardia lamblia</i>, <i>Giardia intestinalis</i>, <i>Trypanosoma brucei</i>, <i>Trypanosoma cruzi</i>, <i>Leishmania major</i>, <i>Perkinsus marinus</i>, <i>Cryptosporidium muris</i>, <i>Cryptosporidium hominis</i>, <i>Cryptosporidium parvum</i>), plants (<i>Arabidopsis thaliana</i>, <i>Medicago truncatula</i>, <i>Ricinus communis</i>) green algae (<i>Ostreococcus tauri</i>, <i>Chlamydomonas reinhardtii</i>, <i>Volvox carteri</i>) and the diatom <i>Phaeodactylum tricornutum</i> 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.</p

Separases from all species share a similar topology with highly conserved regions, which includes a caspase-like domain in their C-terminal region.

<p>(A) Consolidated secondary structure prediction of separase from <i>C</i>. <i>elegans</i> using both PsiPred and JPred predicts a largely helical N-terminus (dark grey) with an unstructured region around residue 400 and a region of three β-strands from residues 720 to 750 (light grey). The conserved C-terminal half harbours the caspase-like domain (black), residues 900 to 1140. The catalytic dyad is indicated as white lines. (B) Multiple sequence alignment of the caspase-like domain of separase from <i>C</i>. <i>elegans</i> in comparison with human caspase 3, gingipain-R and MALT-1. Alignment was manually adjusted using Jalview to match secondary structure elements from predictions (PsiPred) of separases to structural elements as observed in caspase 3 (3EDQ), MALT-1 (3UO8) and gingipain R (1CVR). α-helices are shown in dark grey and β-strands in light grey. The conserved catalytic dyad (C, H) is shown in bold letters.</p

Binding of fascaplysin and carbofascaplyin in CDK4 and CDK2.

<p>(<b>A</b>) Distances between the carbonyl oxygen (black), and amide hydrogen (red) of V96^CDK4 and the indoyl hydrogen and carbonyl oxygen of FAS, respectively. (<b>B</b>) Distances between the carbonyl oxygen (black) and amide hydrogen (red) of V96^CDK4 and the indoyl hydrogen and carbonyl oxygen of CRB, respectively. (<b>C</b>) Distances between the carbonyl oxygen (black) and amide hydrogen (red) of L83^CDK2 and the indoyl hydrogen and carbonyl oxygen of FAS, respectively. (<b>D</b>) Distances between the carbonyl oxygen (black) and amide hydrogen (red) of L83^CDK2 and the indoyl hydrogen and carbonyl oxygen of CRB, respectively. (<b>E</b>) Distances between the His95^CDK4 Nδ-H and the carbonyl oxygen of FAS (red) and CRB (black), respectively. (<b>F</b>) Distances between Lys89^CDK2 amine-N and the N+/C of FAS (red) and CRB (black), respectively.</p

Thermodynamic Integration.

<p>Each point (200 ps window) represents the free energy “cost” of the carbofascaplysin to fascaplysin transformation in the CDK4 and CDK2 complex calculated from 19 values for λ. The difference (ΔΔG⁰) between the two plots quantifies the energetic contribution for selectivity that can be attributed to the positive fascaplysin charge.</p

Interactions made between the predicted substrate peptide MEVER and the homology model of the <i>C</i>. <i>elegans</i> separase caspase domain.

<p>(A) Residues in separases that interact with the substrate peptide are highlighted in an alignment. P1-Arg is anchored via hydrogen bonds to Glu917 (~) and Asp1082 towards the N-terminus of helix α4 (+). The main chain amide-NH of residue P1 interacts with main chain oxygen atom of Thr1075 (#). The side chain of P2-Glu forms hydrogen bonds to Arg1120 (which is located in a well-conserved region preceding β6) and Arg1044 (which is located in a loop region between helix α3’ and β4) which are both highlighted with * in the alignment. Aside from hydrophobic interactions, P3-Val interacts with both the main chain and side chain oxygen atoms of Thr1077 (^). P4-Glu forms hydrogen bonds to the main chain amide of Lys1118 and guanidino group of Arg1116 (–). α-helices are shown in dark grey and β-strands in light grey. The catalytic dyad (C, H) is shown in bold letters. (B) Schematic representation of interactions between <i>C</i>. <i>elegans</i> separase and the proposed substrate peptide EVER. Corresponding interacting residues in separases from other species are taken from Fig 4A. Core recognition sites of Scc1 proteins are shown in the boxed inset.</p

Predicted binding modes for the fascaplysin/CDK4 and the fascaplysin/CDK2 complexes.

<p>The predicted binding modes for CDK4 (<b>A</b>) and CDK2 (<b>B</b>) show two hydrogen bonds between NH and carbonyl groups of fascaplysin and the backbone carbonyl and NH of Val96 in CDK4 and of Leu83 in CDK2, respectively. In the CDK4/fascaplysin binding mode an additional hydrogen bond between the Nδ-H of the His95^CDK4 imidazole side chain and fascaplysin is possible.</p

Active site conservation in CDK2, CDK4 and CDK6.

<p>(<b>A</b>) Structural overlay of active site residues for CDK2 (green, PDB-ID: 1FIN), CDK4 (blue, PDB-ID: 2W96) and CDK6 (red, PDB-ID: 1G3N). (<b>B</b>) Corresponding sequence alignment of the active site residues, the colour scheme is as in (<b>A</b>).</p

Mapping the site of action of the human P2X7 receptor antagonists AZ11645373, brilliant blue G, KN-62, calmidazolium and ZINC58368839 to the inter-subunit allosteric pocket.

The P2X7 receptor (P2X7R) is a trimeric ligand-gated ion channel which is activated by ATP. It is implicated in the cellular response to trauma/disease and considered to have significant therapeutic potential. Using chimeras and point mutants we have mapped the binding site of the P2X7R selective antagonist AZ11645373 to the known allosteric binding pocket at the interface between two subunits, in proximity to, but separated from the ATP binding site. Our structural model of AZ11645373 binding is consistent with effects of mutations on antagonist sensitivity, and the proposed binding mode explains variation in antagonist sensitivity between the human and rat P2X7 receptors. We have also determined the site of action for the P2X7R selective antagonists ZINC58368839, brilliant blue G, KN-62 and calmidazolium. The effect of inter-subunit allosteric pocket "signature mutants" F88A, T90V, D92A, F103A and V312A on antagonist sensitivity suggests that ZINC58368839 comprises a similar binding mode as AZ11645373 and other previously characterised antagonists. For the larger antagonists brilliant blue G, KN-62 and calmidazolium our data imply an overlapping, but distinct binding mode involving the central upper vestibule of the receptor in addition to the inter-subunit allosteric pocket. Our work explains the site of action for a series of P2X7R antagonists, and establishes "signature mutants" for P2X7R binding mode characterization. SIGNIFICANCE STATEMENT: The P2X7 receptor is a trimeric ligand-gated ion channel activated by ATP. The receptor is implicated in the cellular response to trauma/disease, and considered to have significant therapeutic potential. Using chimeras, point mutants and molecular modelling we have mapped the binding site of the P2X7 receptor selective antagonists AZ11645373, ZINC58368839, brilliant blue G, KN-62 and calmidazolium to a known inter-subunit binding pocket distinct from the ATP binding site. Our work explains the site of action for a series of P2X7R antagonists, and establishes signature mutants for P2X7R binding mode characterization