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

Winter A, Schmid R, Bayliss R - PLoS Comput. Biol. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Leicester, Leicester, United Kingdom.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Homology modelling of the caspase-like domain of C. elegans separase using caspase 3 as template.(A) The homology model of the caspase-like domain of separase from C. elegans is shown in cartoon view with the proposed substrate peptide MEVER as sticks (magenta). The additional helices α2’ and α3’ that were introduced into the caspase 3 template (cyan) as well as the short helix modelled into the loop between β1 and α1 (red) are highlighted. Core model and loops were generated using MODELLER and energy minimized using GROMACS. (B) Surface electrostatics of the caspase-like domain in separases (shown here without substrate peptide) shows several positive (blue) and negative (red) patches that may be important for interactions with modulators such as securin or other domains within separase itself. Left: front view, same as view in (A). Right: view from back of molecule via vertical rotation by 180°. (C) Proposed interaction of the separase active site with substrate peptide MEVER (in sticks, magenta). Residues interacting with the peptide are labelled and shown in orange. Hydrogen bonds formed between peptide and protein are shown as black dashes. The catalytic residues H1014 and C1040 are indicated as green sticks. (D) Electrostatic properties of the substrate pocket reveal a deep negative pocket that receives the P1-Arg residue and a large positive patch that locks the side chains of P2-Glu and P4-Glu in place. All figures were prepared using PyMol.
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pcbi.1004548.g003: Homology modelling of the caspase-like domain of C. elegans separase using caspase 3 as template.(A) The homology model of the caspase-like domain of separase from C. elegans is shown in cartoon view with the proposed substrate peptide MEVER as sticks (magenta). The additional helices α2’ and α3’ that were introduced into the caspase 3 template (cyan) as well as the short helix modelled into the loop between β1 and α1 (red) are highlighted. Core model and loops were generated using MODELLER and energy minimized using GROMACS. (B) Surface electrostatics of the caspase-like domain in separases (shown here without substrate peptide) shows several positive (blue) and negative (red) patches that may be important for interactions with modulators such as securin or other domains within separase itself. Left: front view, same as view in (A). Right: view from back of molecule via vertical rotation by 180°. (C) Proposed interaction of the separase active site with substrate peptide MEVER (in sticks, magenta). Residues interacting with the peptide are labelled and shown in orange. Hydrogen bonds formed between peptide and protein are shown as black dashes. The catalytic residues H1014 and C1040 are indicated as green sticks. (D) Electrostatic properties of the substrate pocket reveal a deep negative pocket that receives the P1-Arg residue and a large positive patch that locks the side chains of P2-Glu and P4-Glu in place. All figures were prepared using PyMol.

Mentions: For comparative modelling of the catalytic domain of C. elegans separase we chose caspase 3 (PDB code 3EDQ and [26]) as template because firstly the presence of subdomain A causes a slightly open and twisted conformation of subdomain B in gingipain-R, and secondly, caspases present a more compact fold with shorter loops than human MALT-1. Due to the low sequence identity and similarity between the two proteins, 7.33% and 20.33%, respectively (SIAS server), protein structure analysis was used to generate an accurate alignment. First, the positions of the catalytic residues His and Cys as well as residues predicted to represent the buried β-strands were aligned. Then the positions of other secondary structure elements were aligned and residues thought to interact with the substrate were matched (Fig 3A). Helices α2’ and α3’ were inserted into the template structure file as separate helices and positions adjusted by iterative modelling rounds. The domain boundaries of the caspase-like domain in C. elegans separase were determined to be Tyr902 and Gln1140. This initial model was enhanced by adding a proposed substrate peptide, which enabled detailed analysis of the substrate pocket and its recognition of cleavage sites. Reports in the literature suggest that the consensus sequence for recognition by separases is D/ExxR with the P1 residue being an arginine, similar to gingipain-R and MALT-1, but in contrast to caspases where the recognition sequence is D/ExxD. In-depth substrate specificity analysis on S. cerevisiae separase revealed explicit restraints for certain amino acids in positions P2 to P6 and a consensus sequence of SIEVGR [36]. Sullivan and co-workers also suggested that the difference in specificity between budding yeast and human separase is likely due to the absence of a hydrophobic residue in the P5 position in human Scc1-consensus sequence, which is not tolerated by budding yeast separase. Additionally, positions P2 and P3 are occupied by larger hydrophobic residues in human Scc1. This might contribute towards discrimination of these two homologues by separases from budding yeast and human, respectively. The cleavage sites in human Scc1 are DREIMRE and IEEPSRL, in X. laevis DREMMRE (putative), in D. melanogaster TPEIIRC (putative) and DREIMRE in mouse (putative) [31]. The recognition sites in S. cerevisiae are SLEVGRR and SVEQGRR [7]. We aligned Scc1 sequences from different species (S2 File) and determined that the likely recognition sites present in Scc1 in C. elegans are LMEVERD200 or EVERDRD202 (Table 1). Interestingly, both proposed cleavage sites in C. elegans Scc1 possess an acidic residue in position P2, which is not present in other recognition sequences and might be important for the recognition of this sequence by separase from C. elegans. We therefore included the proposed substrate peptide MEVER in our homology model.


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

Winter A, Schmid R, Bayliss R - PLoS Comput. Biol. (2015)

Homology modelling of the caspase-like domain of C. elegans separase using caspase 3 as template.(A) The homology model of the caspase-like domain of separase from C. elegans is shown in cartoon view with the proposed substrate peptide MEVER as sticks (magenta). The additional helices α2’ and α3’ that were introduced into the caspase 3 template (cyan) as well as the short helix modelled into the loop between β1 and α1 (red) are highlighted. Core model and loops were generated using MODELLER and energy minimized using GROMACS. (B) Surface electrostatics of the caspase-like domain in separases (shown here without substrate peptide) shows several positive (blue) and negative (red) patches that may be important for interactions with modulators such as securin or other domains within separase itself. Left: front view, same as view in (A). Right: view from back of molecule via vertical rotation by 180°. (C) Proposed interaction of the separase active site with substrate peptide MEVER (in sticks, magenta). Residues interacting with the peptide are labelled and shown in orange. Hydrogen bonds formed between peptide and protein are shown as black dashes. The catalytic residues H1014 and C1040 are indicated as green sticks. (D) Electrostatic properties of the substrate pocket reveal a deep negative pocket that receives the P1-Arg residue and a large positive patch that locks the side chains of P2-Glu and P4-Glu in place. All figures were prepared using PyMol.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4626109&req=5

pcbi.1004548.g003: Homology modelling of the caspase-like domain of C. elegans separase using caspase 3 as template.(A) The homology model of the caspase-like domain of separase from C. elegans is shown in cartoon view with the proposed substrate peptide MEVER as sticks (magenta). The additional helices α2’ and α3’ that were introduced into the caspase 3 template (cyan) as well as the short helix modelled into the loop between β1 and α1 (red) are highlighted. Core model and loops were generated using MODELLER and energy minimized using GROMACS. (B) Surface electrostatics of the caspase-like domain in separases (shown here without substrate peptide) shows several positive (blue) and negative (red) patches that may be important for interactions with modulators such as securin or other domains within separase itself. Left: front view, same as view in (A). Right: view from back of molecule via vertical rotation by 180°. (C) Proposed interaction of the separase active site with substrate peptide MEVER (in sticks, magenta). Residues interacting with the peptide are labelled and shown in orange. Hydrogen bonds formed between peptide and protein are shown as black dashes. The catalytic residues H1014 and C1040 are indicated as green sticks. (D) Electrostatic properties of the substrate pocket reveal a deep negative pocket that receives the P1-Arg residue and a large positive patch that locks the side chains of P2-Glu and P4-Glu in place. All figures were prepared using PyMol.
Mentions: For comparative modelling of the catalytic domain of C. elegans separase we chose caspase 3 (PDB code 3EDQ and [26]) as template because firstly the presence of subdomain A causes a slightly open and twisted conformation of subdomain B in gingipain-R, and secondly, caspases present a more compact fold with shorter loops than human MALT-1. Due to the low sequence identity and similarity between the two proteins, 7.33% and 20.33%, respectively (SIAS server), protein structure analysis was used to generate an accurate alignment. First, the positions of the catalytic residues His and Cys as well as residues predicted to represent the buried β-strands were aligned. Then the positions of other secondary structure elements were aligned and residues thought to interact with the substrate were matched (Fig 3A). Helices α2’ and α3’ were inserted into the template structure file as separate helices and positions adjusted by iterative modelling rounds. The domain boundaries of the caspase-like domain in C. elegans separase were determined to be Tyr902 and Gln1140. This initial model was enhanced by adding a proposed substrate peptide, which enabled detailed analysis of the substrate pocket and its recognition of cleavage sites. Reports in the literature suggest that the consensus sequence for recognition by separases is D/ExxR with the P1 residue being an arginine, similar to gingipain-R and MALT-1, but in contrast to caspases where the recognition sequence is D/ExxD. In-depth substrate specificity analysis on S. cerevisiae separase revealed explicit restraints for certain amino acids in positions P2 to P6 and a consensus sequence of SIEVGR [36]. Sullivan and co-workers also suggested that the difference in specificity between budding yeast and human separase is likely due to the absence of a hydrophobic residue in the P5 position in human Scc1-consensus sequence, which is not tolerated by budding yeast separase. Additionally, positions P2 and P3 are occupied by larger hydrophobic residues in human Scc1. This might contribute towards discrimination of these two homologues by separases from budding yeast and human, respectively. The cleavage sites in human Scc1 are DREIMRE and IEEPSRL, in X. laevis DREMMRE (putative), in D. melanogaster TPEIIRC (putative) and DREIMRE in mouse (putative) [31]. The recognition sites in S. cerevisiae are SLEVGRR and SVEQGRR [7]. We aligned Scc1 sequences from different species (S2 File) and determined that the likely recognition sites present in Scc1 in C. elegans are LMEVERD200 or EVERDRD202 (Table 1). Interestingly, both proposed cleavage sites in C. elegans Scc1 possess an acidic residue in position P2, which is not present in other recognition sequences and might be important for the recognition of this sequence by separase from C. elegans. We therefore included the proposed substrate peptide MEVER in our homology model.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Leicester, Leicester, United Kingdom.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus