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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

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.
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pcbi.1004548.g005: 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.

Mentions: Secondary structure prediction on sequences immediately N-terminal of the caspase domain predicted six α-helices, and multiple sequence alignment of the respective regions in different separases reveals conservation of hydrophobic residues at certain positions (S3 Fig). In separase from C. elegans, helices were predicted from residues 755 to 772 (α1), 780 to 812 (α2), 821 to 835 (α3), 842 to 849 (α4), 855 to 866 (α5) and 872 to 890 (α6) (Fig 5A). Interestingly, this region is also identified as part of the peptidase_C50 family (separases, PF03568, E-value of 2.3e-08) when submitted to the Interpro server [49] indicating that this region is well conserved despite the fact that it is not part of the protease domain. This led us to conclude that separases possess an α -helical domain immediately N-terminal to the caspase-like domain (Fig 5A). This domain may interact with and possibly stabilise the catalytically active protease domain as seen in related proteins. For instance, human MALT-1 is a multi-domain protein that contains both a death domain and two immunoglobulin-like domains (Ig) N-terminal to the caspase-like protease domain (Casp) and a third immunoglobulin-like domain C-terminal. The isolated caspase domain hMALT1Casp (329–566) yielded only oligomeric, poorly folded protein whereas the truncation variant hMALT1Casp-Ig3 (334–719) could be expressed in E. coli and was used to solve the hMALT1Casp-Ig3 apostructure [27]. The catalytically active caspase domain in gingipain is preceded by an inactivated domain (subdomain A) that forms a covalently linked dimer with the C-terminal domain, in an arrangement that resembles caspase dimers [25]. Moreover, some of both inflammatory and initiator caspases contain one or more death domains (DD), death effector domain (DED) or caspase recruitment domain (CARD) in their N-terminal regions [39, 50] and oligomerise for activity [51]. These domains are collectively known as death domain superfamily, different subclasses of which have very different sequences but share a common, globular fold made up of six anti-parallel α-helices. Conserved hydrophobic residues at certain positions compose the hydrophobic core of these domains. Considering the importance of death domains for recruitment and activation of caspases and the fact that we predicted six α-helices N-terminal of the caspase-like protease domain led us to investigate the possibility of the presence of a death domain in separases, despite the lack of significant sequence similarity to any known member of the superfamily. As structurally this region might represent a subclass of the death fold domain superfamily, we modelled this region on the CARD domain of human procaspase 9 (PDB code 3YGS) (Fig 5B) to gain further structural and functional insight. Helix α2 was extended in accordance with secondary structure predictions. The model was subsequently adjusted to satisfy positions of amino acids thought to be part of the hydrophobic core: Leu10 (α1), Trp27, Leu30, Leu31 Leu35 and Trp36 (α2), Ile44 (α3), Leu59 and Ile60 (α4), Phe74 and Leu78 (α5), Leu86 and Leu90 (α6) (numbering according to the CARD domain of human procaspase 9). The model shows a six-helix bundle in which the second helix is slightly bent to allow shielding of hydrophobic residues (Fig 5B). Analysis of dihedral angles using a Ramachandran plot revealed that 91% of amino acids (123) were in preferred regions, 5% (7) in additionally allowed regions and 4% (5) in disallowed regions.


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)

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.
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pcbi.1004548.g005: 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.
Mentions: Secondary structure prediction on sequences immediately N-terminal of the caspase domain predicted six α-helices, and multiple sequence alignment of the respective regions in different separases reveals conservation of hydrophobic residues at certain positions (S3 Fig). In separase from C. elegans, helices were predicted from residues 755 to 772 (α1), 780 to 812 (α2), 821 to 835 (α3), 842 to 849 (α4), 855 to 866 (α5) and 872 to 890 (α6) (Fig 5A). Interestingly, this region is also identified as part of the peptidase_C50 family (separases, PF03568, E-value of 2.3e-08) when submitted to the Interpro server [49] indicating that this region is well conserved despite the fact that it is not part of the protease domain. This led us to conclude that separases possess an α -helical domain immediately N-terminal to the caspase-like domain (Fig 5A). This domain may interact with and possibly stabilise the catalytically active protease domain as seen in related proteins. For instance, human MALT-1 is a multi-domain protein that contains both a death domain and two immunoglobulin-like domains (Ig) N-terminal to the caspase-like protease domain (Casp) and a third immunoglobulin-like domain C-terminal. The isolated caspase domain hMALT1Casp (329–566) yielded only oligomeric, poorly folded protein whereas the truncation variant hMALT1Casp-Ig3 (334–719) could be expressed in E. coli and was used to solve the hMALT1Casp-Ig3 apostructure [27]. The catalytically active caspase domain in gingipain is preceded by an inactivated domain (subdomain A) that forms a covalently linked dimer with the C-terminal domain, in an arrangement that resembles caspase dimers [25]. Moreover, some of both inflammatory and initiator caspases contain one or more death domains (DD), death effector domain (DED) or caspase recruitment domain (CARD) in their N-terminal regions [39, 50] and oligomerise for activity [51]. These domains are collectively known as death domain superfamily, different subclasses of which have very different sequences but share a common, globular fold made up of six anti-parallel α-helices. Conserved hydrophobic residues at certain positions compose the hydrophobic core of these domains. Considering the importance of death domains for recruitment and activation of caspases and the fact that we predicted six α-helices N-terminal of the caspase-like protease domain led us to investigate the possibility of the presence of a death domain in separases, despite the lack of significant sequence similarity to any known member of the superfamily. As structurally this region might represent a subclass of the death fold domain superfamily, we modelled this region on the CARD domain of human procaspase 9 (PDB code 3YGS) (Fig 5B) to gain further structural and functional insight. Helix α2 was extended in accordance with secondary structure predictions. The model was subsequently adjusted to satisfy positions of amino acids thought to be part of the hydrophobic core: Leu10 (α1), Trp27, Leu30, Leu31 Leu35 and Trp36 (α2), Ile44 (α3), Leu59 and Ile60 (α4), Phe74 and Leu78 (α5), Leu86 and Leu90 (α6) (numbering according to the CARD domain of human procaspase 9). The model shows a six-helix bundle in which the second helix is slightly bent to allow shielding of hydrophobic residues (Fig 5B). Analysis of dihedral angles using a Ramachandran plot revealed that 91% of amino acids (123) were in preferred regions, 5% (7) in additionally allowed regions and 4% (5) in disallowed regions.

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