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A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

Rendón-Ramírez A, Shukla M, Oda M, Chakraborty S, Minda R, Dandekar AM, Ásgeirsson B, Goñi FM, Rao BJ - PLoS ONE (2013)

Bottom Line: This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM).Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase.Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

View Article: PubMed Central - PubMed

Affiliation: Unidad de Biofísica, Consejo Superior de Investigaciones Científicas, Universidad del Pais Vasco/Euskal Herriko Unibertsitatea and Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain.

ABSTRACT
Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

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Related in: MedlinePlus

CLASP detects a glutamic protease motif in PC-PLC (PDBid:1AH7).The residues predicted to be responsible for the protease activity (Gln39, Glu42, and Trp43, in shades of red) does not coincide with its native active site (Trp1, His14, Asp122, HiS128, Glu146, Asp55, and His69, in shades of green). The motif is selected from a protein from the Eqolisin family of peptidases: PDBid:1S2B.
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pone-0070923-g003: CLASP detects a glutamic protease motif in PC-PLC (PDBid:1AH7).The residues predicted to be responsible for the protease activity (Gln39, Glu42, and Trp43, in shades of red) does not coincide with its native active site (Trp1, His14, Asp122, HiS128, Glu146, Asp55, and His69, in shades of green). The motif is selected from a protein from the Eqolisin family of peptidases: PDBid:1S2B.

Mentions: The PC-PLC proteolytic activity could also be an artifact of metallo-protease contamination, which is difficult to remove. CLASP detects in this protein a glutamic protease motif from the Eqolisin family of peptidases, PDBid:1S2B (Table 5), which does not coincide with its native active site (Fig. 3). While this protein’s lack of inhibition by serine and metallo-protease inhibitors is consistent with CLASP analysis, mutational studies would be required to confirm the moonlighting glutamic protease scaffold [34]. Thus, the protease activities of CPA and PC-PLC remain open to debate.


A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

Rendón-Ramírez A, Shukla M, Oda M, Chakraborty S, Minda R, Dandekar AM, Ásgeirsson B, Goñi FM, Rao BJ - PLoS ONE (2013)

CLASP detects a glutamic protease motif in PC-PLC (PDBid:1AH7).The residues predicted to be responsible for the protease activity (Gln39, Glu42, and Trp43, in shades of red) does not coincide with its native active site (Trp1, His14, Asp122, HiS128, Glu146, Asp55, and His69, in shades of green). The motif is selected from a protein from the Eqolisin family of peptidases: PDBid:1S2B.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3733634&req=5

pone-0070923-g003: CLASP detects a glutamic protease motif in PC-PLC (PDBid:1AH7).The residues predicted to be responsible for the protease activity (Gln39, Glu42, and Trp43, in shades of red) does not coincide with its native active site (Trp1, His14, Asp122, HiS128, Glu146, Asp55, and His69, in shades of green). The motif is selected from a protein from the Eqolisin family of peptidases: PDBid:1S2B.
Mentions: The PC-PLC proteolytic activity could also be an artifact of metallo-protease contamination, which is difficult to remove. CLASP detects in this protein a glutamic protease motif from the Eqolisin family of peptidases, PDBid:1S2B (Table 5), which does not coincide with its native active site (Fig. 3). While this protein’s lack of inhibition by serine and metallo-protease inhibitors is consistent with CLASP analysis, mutational studies would be required to confirm the moonlighting glutamic protease scaffold [34]. Thus, the protease activities of CPA and PC-PLC remain open to debate.

Bottom Line: This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM).Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase.Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

View Article: PubMed Central - PubMed

Affiliation: Unidad de Biofísica, Consejo Superior de Investigaciones Científicas, Universidad del Pais Vasco/Euskal Herriko Unibertsitatea and Departamento de Bioquímica, Universidad del País Vasco, Bilbao, Spain.

ABSTRACT
Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

Show MeSH
Related in: MedlinePlus