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A sequence and structure based method to predict putative substrates, functions and regulatory networks of endo proteases.

Venkatraman P, Balakrishnan S, Rao S, Hooda Y, Pol S - PLoS ONE (2009)

Bottom Line: In addition, by using functional annotations, we have demonstrated how normal and unknown functions of a protease can be envisaged.We have developed a network which can be integrated to create a proteolytic world.This network can in turn be extended to integrate other regulatory networks to build a system wide knowledge of the proteome.

View Article: PubMed Central - PubMed

Affiliation: Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, Maharashtra, India. vprasanna@actrec.gov.in

ABSTRACT

Background: Proteases play a central role in cellular homeostasis and are responsible for the spatio-temporal regulation of function. Many putative proteases have been recently identified through genomic approaches, leading to a surge in global profiling attempts to characterize their function. Through such efforts and others it has become evident that many proteases play non-traditional roles. Accordingly, the number and the variety of the substrate repertoire of proteases are expected to be much larger than previously assumed. In line with such global profiling attempts, we present here a method for the prediction of natural substrates of endo proteases (human proteases used as an example) by employing short peptide sequences as specificity determinants.

Methodology/principal findings: Our method incorporates specificity determinants unique to individual enzymes and physiologically relevant dual filters namely, solvent accessible surface area--a parameter dependent on protein three-dimensional structure and subcellular localization. By incorporating such hitherto unused principles in prediction methods, a novel ligand docking strategy to mimic substrate binding at the active site of the enzyme, and GO functions, we identify and perform subjective validation on putative substrates of matriptase and highlight new functions of the enzyme. Using relative solvent accessibility to rank order we show how new protease regulatory networks and enzyme cascades can be created.

Conclusion: We believe that our physiologically relevant computational approach would be a very useful complementary method in the current day attempts to profile proteases (endo proteases in particular) and their substrates. In addition, by using functional annotations, we have demonstrated how normal and unknown functions of a protease can be envisaged. We have developed a network which can be integrated to create a proteolytic world. This network can in turn be extended to integrate other regulatory networks to build a system wide knowledge of the proteome.

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Related in: MedlinePlus

Structure of matriptase docked with a model peptide substrate.A) AEGRS (spheres) was docked to the matriptase structure (2GV6; light blue) using various components of Mastero (Schrödinger) as described under Text S1. Residues that are 4 Å distance from the ligand are shown as sticks. Polar interactions of the ligand with active site residues are indicated as dashes (yellow).
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pone-0005700-g002: Structure of matriptase docked with a model peptide substrate.A) AEGRS (spheres) was docked to the matriptase structure (2GV6; light blue) using various components of Mastero (Schrödinger) as described under Text S1. Residues that are 4 Å distance from the ligand are shown as sticks. Polar interactions of the ligand with active site residues are indicated as dashes (yellow).

Mentions: To obtain structural insights about the binding pocket in matriptase, we have docked a common scaffold EGRS with Arg (REGRS) and Ala (AEGRS) at the P4 position. Both peptides fitted well within the binding pocket. The pentapeptide AEGRS (GlideScore of-9.035 kcal/mole) seems to bind tighter than REGRS (GlideScore of −6.730 kcal/mole), as can be seen from its compact positioning in the matriptase cavity (Figure 2). The guanidinium group of P1 Arg is set deep into the S1 pocket of the protein and is hydrogen bonded to Ser190 and Gly219. These interacting residues and the P1 Arg are held in position within the pocket made of Cys191, Val213, Gly216, Trp215 and Phe99. A salt bridge between P1 Arg and Asp189 reinforces the enzyme substrate interaction. Asp184 and Gly193 interact with the carbonyl carbon of the scissile bond which is covalently bound to the catalytic serine (Ser 195). Ser at the P1′ position is held within the binding pocket via long range van der Waals and electrostatic interactions with the His57 side chain and Ile41 backbone carbonyl oxygen. The P3 glutamate side chain carboxylate is hydrogen bonded to the side chain amide of Gln192 and P4 Ala is in energetically stable hydrophobic contact with Ile60 isobutyl side chain. In addition, interactions between the carboxylate moiety of the P3 Glu side chain of the ligand and the phenolic side chain of Tyr146 can be potentially mediated by a water molecule. When P4 is an Arg, additional hydrogen bonding interactions are made by the P4 side chain guanidinium group with Ile60 and Cys58 backbone carbonyl oxygen. Nevertheless, a lower binding affinity is predicted for this sequence, possibly due to the assessment of energetic penalty for the solvent exposure of the trimethylene chain formed by Cβ, Cγ and Cδ atoms in the side chain of the P1 Arg. Additional docking studies showed that the pocket holding the P1′ residue was able to accommodate multiple amino acids (data not shown). Also P3 could be changed to Gln with no drastic change in the binding geometry as the glutamine side chain amide is able to engage the side chain of Gln192 (similar to P3 Glu). Non-specific peptides like AAADS (GlideScore of 0.23 kcal/mole) demonstrate considerably reduced binding to the matriptase active site with GlideScore values being nearly 10 kcal/mole higher than the specific sequence AEGRS.


A sequence and structure based method to predict putative substrates, functions and regulatory networks of endo proteases.

Venkatraman P, Balakrishnan S, Rao S, Hooda Y, Pol S - PLoS ONE (2009)

Structure of matriptase docked with a model peptide substrate.A) AEGRS (spheres) was docked to the matriptase structure (2GV6; light blue) using various components of Mastero (Schrödinger) as described under Text S1. Residues that are 4 Å distance from the ligand are shown as sticks. Polar interactions of the ligand with active site residues are indicated as dashes (yellow).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2683571&req=5

pone-0005700-g002: Structure of matriptase docked with a model peptide substrate.A) AEGRS (spheres) was docked to the matriptase structure (2GV6; light blue) using various components of Mastero (Schrödinger) as described under Text S1. Residues that are 4 Å distance from the ligand are shown as sticks. Polar interactions of the ligand with active site residues are indicated as dashes (yellow).
Mentions: To obtain structural insights about the binding pocket in matriptase, we have docked a common scaffold EGRS with Arg (REGRS) and Ala (AEGRS) at the P4 position. Both peptides fitted well within the binding pocket. The pentapeptide AEGRS (GlideScore of-9.035 kcal/mole) seems to bind tighter than REGRS (GlideScore of −6.730 kcal/mole), as can be seen from its compact positioning in the matriptase cavity (Figure 2). The guanidinium group of P1 Arg is set deep into the S1 pocket of the protein and is hydrogen bonded to Ser190 and Gly219. These interacting residues and the P1 Arg are held in position within the pocket made of Cys191, Val213, Gly216, Trp215 and Phe99. A salt bridge between P1 Arg and Asp189 reinforces the enzyme substrate interaction. Asp184 and Gly193 interact with the carbonyl carbon of the scissile bond which is covalently bound to the catalytic serine (Ser 195). Ser at the P1′ position is held within the binding pocket via long range van der Waals and electrostatic interactions with the His57 side chain and Ile41 backbone carbonyl oxygen. The P3 glutamate side chain carboxylate is hydrogen bonded to the side chain amide of Gln192 and P4 Ala is in energetically stable hydrophobic contact with Ile60 isobutyl side chain. In addition, interactions between the carboxylate moiety of the P3 Glu side chain of the ligand and the phenolic side chain of Tyr146 can be potentially mediated by a water molecule. When P4 is an Arg, additional hydrogen bonding interactions are made by the P4 side chain guanidinium group with Ile60 and Cys58 backbone carbonyl oxygen. Nevertheless, a lower binding affinity is predicted for this sequence, possibly due to the assessment of energetic penalty for the solvent exposure of the trimethylene chain formed by Cβ, Cγ and Cδ atoms in the side chain of the P1 Arg. Additional docking studies showed that the pocket holding the P1′ residue was able to accommodate multiple amino acids (data not shown). Also P3 could be changed to Gln with no drastic change in the binding geometry as the glutamine side chain amide is able to engage the side chain of Gln192 (similar to P3 Glu). Non-specific peptides like AAADS (GlideScore of 0.23 kcal/mole) demonstrate considerably reduced binding to the matriptase active site with GlideScore values being nearly 10 kcal/mole higher than the specific sequence AEGRS.

Bottom Line: In addition, by using functional annotations, we have demonstrated how normal and unknown functions of a protease can be envisaged.We have developed a network which can be integrated to create a proteolytic world.This network can in turn be extended to integrate other regulatory networks to build a system wide knowledge of the proteome.

View Article: PubMed Central - PubMed

Affiliation: Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, Maharashtra, India. vprasanna@actrec.gov.in

ABSTRACT

Background: Proteases play a central role in cellular homeostasis and are responsible for the spatio-temporal regulation of function. Many putative proteases have been recently identified through genomic approaches, leading to a surge in global profiling attempts to characterize their function. Through such efforts and others it has become evident that many proteases play non-traditional roles. Accordingly, the number and the variety of the substrate repertoire of proteases are expected to be much larger than previously assumed. In line with such global profiling attempts, we present here a method for the prediction of natural substrates of endo proteases (human proteases used as an example) by employing short peptide sequences as specificity determinants.

Methodology/principal findings: Our method incorporates specificity determinants unique to individual enzymes and physiologically relevant dual filters namely, solvent accessible surface area--a parameter dependent on protein three-dimensional structure and subcellular localization. By incorporating such hitherto unused principles in prediction methods, a novel ligand docking strategy to mimic substrate binding at the active site of the enzyme, and GO functions, we identify and perform subjective validation on putative substrates of matriptase and highlight new functions of the enzyme. Using relative solvent accessibility to rank order we show how new protease regulatory networks and enzyme cascades can be created.

Conclusion: We believe that our physiologically relevant computational approach would be a very useful complementary method in the current day attempts to profile proteases (endo proteases in particular) and their substrates. In addition, by using functional annotations, we have demonstrated how normal and unknown functions of a protease can be envisaged. We have developed a network which can be integrated to create a proteolytic world. This network can in turn be extended to integrate other regulatory networks to build a system wide knowledge of the proteome.

Show MeSH
Related in: MedlinePlus