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The Importance of Exosite Interactions for Substrate Cleavage by Human Thrombin.

Chahal G, Thorpe M, Hellman L - PLoS ONE (2015)

Bottom Line: The results showed that addition of these regions enhanced the cleavage rate by more than fifty fold.The possibility to mimic this interaction for the sites in factor V and factor VIII by recombinant substrates, which do not have the same folding as the full size target, indicates that the enhancement was primarily dependent on a relatively simple electrostatic interaction.However, the situation was very different for fibrinogen and protein C where other factors than only charge is of major importance.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Uppsala University, BMC, Box 596, SE-751 24, Uppsala, Sweden.

ABSTRACT
Thrombin is a serine protease of the chymotrypsin family that acts both as a procoagulant and as an anticoagulant by cleaving either factor VIII, factor V and fibrinogen or protein C, respectively. Numerous previous studies have shown that electropositive regions at a distance from the active site, so called exosites, are of major importance for the cleavage by human thrombin. Upstream of all the known major cleavage sites for thrombin in factor VIII, factor V and fibrinogen are clusters of negatively charged amino acids. To study the importance of these sites for the interaction with the exosites and thereby the cleavage by thrombin, we have developed a new type of recombinant substrate. We have compared the cleavage rate of the minimal cleavage site, involving only 8-9 amino acids (typically the P4-P4' positions) surrounding the cleavage site, with the substrates also containing the negatively charged regions upstream of the cleavage sites. The results showed that addition of these regions enhanced the cleavage rate by more than fifty fold. However, the enhancement was highly dependent on the sequence of the actual cleavage site. A minimal site that showed poor activity by itself could be cleaved as efficiently as an optimal cleavage site when presented together with these negatively charged regions. Whereas sites conforming closely to the optimal site were only minimally enhanced by the addition of these regions. The possibility to mimic this interaction for the sites in factor V and factor VIII by recombinant substrates, which do not have the same folding as the full size target, indicates that the enhancement was primarily dependent on a relatively simple electrostatic interaction. However, the situation was very different for fibrinogen and protein C where other factors than only charge is of major importance.

No MeSH data available.


Related in: MedlinePlus

Schematic 3-D models of human thrombin showing the position of the N-terminal region of fibrinogen α chain.Panel A shows a space-filling model with the alpha chain peptide in purple. Panel B shows the interaction between thrombin (ribbon structure in beige) and the N-terminal region of fibrinogen α chain (ball and stick structure in purple) in detail. The same orientation as panel A is shown with the catalytic residues His57, Asp102 and Ser195 together with the S1 pocket residues Asp 189, Gly216 and 226 in green. Thrombin structure from PDB, code 1DM4 run using UCSF Chimera v1.8 and annotated in Adobe Illustrator CS5.
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pone.0129511.g007: Schematic 3-D models of human thrombin showing the position of the N-terminal region of fibrinogen α chain.Panel A shows a space-filling model with the alpha chain peptide in purple. Panel B shows the interaction between thrombin (ribbon structure in beige) and the N-terminal region of fibrinogen α chain (ball and stick structure in purple) in detail. The same orientation as panel A is shown with the catalytic residues His57, Asp102 and Ser195 together with the S1 pocket residues Asp 189, Gly216 and 226 in green. Thrombin structure from PDB, code 1DM4 run using UCSF Chimera v1.8 and annotated in Adobe Illustrator CS5.

Mentions: To study the involvement of the exosites for a number of important activation sites for human thrombin, we designed and ordered complementary single-stranded oligonucleotides encoding the region covering the thrombin cleavage sites with four amino acids up- and downstream of the cleavage sites in human FV, FVIII, fibrinogen α chain, fibrinogen β chain and protein C (Fig 3). This region is named the P4-P4’region or the minimal site. After mixing the paired oligonucleotides they form a stable double-stranded oligonucleotide with BamHI and SalI sticky ends. These were cloned into the Bam HI and SalI sites of a cleaved bacterial expression, pET21-2x trx, vector. This vector contains two E. coli trx genes with a linker region in between where different sequences can be introduced followed by a downstream six-His tag. (Fig 3). For the longer sequences also covering the negatively charged regions N-terminal, or both N- and C-terminal of the cleavage site, these regions were ordered as designer genes, cloned and sequence-verified from Genscript. In total, 28 different constructs were produced: FVIII-Arg372 minimal, FVIII-Arg372 N-region, FVIII-Arg372 N-region mutated, FVIII-Arg372 N+C-regions, FVIII-Arg740 minimal, FVIII-Arg740 N-region, FVIII-Arg740 N-region mutated, FVIII-Arg740 N+C-regions, FVIII-Arg1689 minimal, FVIII-Arg1689 N-region, FVIII-Arg1689 N-region mutated, FVIII-Arg1689 N+C-regions, FV-Arg709 minimal, FV-Arg709 N+C-regions, FV-Arg1018 minimal, FV-Arg1018 N+C-regions, FV-Arg1545 minimal, FV-Arg1545 N-region, FV-Arg1545 N+C-regions, fibrinogen α chain-Arg16 minimal, fibrinogen α chain-Arg16 N-region, fibrinogen β chain minimal, fibrinogen β chain N-region, protein C-Arg168 minimal and protein C-Arg168 N-region. The sequences of all the individual inserts are shown above each SDS-PAGE gel with the cleavage reaction for FVIII, FV, fibrinogen α chain, fibrinogen β chain and protein C respectively (as shown in Figs 3–6). Four different mutated variants of the N-terminal region of the fibrinogen α chain-Arg16 were also produced to study the importance of individual amino acid positions in this region of the target molecule (Figs 7 and 8).


The Importance of Exosite Interactions for Substrate Cleavage by Human Thrombin.

Chahal G, Thorpe M, Hellman L - PLoS ONE (2015)

Schematic 3-D models of human thrombin showing the position of the N-terminal region of fibrinogen α chain.Panel A shows a space-filling model with the alpha chain peptide in purple. Panel B shows the interaction between thrombin (ribbon structure in beige) and the N-terminal region of fibrinogen α chain (ball and stick structure in purple) in detail. The same orientation as panel A is shown with the catalytic residues His57, Asp102 and Ser195 together with the S1 pocket residues Asp 189, Gly216 and 226 in green. Thrombin structure from PDB, code 1DM4 run using UCSF Chimera v1.8 and annotated in Adobe Illustrator CS5.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4482499&req=5

pone.0129511.g007: Schematic 3-D models of human thrombin showing the position of the N-terminal region of fibrinogen α chain.Panel A shows a space-filling model with the alpha chain peptide in purple. Panel B shows the interaction between thrombin (ribbon structure in beige) and the N-terminal region of fibrinogen α chain (ball and stick structure in purple) in detail. The same orientation as panel A is shown with the catalytic residues His57, Asp102 and Ser195 together with the S1 pocket residues Asp 189, Gly216 and 226 in green. Thrombin structure from PDB, code 1DM4 run using UCSF Chimera v1.8 and annotated in Adobe Illustrator CS5.
Mentions: To study the involvement of the exosites for a number of important activation sites for human thrombin, we designed and ordered complementary single-stranded oligonucleotides encoding the region covering the thrombin cleavage sites with four amino acids up- and downstream of the cleavage sites in human FV, FVIII, fibrinogen α chain, fibrinogen β chain and protein C (Fig 3). This region is named the P4-P4’region or the minimal site. After mixing the paired oligonucleotides they form a stable double-stranded oligonucleotide with BamHI and SalI sticky ends. These were cloned into the Bam HI and SalI sites of a cleaved bacterial expression, pET21-2x trx, vector. This vector contains two E. coli trx genes with a linker region in between where different sequences can be introduced followed by a downstream six-His tag. (Fig 3). For the longer sequences also covering the negatively charged regions N-terminal, or both N- and C-terminal of the cleavage site, these regions were ordered as designer genes, cloned and sequence-verified from Genscript. In total, 28 different constructs were produced: FVIII-Arg372 minimal, FVIII-Arg372 N-region, FVIII-Arg372 N-region mutated, FVIII-Arg372 N+C-regions, FVIII-Arg740 minimal, FVIII-Arg740 N-region, FVIII-Arg740 N-region mutated, FVIII-Arg740 N+C-regions, FVIII-Arg1689 minimal, FVIII-Arg1689 N-region, FVIII-Arg1689 N-region mutated, FVIII-Arg1689 N+C-regions, FV-Arg709 minimal, FV-Arg709 N+C-regions, FV-Arg1018 minimal, FV-Arg1018 N+C-regions, FV-Arg1545 minimal, FV-Arg1545 N-region, FV-Arg1545 N+C-regions, fibrinogen α chain-Arg16 minimal, fibrinogen α chain-Arg16 N-region, fibrinogen β chain minimal, fibrinogen β chain N-region, protein C-Arg168 minimal and protein C-Arg168 N-region. The sequences of all the individual inserts are shown above each SDS-PAGE gel with the cleavage reaction for FVIII, FV, fibrinogen α chain, fibrinogen β chain and protein C respectively (as shown in Figs 3–6). Four different mutated variants of the N-terminal region of the fibrinogen α chain-Arg16 were also produced to study the importance of individual amino acid positions in this region of the target molecule (Figs 7 and 8).

Bottom Line: The results showed that addition of these regions enhanced the cleavage rate by more than fifty fold.The possibility to mimic this interaction for the sites in factor V and factor VIII by recombinant substrates, which do not have the same folding as the full size target, indicates that the enhancement was primarily dependent on a relatively simple electrostatic interaction.However, the situation was very different for fibrinogen and protein C where other factors than only charge is of major importance.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Uppsala University, BMC, Box 596, SE-751 24, Uppsala, Sweden.

ABSTRACT
Thrombin is a serine protease of the chymotrypsin family that acts both as a procoagulant and as an anticoagulant by cleaving either factor VIII, factor V and fibrinogen or protein C, respectively. Numerous previous studies have shown that electropositive regions at a distance from the active site, so called exosites, are of major importance for the cleavage by human thrombin. Upstream of all the known major cleavage sites for thrombin in factor VIII, factor V and fibrinogen are clusters of negatively charged amino acids. To study the importance of these sites for the interaction with the exosites and thereby the cleavage by thrombin, we have developed a new type of recombinant substrate. We have compared the cleavage rate of the minimal cleavage site, involving only 8-9 amino acids (typically the P4-P4' positions) surrounding the cleavage site, with the substrates also containing the negatively charged regions upstream of the cleavage sites. The results showed that addition of these regions enhanced the cleavage rate by more than fifty fold. However, the enhancement was highly dependent on the sequence of the actual cleavage site. A minimal site that showed poor activity by itself could be cleaved as efficiently as an optimal cleavage site when presented together with these negatively charged regions. Whereas sites conforming closely to the optimal site were only minimally enhanced by the addition of these regions. The possibility to mimic this interaction for the sites in factor V and factor VIII by recombinant substrates, which do not have the same folding as the full size target, indicates that the enhancement was primarily dependent on a relatively simple electrostatic interaction. However, the situation was very different for fibrinogen and protein C where other factors than only charge is of major importance.

No MeSH data available.


Related in: MedlinePlus