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Isolation, cloning and structural characterisation of boophilin, a multifunctional Kunitz-type proteinase inhibitor from the cattle tick.

Macedo-Ribeiro S, Almeida C, Calisto BM, Friedrich T, Mentele R, Stürzebecher J, Fuentes-Prior P, Pereira PJ - PLoS ONE (2008)

Bottom Line: Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin.The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P(1) residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints.This finding explains the formation of a ternary thrombin.boophilin.trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo.

View Article: PubMed Central - PubMed

Affiliation: Centro de Neurociências e Biologia Celular (CNC), Coimbra, Portugal.

ABSTRACT
Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine alpha-thrombin.boophilin complex, refined at 2.35 A resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S(1) pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9 degrees and is displaced by 6 A, while the C-terminal domain rotates almost 6 degrees accompanied by a 3 A displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P(1) residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin.boophilin.trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo.

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Thrombin-bound boophilin retains the ability to interact with other serine proteinases.(A) Schematic representation of two hypothetical thrombin·boophilin complexes. In the upper, BPTI-like mechanism, binding of the C-terminal boophilin domain to exosite I promotes extensive rearrangements of loops surrounding the active site to allow insertion of the N-terminal domain in a canonical manner. In the alternative, ornithodorin-like mechanism, exosite engagement is not associated with important modifications of the thrombin active site region, which is occupied by the N-terminal peptide of the inhibitor in a parallel manner. (B) Demonstration of thrombin·boophilin·trypsin ternary complex formation via native gel electrophoresis. One µg human α-thrombin·boophilin complex was incubated with increasing amounts of bovine trypsin (from ≈300 ng, lane 3, to ≈6 µg, lane 9), and samples were resolved in an 8% polyacrylamide gel. Lanes 1 and 2 contain 1 µg thrombin and 1 µg thrombin·boophilin complex, respectively; the newly formed species corresponds to the ternary complex.
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pone-0001624-g004: Thrombin-bound boophilin retains the ability to interact with other serine proteinases.(A) Schematic representation of two hypothetical thrombin·boophilin complexes. In the upper, BPTI-like mechanism, binding of the C-terminal boophilin domain to exosite I promotes extensive rearrangements of loops surrounding the active site to allow insertion of the N-terminal domain in a canonical manner. In the alternative, ornithodorin-like mechanism, exosite engagement is not associated with important modifications of the thrombin active site region, which is occupied by the N-terminal peptide of the inhibitor in a parallel manner. (B) Demonstration of thrombin·boophilin·trypsin ternary complex formation via native gel electrophoresis. One µg human α-thrombin·boophilin complex was incubated with increasing amounts of bovine trypsin (from ≈300 ng, lane 3, to ≈6 µg, lane 9), and samples were resolved in an 8% polyacrylamide gel. Lanes 1 and 2 contain 1 µg thrombin and 1 µg thrombin·boophilin complex, respectively; the newly formed species corresponds to the ternary complex.

Mentions: The functional evidence presented above, combined with structural information for thrombin·ornithodorin [25] and thrombin(E192Q)·BPTI complexes [23], immediately suggested two possible modes for boophilin binding to cognate thrombin (Figure 4A). In both cases, the highly acidic boophilin C-terminal domain (11 aspartate/glutamate residues outbalance four basic residues) would interact with thrombin's positively charged exosite I. However, the two models differ with regard to the region of the N-terminal domain that blocks the active site of the proteinase. Binding in a canonical manner, i.e. via reactive-site loop requires major rearrangements of loops surrounding the active centre, and in particular of the 60-loop. By contrast, in the ornithodorin-like conformation only the N-terminal peptide of the inhibitor interacts with thrombin active site, which is essentially unmodified compared to the free enzyme. Preliminary modelling experiments indicated that both conformations could be plausibly adopted in the binary thrombin·boophilin complex, given the considerable length of the interdomain linker.


Isolation, cloning and structural characterisation of boophilin, a multifunctional Kunitz-type proteinase inhibitor from the cattle tick.

Macedo-Ribeiro S, Almeida C, Calisto BM, Friedrich T, Mentele R, Stürzebecher J, Fuentes-Prior P, Pereira PJ - PLoS ONE (2008)

Thrombin-bound boophilin retains the ability to interact with other serine proteinases.(A) Schematic representation of two hypothetical thrombin·boophilin complexes. In the upper, BPTI-like mechanism, binding of the C-terminal boophilin domain to exosite I promotes extensive rearrangements of loops surrounding the active site to allow insertion of the N-terminal domain in a canonical manner. In the alternative, ornithodorin-like mechanism, exosite engagement is not associated with important modifications of the thrombin active site region, which is occupied by the N-terminal peptide of the inhibitor in a parallel manner. (B) Demonstration of thrombin·boophilin·trypsin ternary complex formation via native gel electrophoresis. One µg human α-thrombin·boophilin complex was incubated with increasing amounts of bovine trypsin (from ≈300 ng, lane 3, to ≈6 µg, lane 9), and samples were resolved in an 8% polyacrylamide gel. Lanes 1 and 2 contain 1 µg thrombin and 1 µg thrombin·boophilin complex, respectively; the newly formed species corresponds to the ternary complex.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230226&req=5

pone-0001624-g004: Thrombin-bound boophilin retains the ability to interact with other serine proteinases.(A) Schematic representation of two hypothetical thrombin·boophilin complexes. In the upper, BPTI-like mechanism, binding of the C-terminal boophilin domain to exosite I promotes extensive rearrangements of loops surrounding the active site to allow insertion of the N-terminal domain in a canonical manner. In the alternative, ornithodorin-like mechanism, exosite engagement is not associated with important modifications of the thrombin active site region, which is occupied by the N-terminal peptide of the inhibitor in a parallel manner. (B) Demonstration of thrombin·boophilin·trypsin ternary complex formation via native gel electrophoresis. One µg human α-thrombin·boophilin complex was incubated with increasing amounts of bovine trypsin (from ≈300 ng, lane 3, to ≈6 µg, lane 9), and samples were resolved in an 8% polyacrylamide gel. Lanes 1 and 2 contain 1 µg thrombin and 1 µg thrombin·boophilin complex, respectively; the newly formed species corresponds to the ternary complex.
Mentions: The functional evidence presented above, combined with structural information for thrombin·ornithodorin [25] and thrombin(E192Q)·BPTI complexes [23], immediately suggested two possible modes for boophilin binding to cognate thrombin (Figure 4A). In both cases, the highly acidic boophilin C-terminal domain (11 aspartate/glutamate residues outbalance four basic residues) would interact with thrombin's positively charged exosite I. However, the two models differ with regard to the region of the N-terminal domain that blocks the active site of the proteinase. Binding in a canonical manner, i.e. via reactive-site loop requires major rearrangements of loops surrounding the active centre, and in particular of the 60-loop. By contrast, in the ornithodorin-like conformation only the N-terminal peptide of the inhibitor interacts with thrombin active site, which is essentially unmodified compared to the free enzyme. Preliminary modelling experiments indicated that both conformations could be plausibly adopted in the binary thrombin·boophilin complex, given the considerable length of the interdomain linker.

Bottom Line: Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin.The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P(1) residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints.This finding explains the formation of a ternary thrombin.boophilin.trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo.

View Article: PubMed Central - PubMed

Affiliation: Centro de Neurociências e Biologia Celular (CNC), Coimbra, Portugal.

ABSTRACT
Inhibitors of coagulation factors from blood-feeding animals display a wide variety of structural motifs and inhibition mechanisms. We have isolated a novel inhibitor from the cattle tick Boophilus microplus, one of the most widespread parasites of farm animals. The inhibitor, which we have termed boophilin, has been cloned and overexpressed in Escherichia coli. Mature boophilin is composed of two canonical Kunitz-type domains, and inhibits not only the major procoagulant enzyme, thrombin, but in addition, and by contrast to all other previously characterised natural thrombin inhibitors, significantly interferes with the proteolytic activity of other serine proteinases such as trypsin and plasmin. The crystal structure of the bovine alpha-thrombin.boophilin complex, refined at 2.35 A resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, binds in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S(1) pocket, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite I of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distorted Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithodorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9 degrees and is displaced by 6 A, while the C-terminal domain rotates almost 6 degrees accompanied by a 3 A displacement. The reactive-site loop of the N-terminal Kunitz domain of boophilin with its P(1) residue, K31, is fully solvent exposed and could thus bind a second trypsin-like proteinase without sterical restraints. This finding explains the formation of a ternary thrombin.boophilin.trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo.

Show MeSH
Related in: MedlinePlus