Isolation, cloning and structural characterisation of boophilin, a multifunctional Kunitz-type proteinase inhibitor from the cattle tick.
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vor 16 Jahren
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.
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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