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

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 doned 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 α-thrombin boophilin complex, refined at 2.35 Å resolution reveals a non-canonical binding mode to the proteinase. The N-terminal region of the mature inhibitor, Q16-R17-N18, birics in a parallel manner across the active site of the proteinase, with the guanidinium group of R17 anchored in the S, pockers, while the C-terminal Kunitz domain is negatively charged and docks into the basic exosite 1 of thrombin. This binding mode resembles the previously characterised thrombin inhibitor, ornithodorin which, unlike boophilin, is composed of two distored Kunitz modules. Unexpectedly, both boophilin domains adopt markedly different orientations when compared to those of ornithadorin, in its complex with thrombin. The N-terminal boophilin domain rotates 9° and its diaplaced by 6 Å, while the C-terminal domain rotates almost 6° accompained by a 3 Å displacement. The reactive-site loop of the N-terminal Kunitz domain of sterical testraints. This finding explains the formation of a ternary thrombin boophilin trypsin complex, and suggests a mechanism for prothrombinase inhibition in vivo. © 2008 Macedo-Ribeiro et al.