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Ra-KLP, a 75 amino acid protein secreted by the salivary gland of the brown ear tick Rhipicephalus appendiculatus has a sequence resembling those of Kunitz/BPTI proteins. We report the detection, purification and characterization of the function of Ra-KLP. In addition, determination of the three-dimensional crystal structure of Ra-KLP at 1.6 A resolution using sulphur single-wavelength anomalous dispersion reveals that much of the loop structure of classical Kunitz domains, including the protruding protease-binding loop, has been replaced by beta-strands. Even more unusually, the N-terminal portion of the polypeptide chain is pinned to the "Kunitz head" by two disulphide bridges not found in classical Kunitz/BPTI proteins. The disulphide bond pattern has been further altered by the loss of the bridge that normally stabilizes the protease-binding loop. Consistent with the conversion of this loop into a beta-strand, Ra-KLP shows no significant anti-protease activity; however, it activates maxiK channels in an in vitro system, suggesting a potential mechanism for regulating host blood supply during feeding.

Original publication

DOI

10.1016/j.jmb.2009.04.045

Type

Journal article

Journal

J Mol Biol

Publication Date

19/06/2009

Volume

389

Pages

734 - 747

Keywords

Amino Acid Sequence, Animals, Aprotinin, Cell Line, Crystallography, X-Ray, Dimerization, Evolution, Molecular, Humans, Ion Channels, Models, Molecular, Molecular Sequence Data, Patch-Clamp Techniques, Phylogeny, Protein Structure, Secondary, Protein Structure, Tertiary, Rhipicephalus, Saliva, Sequence Alignment, Sequence Homology, Amino Acid