Paesen Guido C, Siebold Christian, Dallas Mark L, Peers Chris, Harlos Karl, Nuttall Patricia A, Nunn Miles A, Stuart David I, Esnouf Robert M
CEH Oxford, Mansfield Road, Oxford OX1 3SR, UK.
J Mol Biol. 2009 Jun 19;389(4):734-47. doi: 10.1016/j.jmb.2009.04.045. Epub 2009 Apr 24.
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that much of the loop structure of classical Kunitz domains, including the protruding protease-binding loop, has been replaced by beta-strands.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.
Ra-KLP是一种由棕色耳蜱（Rhipicephalus appendiculatus）唾液腺分泌的75个氨基酸的蛋白质，其序列与库尼茨/抑肽酶（Kunitz/BPTI）蛋白相似。我们报告了Ra-KLP功能的检测、纯化及特性鉴定。此外，利用硫单波长反常色散在1.6埃分辨率下测定Ra-KLP的三维晶体结构，结果显示经典库尼茨结构域的许多环结构，包括突出的蛋白酶结合环，已被β链取代。更不寻常的是，多肽链的N端部分通过两个在经典库尼茨/抑肽酶蛋白中未发现的二硫键固定在“库尼茨头部”。由于通常稳定蛋白酶结合环的桥键缺失，二硫键模式进一步改变。与该环转变为β链一致，Ra-KLP没有显著的抗蛋白酶活性；然而，它在体外系统中激活大电导钙激活钾通道（maxiK通道），提示了在进食过程中调节宿主血液供应的潜在机制。
