Hemmi Hikaru, Kumazaki Takashi, Yoshizawa-Kumagaye Kumiko, Nishiuchi Yuji, Yoshida Takuya, Ohkubo Tadayasu, Kobayashi Yuji
National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
Biochemistry. 2005 Jul 19;44(28):9626-36. doi: 10.1021/bi0472806.
Anemonia elastase inhibitor (AEI) is a "nonclassical" Kazal-type elastase inhibitor from Anemonia sulcata. Unlike many nonclassical inhibitors, AEI does not have a cystine-stabilized alpha-helical (CSH) motif in the sequence. We chemically synthesized AEI and determined its three-dimensional solution structure by two-dimensional NMR spectroscopy. The resulting structure of AEI was characterized by a central alpha-helix and a three-stranded antiparallel beta-sheet of a typical Kazal-type inhibitor such as silver pheasant ovomucoid third domain (OMSVP3), even though the first and fifth half-cystine residues forming a disulfide bond in AEI are shifted both toward the C-terminus in comparison with those of OMSVP3. Synthesized AEI exhibited unexpected strong inhibition toward Streptomyces griseus protease B (SGPB). Our previous study [Hemmi, H., et al. (2003) Biochemistry 42, 2524-2534] demonstrated that the site-specific introduction of the engineered disulfide bond into the OMSVP3 molecule to form the CSH motif could produce an inhibitor with a narrower specificity. Thus, the CSH motif-containing derivative of AEI (AEI analogue) was chemically synthesized when a Cys(4)-Cys(34) bond was changed to a Cys(6)-Cys(31) bond. The AEI analogue scarcely inhibited porcine pancreatic elastase (PPE), even though it exhibited almost the same potent inhibitory activity toward SGPB. For the molecular scaffold, essentially no structural difference was detected between the two, but the N-terminal loop from Pro(5) to Ile(7) near the putative reactive site (Met(10)-Gln(11)) in the analogue moved by 3.7 A toward the central helix to form the introduced Cys(6)-Cys(31) bond. Such a conformational change in the restricted region correlates with the specificity change of the inhibitor.
海葵弹性蛋白酶抑制剂（AEI）是一种来自沟迎风海葵的“非经典”卡扎尔型弹性蛋白酶抑制剂。与许多非经典抑制剂不同，AEI在序列中没有胱氨酸稳定的α-螺旋（CSH）基序。我们化学合成了AEI，并通过二维核磁共振光谱确定了其三维溶液结构。AEI的最终结构的特征是具有一个中心α-螺旋和一个典型卡扎尔型抑制剂（如白鹇卵类粘蛋白第三结构域（OMSVP3））的三链反平行β-折叠，尽管在AEI中形成二硫键的第一个和第五个半胱氨酸残基与OMSVP3相比都向C端移动。合成的AEI对灰色链霉菌蛋白酶B（SGPB）表现出意想不到的强抑制作用。我们之前的研究[Hemmi, H., 等人（2003年）《生物化学》42, 2524 - 2534]表明，在OMSVP3分子中位点特异性引入工程化二硫键以形成CSH基序可以产生特异性更窄的抑制剂。因此，当将Cys(4)-Cys(34)键改为Cys(6)-Cys(31)键时，化学合成了含CSH基序的AEI衍生物（AEI类似物）。AEI类似物几乎不抑制猪胰弹性蛋白酶（PPE），尽管它对SGPB表现出几乎相同的强效抑制活性。对于分子支架，两者之间基本上没有检测到结构差异，但类似物中靠近假定反应位点（Met(10)-Gln(11)）的从Pro(5)到Ile(7)的N端环向中心螺旋移动了3.7 Å以形成引入的Cys(6)-Cys(31)键。受限区域的这种构象变化与抑制剂的特异性变化相关。
