Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.
Biochem J. 2012 Mar 15;442(3):563-72. doi: 10.1042/BJ20111360.
Drosophila melanogaster crammer is a novel cathepsin inhibitor that is involved in LTM (long-term memory) formation. The mechanism by which the inhibitory activity is regulated remains unclear. In the present paper we have shown that the oligomeric state of crammer is pH dependent. At neutral pH, crammer is predominantly dimeric in vitro as a result of disulfide bond formation, and is monomeric at acidic pH. Our inhibition assay shows that monomeric crammer, not disulfide-bonded dimer, is a strong competitive inhibitor of cathepsin L. Crammer is a monomeric molten globule in acidic solution, a condition that is similar to the environment in the lysosome where crammer is probably located. Upon binding to cathepsin L, however, crammer undergoes a molten globule-to-ordered structural transition. Using high-resolution NMR spectroscopy, we have shown that a cysteine-to-serine point mutation at position 72 (C72S) renders crammer monomeric at pH 6.0 and that the structure of the C72S variant highly resembles that of wild-type crammer in complex with cathepsin L at pH 4.0. We have determined the first solution structure of propeptide-like protease inhibitor in its active form and examined in detail using a variety of spectroscopic methods the folding properties of crammer in order to delineate its biomolecular recognition of cathepsin.
果蝇黑素体紧张是一种新的组织蛋白酶抑制剂，参与 LTM（长时程记忆）的形成。调节抑制活性的机制尚不清楚。在本文中，我们已经表明，紧张的寡聚状态是 pH 依赖性的。在中性 pH 下，紧张主要以二聚体形式存在于体外，这是由于二硫键的形成，而在酸性 pH 下则是单体形式。我们的抑制试验表明，单体紧张，而不是二硫键结合的二聚体，是组织蛋白酶 L 的强竞争性抑制剂。紧张在酸性溶液中是单体无规卷曲，这种条件类似于溶酶体中的环境，紧张可能位于溶酶体中。然而，在与组织蛋白酶 L 结合后，紧张经历了无规卷曲到有序结构的转变。使用高分辨率 NMR 光谱，我们已经表明，位置 72 处的半胱氨酸到丝氨酸点突变（C72S）使紧张在 pH 6.0 时呈单体形式，并且 C72S 变体的结构在 pH 4.0 时与野生型紧张与组织蛋白酶 L 复合物非常相似。我们已经确定了原肽样蛋白酶抑制剂在其活性形式下的第一个溶液结构，并使用各种光谱方法详细检查了紧张的折叠性质，以描绘其对组织蛋白酶的生物分子识别。
