Stability and global fold of the mouse prohormone convertase 1 pro-domain.
Stability and global fold of the mouse prohormone convertase 1 pro-domain.
复制标题
小鼠激素原转化酶 1 前结构域的稳定性和全局折叠。
作者：
Tangrea,MA;Alexander,P;Bryan,PN;Eisenstein,E;Toedt,J;Orban,J
我们纯化了在大肠杆菌中表达的小鼠激素原转化酶 1 (PC1) 前结构域，并使用多种生物物理方法证明该结构域是一个独立的折叠单元，在蛋白质浓度为 20 μM、pH 7.0 时，aTm 为 39 °C。这与细菌和人类弗林蛋白酶中类似的前结构域显着不同，后者在 25°C 下展开，并且需要催化结构域才能结构化 [Bryan 等人，2017]。 (1995)生物化学 34, 10310−10318;巴塔查尔贾等人。 (2000)J。生物分子。核磁共振 16, 275−276]。使用异核核磁共振波谱，我们确定了 PC1 前结构域的主链 1H、13C 和 15N 分配。根据1H/13C化学位移指数、NOE分析和氢交换测量，前结构域显示由四链β-折叠和两个α-螺旋组成。这里给出的结果表明，与枯草杆菌蛋白酶复合的细菌前结构域和未复合的小鼠 PC1 前结构域具有非常相似的整体折叠，尽管缺乏序列同源性。结构数据有助于解释PC家族前结构域中二次加工位点的位置，并提出了与催化结构域结合的共有序列。
We have purified the mouse prohormone convertase 1 (PC1) pro-domain expressed inEscherichia colicells and demonstrated, using a number of biophysical methods, that this domain is an independent folding unit with aTmof 39 °C at a protein concentration of 20 μM and pH 7.0. This differs significantly from similar pro-domains in bacteria and human furin, which are unfolded at 25 °C and require the catalytic domain in order to be structured [Bryan et al. (1995)Biochemistry 34, 10310−10318; Bhattacharjya et al. (2000)J. Biomol. NMR 16, 275−276]. Using heteronuclear NMR spectroscopy, we have determined the backbone1H,13C, and15N assignments for the pro-domain of PC1. On the basis of1H/13C chemical shift indices, NOE analysis, and hydrogen exchange measurements, the pro-domain is shown to consist of a four-stranded β-sheet and two α-helices. The results presented here show that both the bacterial pro-domain in complex with subtilisin and the uncomplexed mouse PC1 pro-domain have very similar overall folds despite a lack of sequence homology. The structural data help to explain the location of the secondary processing sites in the pro-domains of the PC family, and a consensus sequence for binding to the catalytic domain is proposed.