H-1,N-15 and C-13 NMR resonance assignment, secondary structure and global fold of the FMN-binding domain of human cytochrome P450 reductase
H-1,N-15 and C-13 NMR resonance assignment, secondary structure and global fold of the FMN-binding domain of human cytochrome P450 reductase
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DOI：
10.1023/a:1018313830207
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发表时间：
1997-07-01
影响因子：
2.7
通讯作者：
Roberts, GCK
中科院分区：
文献类型：
--
作者：
Barsukov, I;Modi, S;Roberts, GCK
人NADPH-细胞色素P450还原酶的FMN结合区，对应于外显子3-7，以活性形式高水平表达，并用C-13和N-15标记。大多数主链和脂肪族侧链H-1，N-15和C-13的共振峰已经用异核双共振法和三重共振法结合半自动指认策略进行了归属。根据化学位移指数和NOE连接性估计的二级结构由6个α-螺旋和5个β-链组成。全局折叠是从4DC-13分辨HMQC-NOESY-HMQC谱中明确指定的长程NOE推导出来的。折叠是交替的阿尔法和贝塔类型，五条贝塔链排列成一个平行的贝塔折叠。二级结构和整体折叠非常类似于细菌黄毒素的二级结构和全局折叠，但FMN结合结构域在N末端有一个额外的短螺旋来代替环，并且在N-末端有一个额外的螺旋(导致完整的P450还原酶中的膜锚定结构域)。将实验约束与同源建模相结合，得到满足观察到的NOE约束的FMN结合结构域的结构。化学位移比较表明，FMN结合和FMN还原的影响主要局限于结合部位。
The FMN-binding domain of human NADPH-cytochrome P450 reductase, corresponding to exons 3-7, has been expressed at high level in an active form and labelled with C-13 and N-15. Most of the backbone and aliphatic side-chain H-1, N-15 and C-13 resonances have been assigned using heteronuclear double-and triple-resonance methods, together with a semiautomatic assignment strategy. The secondary structure as estimated from the chemical shift index and NOE connectivities consists of six alpha-helices and five beta-strands. The global fold was deduced from the long-range NOEs unambiguously assigned in a 4D C-13-resolved HMQC-NOESY-HMQC spectrum. The fold is of the alternating alpha/beta type, with the five beta-strands arranged into a parallel beta-sheet. The secondary structure and global fold are very similar to those of the bacterial flavodoxins, but the FMN-binding domain has an extra short helix in place of a loop, and an extra helix at the N-terminus (leading to the membrane anchor domain in the intact P450 reductase). The experimental constraints were combined with homology modelling to obtain a structure of the FMN-binding domain satisfying the observed NOE constraints. Chemical shift comparisons showed that the effects of FMN binding and of FMN reduction are largely localised at the binding site.