Wang Lijun, Cowley Aaron B, Terzyan Simon, Zhang Xuejun, Benson David R
Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, USA.
Proteins. 2007 May 1;67(2):293-304. doi: 10.1002/prot.21250.
We report a 1.55 A X-ray crystal structure of the heme-binding domain of cytochrome b(5) from Musca domestica (house fly; HF b(5)), and compare it with previously published structures of the heme-binding domains of bovine microsomal cytochrome b(5) (bMc b(5)) and rat outer mitochondrial membrane cytochrome b(5) (rOM b(5)). The structural comparison was done in the context of amino acid sequences of all known homologues of the proteins under study. We show that insect b(5)s contain an extended hydrophobic patch at the base of the heme binding pocket, similar to the one previously shown to stabilize mammalian OM b(5)s relative to their Mc counterparts. The hydrophobic patch in insects includes a residue with a bulky hydrophobic side chain at position 71 (Met). Replacing Met71 in HF b(5) with Ser, the corresponding residue in all known mammalian Mc b(5)s, is found to substantially destabilize the holoprotein. The destabilization is a consequence of two related factors: (1) a large decrease in apoprotein stability and (2) extension of conformational disruption in the apoprotein beyond the empty heme binding pocket (core 1) and into the heme-independent folding core (core 2). Analogous changes have previously been shown to accompany replacement of Leu71 in rOM b(5) with Ser. That the stabilizing role of Met71 in HF b(5) is manifested primarily in the apo state is highlighted by the fact that its crystallographic Calpha B factor is modestly larger than that of Ser71 in bMc b(5), indicating that it slightly destabilizes local polypeptide conformation when heme is in its binding pocket. Finally, we show that the final unit of secondary structure in the cytochrome b(5) heme-binding domain, a 3(10) helix known as alpha6, differs substantially in length and packing interactions not only for different protein isoforms but also for given isoforms from different species.
我们报道了家蝇细胞色素b(5)血红素结合结构域的1.55 Å X射线晶体结构，并将其与先前发表的牛微粒体细胞色素b(5)（bMc b(5)）和大鼠线粒体外膜细胞色素b(5)（rOM b(5)）的血红素结合结构域结构进行比较。结构比较是在研究的蛋白质所有已知同源物的氨基酸序列背景下进行的。我们发现昆虫的b(5)在血红素结合口袋底部含有一个延伸的疏水区域，类似于先前显示的相对于其微粒体对应物稳定哺乳动物线粒体外膜b(5)的区域。昆虫中的疏水区域包括在第71位具有大的疏水侧链的残基（甲硫氨酸）。在家蝇b(5)中将第71位的甲硫氨酸替换为所有已知哺乳动物微粒体b(5)中的对应残基丝氨酸，发现这会使全蛋白显著不稳定。这种不稳定是两个相关因素的结果：（1）脱辅基蛋白稳定性大幅下降，（2）脱辅基蛋白中的构象破坏超出空的血红素结合口袋（核心1）并延伸到不依赖血红素的折叠核心（核心2）。先前已表明，将大鼠线粒体外膜b(5)中的第71位亮氨酸替换为丝氨酸会伴随类似变化。家蝇b(5)中甲硫氨酸71的稳定作用主要在脱辅基状态下体现，这一事实通过其晶体学Cα B因子略大于牛微粒体b(5)中丝氨酸71的Cα B因子得以突出，表明当血红素在其结合口袋中时，它会使局部多肽构象略有不稳定。最后，我们表明细胞色素b(5)血红素结合结构域中的二级结构最终单元，即被称为α6的3(10)螺旋，不仅在不同蛋白质异构体中，而且在来自不同物种的给定异构体中，其长度和堆积相互作用都有很大差异。
