Wagenknecht T, Grassucci R, Berkowitz J, Forneris C
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
J Struct Biol. 1992 Jul-Aug;109(1):70-7. doi: 10.1016/1047-8477(92)90069-m.
The dihydrolipoyl transacetylase (E2p) component of the pyruvate dehydrogenase complex (PDC) of Escherichia coli is a multidomain polypeptide comprising a catalytic domain, a domain that binds dihydrolipoyl dehydrogenase (E3-binding domain), and three domains containing lipoic acid (lipoyl domains). In PDC 24 subunits of E2p associate by means of interactions involving the catalytic domains to form the structural core of PDC. From cryoelectron microscopy and computer image analysis of frozen-hydrated isolated E2p cores it appears that the lipoyl domains are located peripherally about the core complex and do not assume fixed positions. To further test this interpretation the visibility of the lipoyl domains in electron micrographs was enhanced by specifically biotinylating the lipoic acids and labeling them with streptavidin. In agreement with the studies of native, unlabeled E2p cores, cryoelectron microscopy of the streptavidin-labeled E2p cores showed that the lipoic acid moieties are capable of extending approximately 13 nm from the surface of the core. Localization of the E3-binding domains was accomplished by cryoelectron microscopy of E2p-E3 subcomplexes prepared by reconstitution in vitro. Frequently an apparent gap of several nanometers separated the bound E3 from the surface of the core. The third component of PDC, pyruvate dehydrogenase (E1p), appeared to bind to the E2p core in a manner similar to that observed for E3. These results support a structural model of the E2p core in which the catalytic, E3-binding, and three lipoyl domains are interconnected by linker sequences that assume extended and flexible conformations.
大肠杆菌丙酮酸脱氢酶复合体（PDC）的二氢硫辛酰胺转乙酰基酶（E2p）组分是一种多结构域多肽，它包含一个催化结构域、一个结合二氢硫辛酰胺脱氢酶的结构域（E3结合结构域）以及三个含硫辛酸的结构域（硫辛酰结构域）。在PDC中，24个E2p亚基通过涉及催化结构域的相互作用缔合，形成PDC的结构核心。从冷冻水合的分离E2p核心的冷冻电子显微镜和计算机图像分析来看，硫辛酰结构域似乎位于核心复合体的周边，且位置不固定。为了进一步验证这一解释，通过对硫辛酸进行特异性生物素化并用链霉亲和素标记，增强了电子显微照片中硫辛酰结构域的可见性。与对天然未标记E2p核心的研究一致，链霉亲和素标记的E2p核心的冷冻电子显微镜显示，硫辛酸部分能够从核心表面延伸约13纳米。E3结合结构域的定位是通过对体外重组制备的E2p-E3亚复合体进行冷冻电子显微镜观察来完成的。结合的E3与核心表面之间常常有几纳米的明显间隙。PDC的第三个组分，丙酮酸脱氢酶（E1p），似乎以与E3类似的方式结合到E2p核心上。这些结果支持了E2p核心的结构模型，其中催化结构域、E3结合结构域和三个硫辛酰结构域通过呈伸展且灵活构象的连接序列相互连接。
