A multipronged approach unravels unprecedented protein-protein interactions in the human 2-oxoglutarate dehydrogenase multienzyme complex.
A multipronged approach unravels unprecedented protein-protein interactions in the human 2-oxoglutarate dehydrogenase multienzyme complex.
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多管齐下的方法揭示了人类 2-酮戊二酸脱氢酶多酶复合物中前所未有的蛋白质-蛋白质相互作用。
DOI：
10.1074/jbc.ra118.005432
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发表时间：
2018
期刊：
影响因子：
--
通讯作者：
Jordan,Frank
中科院分区：
文献类型：
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作者：
Zhou,Jieyu;Yang,Luying;Ozohanics,Oliver;Zhang,Xu;Wang,Junjie;Ambrus,Attila;Arjunan,Palaniappa;Brukh,Roman;Nemeria,NataliaS;Furey,William;Jordan,Frank
人2-酮基谷氨酸脱氢酶复合物（hOGDHc）在三羧酸（TCA）循环中起关键作用，其活性降低与神经退行性疾病相关。hOGDHc由hE 1 o、hE 2 o和hE 3三种组分组成，我们最近报道了功能活性的E1 o和E2 o组分，从而能够研究它们的组装。目前还没有hE 2 o组分的原子分辨率结构，因此我们首先研究了二元亚复合物（hE 1 o-hE 2 o，hE 1 o-hE 3和hE 2 o-hE 3）中的相互作用，以了解它们相互作用的强度并确定其中的相互作用位点。我们进行了多种物理化学研究，包括荧光，氢氘交换MS（HDX-MS）和化学交联MS（CL-MS）。我们的荧光研究表明hE 10-hE 20亚复合物有很强的相互作用，但hE 10-hE 3亚复合物中的相互作用要弱得多，并且未能鉴定hE 20-hE 3亚复合物中的任何相互作用。HDX-MS研究给出了包含全长组分的hE 10-hE 20和hE 10-hE 3亚复合物中相互作用的证据，鉴定：（i）hE 10的N-末端区域，特别是两个肽18 YVEEM 22和27 ENPKSVHKSWDIF 39，作为构成负责hE 10与hE 20和hE 3组分组装成hOGDHc的结合区域，hOGDHc是在现有X射线结构中不存在的hE 1区域;和（ii）新的hE 2 o区，其包含来自接头区和催化结构域的残基，作为与hE 1 o相互作用的关键区。CL-MS鉴定了hE 1 o和hE 2 o组分中相互作用的基因座。
The human 2-oxoglutaric acid dehydrogenase complex (hOGDHc) plays a pivotal role in the tricarboxylic acid (TCA) cycle, and its diminished activity is associated with neurodegenerative diseases. The hOGDHc comprises three components, hE1o, hE2o, and hE3, and we recently reported functionally active E1o and E2o components, enabling studies on their assembly. No atomic-resolution structure for the hE2o component is currently available, so here we first studied the interactions in the binary subcomplexes (hE1o–hE2o, hE1o–hE3, and hE2o–hE3) to gain insight into the strength of their interactions and to identify the interaction loci in them. We carried out multiple physico-chemical studies, including fluorescence, hydrogen–deuterium exchange MS (HDX-MS), and chemical cross-linking MS (CL-MS). Our fluorescence studies suggested a strong interaction for the hE1o–hE2o subcomplex, but a much weaker interaction in the hE1o–hE3 subcomplex, and failed to identify any interaction in the hE2o–hE3 subcomplex. The HDX-MS studies gave evidence for interactions in the hE1o–hE2o and hE1o–hE3 subcomplexes comprising full-length components, identifying: (i) the N-terminal region of hE1o, in particular the two peptides18YVEEM22and27ENPKSVHKSWDIF39as constituting the binding region responsible for the assembly of the hE1o with both the hE2o and hE3 components into hOGDHc, an hE1 region absent in available X-ray structures; and (ii) a novel hE2o region comprising residues from both a linker region and from the catalytic domain as being a critical region interacting with hE1o. The CL-MS identified the loci in the hE1o and hE2o components interacting with each other.