Assembly of human C-terminal binding protein (CtBP) into tetramers
Assembly of human C-terminal binding protein (CtBP) into tetramers
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DOI：
10.1074/jbc.ra118.002514
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发表时间：
2018-06-08
影响因子：
4.8
通讯作者：
Royer, William E., Jr.
中科院分区：
文献类型：
--
作者：
Bellesis, Andrew G.;Jecrois, Anne M.;Royer, William E., Jr.
c端结合蛋白1 （CtBP1）和CtBP2是转录共调节因子，通过结合转录因子和将染色质重塑酶募集到基因启动子中来抑制许多细胞过程，如凋亡。人类CtBP的NAD(H)连锁寡聚化与其共转录活性相耦合，这与癌症进展有关。然而，CtBP组装的生物学相关水平尚未确定；亚基的立体化学排列也不高于二聚体。在这里，多角度光散射（MALS）数据建立了CtBP1和CtBP2的NAD（+）和nadh依赖性组装成四聚体。对CtBP1和CtBP2晶格内亚基相互作用的研究表明，两者都具有非常相似的四聚体排列，这是由两个二聚体对的组装造成的，特定的相互作用可能对NAD(H)结合敏感。通过创建CtBP1和CtBP2的一系列突变体，我们验证了晶体学观察到的中间二聚体配对稳定溶液四聚体的假设。MALS数据证实，这些突变体破坏了CtBP1和CtBP2的四聚体，二聚体通常保持完整，为CtBP1和CtBP2的四聚体组装提供了第一个立体化学模型。一个微妙的不稳定突变体的晶体结构表明，连接底物和nadd结合域的铰链区域的微小结构扰动足以削弱CtBP1四聚体。这些结果强烈表明，四聚体在CtBP功能中起重要作用，本文报道的一系列CtBP突变体可用于研究四聚体的生理作用。
C-terminal binding protein 1 (CtBP1) and CtBP2 are transcriptional coregulators that repress numerous cellular processes, such as apoptosis, by binding transcription factors and recruiting chromatin-remodeling enzymes to gene promoters. The NAD(H)-linked oligomerization of human CtBP is coupled to its co-transcriptional activity, which is implicated in cancer progression. However, the biologically relevant level of CtBP assembly has not been firmly established; nor has the stereochemical arrangement of the subunits above that of a dimer. Here, multi-angle light scattering (MALS) data established the NAD(+)-and NADH-dependent assembly of CtBP1 and CtBP2 into tetramers. An examination of subunit interactions within CtBP1 and CtBP2 crystal lattices revealed that both share a very similar tetrameric arrangement resulting from assembly of two dimeric pairs, with specific interactions probably being sensitive to NAD(H) binding. Creating a series of mutants of both CtBP1 and CtBP2, we tested the hypothesis that the crystallographically observed interdimer pairing stabilizes the solution tetramer. MALS data confirmed that these mutants disrupt both CtBP1 and CtBP2 tetramers, with the dimer generally remaining intact, providing the first stereochemical models for tetrameric assemblies of CtBP1 and CtBP2. The crystal structure of a subtle destabilizing mutant suggested that small structural perturbations of the hinge region linking the substrate-and NAD-binding domains are sufficient to weaken the CtBP1 tetramer. These results strongly suggest that the tetramer is important in CtBP function, and the series of CtBP mutants reported here can be used to investigate the physiological role of the tetramer.