Pinkas Daniel M, Sanvitale Caroline E, Bufton Joshua C, Sorrell Fiona J, Solcan Nicolae, Chalk Rod, Doutch James, Bullock Alex N
Structural Genomics Consortium, University of Oxford, Old Road Campus, Roosevelt Drive, Oxford OX3 7DQ, U.K.
ISIS Pulsed Neutron and Muon Source, STFC, Harwell Science and Innovation Campus, Didcot OX11 0QX, U.K.
Biochem J. 2017 Nov 1;474(22):3747-3761. doi: 10.1042/BCJ20170527.
Members of the potassium channel tetramerization domain (KCTD) family are soluble non-channel proteins that commonly function as Cullin3 (Cul3)-dependent E3 ligases. Solution studies of the N-terminal BTB domain have suggested that some KCTD family members may tetramerize similarly to the homologous tetramerization domain (T1) of the voltage-gated potassium (Kv) channels. However, available structures of KCTD1, KCTD5 and KCTD9 have demonstrated instead pentameric assemblies. To explore other phylogenetic clades within the KCTD family, we determined the crystal structures of the BTB domains of a further five human KCTD proteins revealing a rich variety of oligomerization architectures, including monomer (SHKBP1), a novel two-fold symmetric tetramer (KCTD10 and KCTD13), open pentamer (KCTD16) and closed pentamer (KCTD17). While these diverse geometries were confirmed by small-angle X-ray scattering (SAXS), only the pentameric forms were stable upon size-exclusion chromatography. With the exception of KCTD16, all proteins bound to Cul3 and were observed to reassemble in solution as 5 : 5 heterodecamers. SAXS data and structural modelling indicate that Cul3 may stabilize closed BTB pentamers by binding across their BTB-BTB interfaces. These extra interactions likely also allow KCTD proteins to bind Cul3 without the expected 3-box motif. Overall, these studies reveal the KCTD family BTB domain to be a highly versatile scaffold compatible with a range of oligomeric assemblies and geometries. This observed interface plasticity may support functional changes in regulation of this unusual E3 ligase family.
钾通道四聚化结构域（KCTD）家族成员是可溶性非通道蛋白，通常作为依赖Cullin3（Cul3）的E3连接酶发挥作用。对N端BTB结构域的溶液研究表明，一些KCTD家族成员可能与电压门控钾（Kv）通道的同源四聚化结构域（T1）类似地形成四聚体。然而，KCTD1、KCTD5和KCTD9的现有结构却显示为五聚体组装。为了探索KCTD家族中的其他系统发育分支，我们确定了另外5种人类KCTD蛋白的BTB结构域的晶体结构，揭示了丰富多样的寡聚化结构，包括单体（SHKBP1）、一种新型的二重对称四聚体（KCTD10和KCTD13）、开放五聚体（KCTD16）和封闭五聚体（KCTD17）。虽然这些不同的几何结构通过小角X射线散射（SAXS）得到了证实，但只有五聚体形式在尺寸排阻色谱中是稳定的。除KCTD16外，所有蛋白都与Cul3结合，并在溶液中重新组装为5:5异源十聚体。SAXS数据和结构建模表明，Cul3可能通过跨其BTB - BTB界面结合来稳定封闭的BTB五聚体。这些额外的相互作用可能还使KCTD蛋白能够在没有预期的3 - 盒基序的情况下结合Cul3。总体而言，这些研究揭示了KCTD家族BTB结构域是一种高度通用的支架，与一系列寡聚体组装和几何结构兼容。这种观察到的界面可塑性可能支持这个不寻常的E3连接酶家族在调节方面的功能变化。
