From the Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, 3052 Parkville, Victoria, Australia.
the Department of Medical Biology, University of Melbourne, 3052 Parkville, Victoria, Australia.
J Biol Chem. 2019 Feb 15;294(7):2470-2485. doi: 10.1074/jbc.RA118.004836. Epub 2018 Dec 15.
The embrane-ssociated ING- (MARCH) family of membrane-bound E3 ubiquitin ligases regulates the levels of cell-surface membrane proteins, many of which are involved in immune responses. Although their role in ubiquitin-dependent endocytosis and degradation of cell-surface proteins is extensively documented, the features of MARCH proteins and their substrates that drive the molecular recognition events leading to ubiquitin transfer remain poorly defined. In this study, we sought to determine the features of human MARCH9 that are required for regulating the surface levels of its substrate proteins. Consistent with previous studies of other MARCH proteins, we found that susceptibility to MARCH9 activity is encoded in the transmembrane (TM) domains of its substrates. Accordingly, substitutions at specific residues and motifs within MARCH9's TM domains resulted in varying degrees of functional impairment. Most notably, a single serine-to-alanine substitution in the first of its two TM domains rendered MARCH9 completely unable to alter the surface levels of two different substrates: the major histocompatibility class I molecule HLA-A2 and the T-cell co-receptor CD4. Solution NMR analysis of a MARCH9 fragment encompassing the two TM domains and extracellular connecting loop revealed that the residues contributing most to MARCH9 activity are located in the α-helical portions of TM1 and TM2 that are closest to the extracellular face of the lipid bilayer. This observation defines a key region required for substrate regulation. In summary, our biochemical and structural findings demonstrate that specific sequences in the α-helical MARCH9 TM domains make crucial contributions to its ability to down-regulate its protein substrates.
膜相关的 ING-（MARCH）家族的膜结合 E3 泛素连接酶调节细胞表面膜蛋白的水平，其中许多蛋白参与免疫反应。尽管它们在泛素依赖的细胞表面蛋白内吞和降解中的作用已被广泛证实，但驱动导致泛素转移的分子识别事件的 MARCH 蛋白及其底物的特征仍未得到明确定义。在这项研究中，我们试图确定人类 MARCH9 调节其底物蛋白表面水平所需的特征。与其他 MARCH 蛋白的先前研究一致，我们发现其底物对 MARCH9 活性的敏感性编码在其跨膜（TM）结构域中。因此，MARCH9 的 TM 结构域中特定残基和基序的取代导致功能障碍的程度不同。最值得注意的是，其两个 TM 结构域中的第一个 TM 结构域中的一个丝氨酸到丙氨酸取代，使 MARCH9 完全无法改变两种不同底物的表面水平：主要组织相容性复合体 I 分子 HLA-A2 和 T 细胞共受体 CD4。包含两个 TM 结构域和细胞外连接环的 MARCH9 片段的溶液 NMR 分析表明，对 MARCH9 活性贡献最大的残基位于最接近脂双层细胞外表面的 TM1 和 TM2 的α-螺旋部分。这一观察结果定义了一个需要进行底物调节的关键区域。总之，我们的生化和结构研究结果表明，α-螺旋 MARCH9 TM 结构域中的特定序列对其下调其蛋白底物的能力做出了至关重要的贡献。
