Duckwitz Wiebke, Hausmann Ralf, Aschrafi Armaz, Schmalzing Günther
Department of Molecular Pharmacology, Rheinisch-Westfaelische Technische Hochschule (RWTH) Aachen, Wendlingweg 2, D-52074 Aachen, Germany.
J Biol Chem. 2006 Dec 22;281(51):39561-72. doi: 10.1074/jbc.M606113200. Epub 2006 Sep 25.
Functional homomeric and heteromeric ATP-gated P2X receptor channels have been shown to display a characteristic trimeric architecture. Of the seven different isoforms (designated P2X(1)-P2X(7)), P2X(5) occurs in humans primarily as a non-functional variant lacking the C-terminal end of the ectodomain and the outer half of the second transmembrane domain. We show that this truncated variant, which results from the splice-skipping of exon 10, is prone to subunit aggregation because the residual transmembrane domain 2 is too short to insert into the membrane. Alleviation of the negative hydrophobic mismatch by the addition of a stretch of moderately hydrophobic residues enabled formation of a second membrane-spanning domain and strictly parallel homotrimerization. Systematic mutagenesis identified only one transmembrane domain 2 residue, Asp(355), which supported homotrimerization in a side chain-specific manner. Our results indicate that transmembrane domain 2 formation contributes 2-fold to hP2X(5) homotrimerization by tethering the end of the ectodomain to the membrane, thereby topologically restricting conformational mobility, and by intramembrane positioning of Asp(355). While transmembrane domain 2 appears to favor assembly by enabling productive subunit interactions in the ectodomain, Asp(355) seems to assist by simultaneously driving intramembrane helix interactions. Overall, these results indicate a complex interplay between topology, helix-helix interactions, and oligomerization to achieve a correctly folded structure.
功能性同聚体和异聚体ATP门控P2X受体通道已被证明具有特征性的三聚体结构。在七种不同的亚型（命名为P2X(1)-P2X(7)）中，P2X(5)在人类中主要以一种非功能性变体的形式存在，该变体缺少胞外域的C末端和第二个跨膜结构域的外半部分。我们发现，这种由外显子10跳跃剪接产生的截短变体易于亚基聚集，因为残留的跨膜结构域2太短而无法插入膜中。通过添加一段适度疏水的残基来减轻负性疏水错配，能够形成第二个跨膜结构域并实现严格平行的同三聚化。系统性诱变仅鉴定出一个跨膜结构域2残基，即天冬氨酸（Asp(355)），它以侧链特异性的方式支持同三聚化。我们的结果表明，跨膜结构域2的形成通过将胞外域末端连接到膜上，从而在拓扑学上限制构象流动性，并通过天冬氨酸（Asp(355)）在膜内的定位，对hP2X(5)同三聚化有两方面的贡献。虽然跨膜结构域2似乎通过在胞外域实现有效的亚基相互作用而有利于组装，但天冬氨酸（Asp(355)）似乎通过同时驱动膜内螺旋相互作用来提供协助。总体而言，这些结果表明拓扑结构、螺旋-螺旋相互作用和寡聚化之间存在复杂的相互作用，以实现正确折叠的结构。
