Koharudin Leonardus M I, Furey William, Liu Hao, Liu Yong-Jian, Gronenborn Angela M
Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260, USA.
J Biol Chem. 2009 Aug 28;284(35):23697-707. doi: 10.1074/jbc.M109.008995. Epub 2009 Jun 24.
Subcellular retrograde transport of cargo receptors from endosomes to the trans-Golgi network is critically involved in a broad range of physiological and pathological processes and highly regulated by a genetically conserved heteropentameric complex, termed retromer. Among the retromer components identified in mammals, sorting nexin 5 and 1 (SNX5; SNX1) have recently been found to interact, possibly controlling the membrane binding specificity of the complex. To elucidate how the unique sequence features of the SNX5 phox domain (SNX5-PX) influence retrograde transport, we have determined the SNX5-PX structure by NMR and x-ray crystallography at 1.5 A resolution. Although the core fold of SNX5-PX resembles that of other known PX domains, we found novel structural features exclusive to SNX5-PX. It is most noteworthy that in SNX5-PX, a long helical hairpin is added to the core formed by a new alpha2'-helix and a much longer alpha3-helix. This results in a significantly altered overall shape of the protein. In addition, the unique double PXXP motif is tightly packed against the rest of the protein, rendering this part of the structure compact, occluding parts of the putative phosphatidylinositol (PtdIns) binding pocket. The PtdIns binding and specificity of SNX5-PX was evaluated by NMR titrations with eight different PtdIns and revealed that SNX5-PX preferentially and specifically binds to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). The distinct structural and PtdIns binding characteristics of SNX5-PX impart specific properties on SNX5, influencing retromer-mediated regulation of retrograde trafficking of transmembrane cargo receptors.
货物受体从内体到反式高尔基体网络的亚细胞逆行运输，在广泛的生理和病理过程中起着关键作用，并受到一种基因保守的异源五聚体复合物（称为回收体）的高度调控。在哺乳动物中鉴定出的回收体成分中，分选连接蛋白5和1（SNX5；SNX1）最近被发现相互作用，可能控制该复合物的膜结合特异性。为了阐明SNX5 phox结构域（SNX5-PX）的独特序列特征如何影响逆行运输，我们通过核磁共振（NMR）和X射线晶体学，以1.5埃的分辨率确定了SNX5-PX的结构。尽管SNX5-PX的核心折叠与其他已知的PX结构域相似，但我们发现了SNX5-PX独有的新结构特征。最值得注意的是，在SNX5-PX中，一个长螺旋发夹结构被添加到由新的α2'-螺旋和长得多的α3-螺旋形成的核心结构上。这导致蛋白质的整体形状发生了显著改变。此外，独特的双PXXP基序紧密堆积在蛋白质的其余部分上，使该结构部分紧凑，封闭了假定的磷脂酰肌醇（PtdIns）结合口袋的部分区域。通过用八种不同的PtdIns进行NMR滴定，评估了SNX5-PX的PtdIns结合和特异性，结果表明SNX5-PX优先且特异性地结合磷脂酰肌醇4,5-二磷酸（PtdIns(4,5)P(2)）。SNX5-PX独特的结构和PtdIns结合特性赋予了SNX5特定的性质，影响了回收体介导的跨膜货物受体逆行运输的调控。
