Novel Biochemical and Structural Insights into the Interaction of Myristoylated Cargo with Unc119 Protein and Their Release by Arl2/3
Novel Biochemical and Structural Insights into the Interaction of Myristoylated Cargo with Unc119 Protein and Their Release by Arl2/3
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DOI：
10.1074/jbc.m116.741827
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发表时间：
2016-09-23
影响因子：
4.8
通讯作者：
Wittinghofer, Alfred
中科院分区：
文献类型：
--
作者：
Jaiswal, Mamta;Fansa, Eyad K.;Wittinghofer, Alfred
初级纤毛是一种高度特化的小天线状细胞突起，从许多真核细胞类型的细胞表面延伸而来。纤毛和细胞质中的蛋白质含量非常不同，但对于大多数纤毛蛋白质来说，分类过程的细节尚不清楚。最近，我们已经表明，异丙基化蛋白质是根据它们与载体蛋白PDE6的亲和力以及Arl3而不是Arl2在纤毛内释放高亲和力货物的能力进行排序的(Fansa，E.K.，Kosling，S.K.，Zent，E.，Wittinghofer，A.，和Ismail，S.(2016)NAT。交警。7,11366)。在这里，我们讨论的问题是，是否有类似的原理管理由载体蛋白Unc119a和Unc119b运输的肉豆蔻酰化货物。因此，我们分析了N-末端肉豆蔻酰化的货运多肽(GNat1、NPHP3、Cystin1、RP2和Src)与Unc119a和Unc119b蛋白的结合强度。肉豆蔻酰化的货物和载体蛋白Unc119之间的亲和力在亚纳摩尔和微摩尔之间变化。来自纤毛定位蛋白的多肽(GNat1、NPHP3和Cystin1)与Unc119蛋白具有高亲和力，而来自非纤毛定位蛋白(Src)的多肽与Unc119蛋白的亲和力较低。具有中等亲和力的多肽(RP2)定位于纤毛过渡区，作为守门人。我们发现，低亲和力的多肽由Arl2GppNHp和Arl3GppNHp共同释放，而高亲和力的多肽仅由Arl3GppNHp释放。对肉豆蔻酰化NPHP3肽与Unc119a络合物的X射线结构的测定揭示了高亲和力结合的分子细节，并表明相对于肉豆蔻化甘氨酸的+2和+3位残基对高亲和力和低亲和力的重要性。在高亲和力和低亲和力多肽之间交换+2和+3位残基的突变分析导致了它们与Unc119a的亲和力逆转，并导致了低亲和力突变体NPHP3的部分错误定位。
Primary cilia are highly specialized small antenna-like cellular protrusions that extend from the cell surface of many eukaryotic cell types. The protein content inside cilia and cytoplasm is very different, but details of the sorting process are not understood for most ciliary proteins. Recently, we have shown that prenylated proteins are sorted according to their affinity to the carrier protein PDE6 and the ability of Arl3 but not Arl2 to release high affinity cargo inside the cilia (Fansa, E. K., Kosling, S. K., Zent, E., Wittinghofer, A., and Ismail, S. (2016) Nat. Commun. 7, 11366). Here we address the question whether a similar principle governs the transport of myristoylated cargo by the carrier proteins Unc119a and Unc119b. We thus analyzed the binding strength of N-terminal myristoylated cargo peptides (GNAT1, NPHP3, Cystin1, RP2, and Src) to Unc119a and Unc119b proteins. The affinity between myristoylated cargo and carrier protein, Unc119, varies between subnanomolar and micromolar. Peptides derived from ciliary localizing proteins (GNAT1, NPHP3, and Cystin1) bind with high affinity to Unc119 proteins, whereas a peptide derived from a non-ciliary localizing protein (Src) has low affinity. The peptide with intermediate affinity (RP2) is localized at the ciliary transition zone as a gate keeper. We show that the low affinity peptides are released by both Arl2GppNHp and Arl3GppNHp, whereas the high affinity peptides are exclusively released by only Arl3GppNHp. Determination of the x-ray structure of myristoylated NPHP3 peptide in complex with Unc119a reveals the molecular details of high affinity binding and suggests the importance of the residues at the +2 and +3 positions relative to the myristoylated glycine for high and low affinities. The mutational analysis of swapping the residues at the +2 and +3 positions between high and low affinity peptides results in reversing their affinities for Unc119a and leads to a partial mislocalization of a low affinity mutant of NPHP3.