Assembly of the SLIP1-SLBP complex on histone mRNA requires heterodimerization and sequential binding of SLBP followed by SLIP1.
Assembly of the SLIP1-SLBP complex on histone mRNA requires heterodimerization and sequential binding of SLBP followed by SLIP1.
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DOI：
10.1021/bi301074r
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发表时间：
2013-01-22
期刊：
影响因子：
2.9
通讯作者：
Thapar R
中科院分区：
文献类型：
--
作者：
Bansal N;Zhang M;Bhaskar A;Itotia P;Lee E;Shlyakhtenko LS;Lam TT;Fritz A;Berezney R;Lyubchenko YL;Stafford WF;Thapar R
SLIP 1-SLBP复合物激活复制依赖性组蛋白mRNA的翻译。在这份报告中，我们描述了SLIP 1-SLBP复合物的活性是如何通过磷酸化和寡聚化来调节的。游离蛋白的生物物理表征表明，尽管SLIP 1是不结合RNA的同源二聚体，但人SLBP是一种内在无序的蛋白，当在真核表达系统如杆状病毒中表达时，其在23个Ser/Thr位点磷酸化。细菌表达的未磷酸化SLIP 1-SLBP复合物形成2：2高亲和力（KD < 0.9 nM）异源四聚体，其也不能结合组蛋白mRNA。相比之下，来自杆状病毒的磷酸化SLBP对SLIP 1具有弱亲和力（KD ~3 µM）。磷酸化的SLBP与组蛋白mRNA茎环的顺序结合，随后与SLIP 1结合，需要形成“活性”三元复合物。SLBP在Thr 171处的磷酸化促进异源四聚体解离成SLIP 1-SLBP异源二聚体。使用丙氨酸扫描诱变，我们证明SLIP 1上的SLBP的结合位点位于接近二聚体界面。SLIP 1同源二聚体界面附近的单点突变体在体外消除了与SLBP的相互作用，并降低了体内组蛋白mRNA的丰度。在这些生物物理研究的基础上，我们提出，寡聚化和SLBP磷酸化可能会调节SLBP-SLIP 1复合物在体内。SLIP 1可用于在体内螯合SLBP，保护其不被蛋白水解降解为无活性的异源四聚体，或者，SLIP 1-SLBP异源四聚体的形成可促进在组蛋白mRNA降解之前从组蛋白mRNA中去除SLBP。
The SLIP1-SLBP complex activates translation of replication-dependent histone mRNAs. In this report, we describe how the activity of the SLIP1-SLBP complex is modulated by phosphorylation and oligomerization. Biophysical characterization of the free proteins shows that whereas SLIP1 is a homodimer that does not bind RNA, human SLBP is an intrinsically disordered protein that is phosphorylated at 23 Ser/Thr sites when expressed in a eukaryotic expression system such as baculovirus. The bacterially expressed unphosphorylated SLIP1-SLBP complex forms a 2:2 high affinity (KD < 0.9 nM) heterotetramer that is also incapable of binding histone mRNA. In contrast, phosphorylated SLBP from baculovirus has weak affinity (KD ~3 µM) for SLIP1. Sequential binding of phosphorylated SLBP to the histone mRNA stem-loop, followed by association with SLIP1 is required to form an “active” ternary complex. Phosphorylation of SLBP at Thr171 promotes dissociation of the heterotetramer to the SLIP1-SLBP heterodimer. Using alanine scanning mutagenesis we demonstrate that the binding site on SLIP1 for SLBP lies close to the dimer interface. A single point mutant near the SLIP1 homodimer interface abolished interaction with SLBP in vitro and reduced histone mRNA abundance in vivo. On the basis of these biophysical studies, we propose that oligomerization and SLBP phosphorylation may regulate the SLBP-SLIP1 complex in vivo. SLIP1 may act to sequester SLBP in vivo protecting it from proteolytic degradation as an inactive hetero-tetramer, or alternatively, formation of the SLIP1-SLBP hetero-tetramer may facilitate removal of SLBP from the histone mRNA prior to histone mRNA degradation.
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