Structural and functional analysis of slit and heparin binding to immunoglobulin-like domains 1 and 2 of Drosophila Robo.
Structural and functional analysis of slit and heparin binding to immunoglobulin-like domains 1 and 2 of Drosophila Robo.
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DOI：
10.1074/jbc.m800688200
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发表时间：
2008-06-06
期刊：
影响因子：
--
通讯作者：
Hohenester E
中科院分区：
文献类型：
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作者：
Fukuhara N;Howitt JA;Hussain SA;Hohenester E
Robo家族的跨膜受体对分泌蛋白Slit的识别为神经系统和其他器官的发育以及肿瘤转移和血管生成提供了重要信号。硫酸乙酰肝素（HS）蛋白聚糖在Slit-Robo信号传导中充当必需的辅助受体。先前的研究表明，Slit的第二个富含亮氨酸的重复结构域D2与Robo的N-末端免疫球蛋白样结构域IG 1 -2结合。在这里，我们提出了两个晶体结构的果蝇机器人IG 1 -2，其中之一含有绑定肝素衍生的寡糖。使用Robo IG 1 -5构建体的基于结构的诱变，我们鉴定了在IG 1表面上形成保守补丁的关键Slit结合残基（Thr 74、Phe 114、Arg 117）; IG 2中类似保守残基的突变对Slit结合没有影响。IG 1（Lys 69，Arg 117，Lys 122，Lys 123）中保守的碱性残基突变，但IG 2中没有，降低了Robo IG 1 -5与肝素的结合，与Robo-肝素共晶体结构完全一致。我们的集体结果，连同最近的最小人类Slit-Robo复合物的晶体结构（Morlot，C.，Thielens，N. M.，拉韦利河B.，Hemrika，W.，罗明河一、Gros，P.，Cusack，S.，和McCarthy，A. A.等人（2007）Proc.Natl. Acad. Sci. U.S.A. 104，14923-14928）揭示了延伸穿过Slit-Robo界面的连续HS/肝素结合表面。基于该复合结合位点的大小，我们预测至少需要5个HS二糖单元来支持Slit-Robo信号传导。
Recognition of the secreted protein Slit by transmembrane receptors of the Robo family provides important signals in the development of the nervous system and other organs, as well as in tumour metastasis and angiogenesis. Heparan sulphate (HS) proteoglycans serve as essential coreceptors in Slit-Robo signalling. Previous studies have shown that the second leucine-rich repeat domain of Slit, D2, binds to the N-terminal immunoglobulin-like domains of Robo, IG1-2. Here we present two crystal structures of Drosophila Robo IG1-2, one of which contains a bound heparin-derived oligosaccharide. Using structure-based mutagenesis of a Robo IG1-5 construct we identified key Slit binding residues (Thr74, Phe114, Arg117) forming a conserved patch on the surface of IG1; mutation of similarly conserved residues in IG2 had no effect on Slit binding. Mutation of conserved basic residues in IG1 (Lys69, Arg117, Lys122, Lys123), but not in IG2, reduced binding of Robo IG1-5 to heparin, in full agreement with the Robo-heparin co-crystal structure. Our collective results, together with a recent crystal structure of a minimal human Slit-Robo complex (Morlot, C., Thielens, N. M., Ravelli, R. B., Hemrika, W., Romijn, R. A., Gros, P., Cusack, S., and McCarthy, A. A. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14923-14928), reveal a contiguous HS/heparin binding surface extending across the Slit-Robo interface. Based on the size of this composite binding site, we predict that at least five HS disaccharide units are required to support Slit-Robo signalling.