Fukuhara Noémi, Howitt Jason A, Hussain Sadaf-Ahmahni, Hohenester Erhard
Department of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom.
J Biol Chem. 2008 Jun 6;283(23):16226-34. doi: 10.1074/jbc.M800688200. Epub 2008 Mar 20.
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 tumor metastasis and angiogenesis. Heparan sulfate (HS) proteoglycans serve as essential co-receptors in Slit-Robo signaling. Previous studies have shown that the second leucinerich 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 (Thr-74, Phe-114, Arg-117) 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 (Lys-69, Arg-117, Lys-122, Lys-123), 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 signaling.
Robo家族的跨膜受体对分泌蛋白Slit的识别在神经系统和其他器官的发育以及肿瘤转移和血管生成过程中提供重要信号。硫酸乙酰肝素（HS）蛋白聚糖是Slit-Robo信号传导中必不可少的共受体。先前的研究表明，Slit的第二个富含亮氨酸重复结构域D2与Robo的N端免疫球蛋白样结构域IG1-2结合。在此，我们展示了果蝇Robo IG1-2的两种晶体结构，其中一种含有结合的肝素衍生寡糖。通过对Robo IG1-5构建体进行基于结构的诱变，我们确定了在IG1表面形成保守区域的关键Slit结合残基（Thr-74、Phe-114、Arg-117）；IG2中类似保守残基的突变对Slit结合没有影响。IG1中保守碱性残基（Lys-69、Arg-117、Lys-122、Lys-123）而非IG2中的突变，降低了Robo IG1-5与肝素的结合，这与Robo-肝素共晶体结构完全一致。我们的总体结果，连同最近最小化的人类Slit-Robo复合物的晶体结构（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），揭示了一个跨越Slit-Robo界面的连续HS/肝素结合表面。基于这个复合结合位点的大小，我们预测至少需要五个HS二糖单元来支持Slit-Robo信号传导。
