Leppänen Veli-Matti, Bespalov Maxim M, Runeberg-Roos Pia, Puurand Ulo, Merits Andres, Saarma Mart, Goldman Adrian
Institute of Biotechnology, University of Helsinki, Helsinki, Finland.
EMBO J. 2004 Apr 7;23(7):1452-62. doi: 10.1038/sj.emboj.7600174. Epub 2004 Mar 25.
Glial cell line-derived neurotrophic factor (GDNF) binds to the GDNF family co-receptor alpha1 (GFRalpha1) and activates RET receptor tyrosine kinase. GFRalpha1 has a putative domain structure of three homologous cysteine-rich domains, where domains 2 and 3 make up a central domain responsible for GDNF binding. We report here the 1.8 A crystal structure of GFRalpha1 domain 3 showing a new protein fold. It is an all-alpha five-helix bundle with five disulfide bridges. The structure was used to model the homologous domain 2, the other half of the GDNF-binding fragment, and to construct the first structural model of the GDNF-GFRalpha1 interaction. Using site-directed mutagenesis, we identified closely spaced residues, Phe213, Arg224, Arg225 and Ile229, comprising a putative GDNF-binding surface. Mutating each one of them had slightly different effects on GDNF binding and RET phosphorylation. In addition, the R217E mutant bound GDNF equally well in the presence and absence of RET. Arg217 may thus be involved in the allosteric properties of GFRalpha1 or in binding RET.
胶质细胞系源性神经营养因子（GDNF）与GDNF家族共同受体α1（GFRα1）结合并激活RET受体酪氨酸激酶。GFRα1具有由三个同源富含半胱氨酸结构域组成的假定结构域结构，其中结构域2和3构成负责GDNF结合的中央结构域。我们在此报告GFRα1结构域3的1.8埃晶体结构，显示出一种新的蛋白质折叠。它是一个具有五个二硫键的全α五螺旋束。该结构用于模拟同源结构域2（GDNF结合片段的另一半）并构建GDNF - GFRα1相互作用的第一个结构模型。使用定点诱变，我们鉴定出紧密间隔的残基苯丙氨酸213、精氨酸224、精氨酸225和异亮氨酸229，它们构成一个假定的GDNF结合表面。对它们中的每一个进行突变对GDNF结合和RET磷酸化有略微不同的影响。此外，R217E突变体在存在和不存在RET的情况下与GDNF结合的效果相同。因此，精氨酸217可能参与GFRα1的变构特性或与RET结合。
