Determinants of Rab5 interaction with the n terminus of early endosome antigen 1
Determinants of Rab5 interaction with the n terminus of early endosome antigen 1
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DOI：
10.1074/jbc.m211514200
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发表时间：
2003-03-07
影响因子：
4.8
通讯作者：
Lambright, DG
中科院分区：
文献类型：
--
作者：
Merithew, E;Stone, C;Lambright, DG
Rab5效应器早期内容体抗原1(EEA1)是一个平行的螺旋二聚体，其N-端为C2H-锌2+指，C-端为FYVE结构域。Rab5与EEA1的N端和C端的独立位点结合。为了进一步了解内体拴系和融合的结构决定因素，我们通过定量结合测量表征了Rab5C与EEA1的截断和定点突变的相互作用。结果表明，C2H2-Zn2+指是N-端与Rab5相互作用的必要条件和充分条件。虽然C2H2锌离子手指上的七肽重复C末端为稳定的同源二聚提供了驱动力，但它并不影响Rab5结合的亲和力或化学计量比。据预测，疏水残基聚集在C2H2锌离子手指的共同表面，在与Rab5的相互作用中起着关键作用。虽然Rab5效应物Rabenosyn的同源C2H2锌2+手指以类似的亲和力与Rab5结合，但酵母同系物Vac1的类似C2H2锌2+手指没有检测到与Rab5的相互作用，反映了关键残基的非保守取代。Rab5的内源性色氨酸荧光发生了很大的变化，伴随着与EEA1的C2H2锌2+手指的结合。这些观察结果可以用一种相互作用模式来解释，在这种模式下，位于Rab5的开关I和II区域之间界面的部分暴露的色氨酸残基位于与EEA1的C2H2锌2+指中的一簇非极性残基的疏水界面内。
The Rab5 effector early endosome antigen 1 (EEA1) is a parallel coiled coil homodimer with an N-terminal C2H2Zn2+ finger and a C-terminal FYVE domain. Rab5 binds to independent sites at the N and C terminus of EEA1. To gain further insight into the structural determinants for endosome tethering and fusion, we have characterized the interaction of Rab5C with truncation and site-specific mutants of EEA1 using quantitative binding measurements. The results demonstrate that the C2H2 Zn2+ finger is both essential and sufficient for the N-terminal interaction with Rab5. Although the heptad repeat C-terminal to the C2H2 Zn2+ finger provides the driving force for stable homodimerization, it does not influence either the affinity or stoichiometry of Rab5 binding. Hydrophobic residues predicted to cluster on a common face of the C2H2 Zn2+ finger play a critical role in the interaction with Rab5. Although the homologous C2H2 Zn2+ finger of the Rab5 effector Rabenosyn binds to Rab5 with comparable affinity, the analogous C2H2Zn2+ finger of the yeast homologue Vac1 shows no detectable interaction with Rab5, reflecting non-conservative substitutions of critical residues. Large changes in the intrinsic tryptophan fluorescence of Rab5 accompany binding to the C2H2 Zn2+ finger of EEA1. These observations can be explained by a mode of interaction in which a partially exposed tryptophan residue located at the interface between the switch I and II regions of Rab5 lies within a hydrophobic interface with a cluster of non-polar residues in the C2H2 Zn2+ finger of EEA1.