期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 278, 期 10, 页码 8494-8500出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M211514200
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资金
- NIGMS NIH HHS [GM56324] Funding Source: Medline
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.
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