Determinants of Rab5 interaction with the N terminus of early endosome antigen 1
Graduate School of Biomedical Sciences; Program in Molecular Medicine
Medical Subject Headings
Amino Acid Sequence; Binding Sites; Membrane Proteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Binding; Sequence Homology, Amino Acid; Surface Plasmon Resonance; Vesicular Transport Proteins; Zinc Fingers; rab5 GTP-Binding Proteins
Life Sciences | Medicine and Health Sciences
The Rab5 effector early endosome antigen 1 (EEA1) is a parallel coiled coil homodimer with an N-terminal C(2)H(2) Zn(2+) 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 C(2)H(2) Zn(2+) finger is both essential and sufficient for the N-terminal interaction with Rab5. Although the heptad repeat C-terminal to the C(2)H(2) Zn(2+) 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 C(2)H(2) Zn(2+) finger play a critical role in the interaction with Rab5. Although the homologous C(2)H(2) Zn(2+) finger of the Rab5 effector Rabenosyn binds to Rab5 with comparable affinity, the analogous C(2)H(2) Zn(2+) 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 C(2)H(2) Zn(2+) 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 C(2)H(2) Zn(2+) finger of EEA1.
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Citation: J Biol Chem. 2003 Mar 7;278(10):8494-500. Epub 2002 Dec 19. Link to article on publisher's site
The Journal of biological chemistry
Merithew, Eric Lee; Stone, Craig; Sudharshan, Eathiraj; and Lambright, David G., "Determinants of Rab5 interaction with the N terminus of early endosome antigen 1" (2002). GSBS Student Publications. 859.