Determinants of Rab5 interaction with the N terminus of early endosome antigen 1.
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.
Merithew
Eric
E
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester 01605, USA.
Stone
Craig
C
Eathiraj
Sudharshan
S
Lambright
David G
DG
D000595:Amino Acid Sequence; D001665:Binding Sites; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D020349:Surface Plasmon Resonance; D033921:Vesicular Transport Proteins; D016335:Zinc Fingers; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism*
D008565:Membrane Proteins; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D020696:rab5 GTP-Binding Proteins