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dc.contributor.authorMerithew, Eric Lee
dc.contributor.authorStone, Craig
dc.contributor.authorSudharshan, Eathiraj
dc.contributor.authorLambright, David G.
dc.date2022-08-11T08:09:02.000
dc.date.accessioned2022-08-23T16:15:54Z
dc.date.available2022-08-23T16:15:54Z
dc.date.issued2002-12-21
dc.date.submitted2008-11-21
dc.identifier.citationJ Biol Chem. 2003 Mar 7;278(10):8494-500. Epub 2002 Dec 19. <a href="http://dx.doi.org/10.1074/jbc.M211514200 ">Link to article on publisher's site</a>
dc.identifier.issn0021-9258 (Print)
dc.identifier.doi10.1074/jbc.M211514200
dc.identifier.pmid12493736
dc.identifier.urihttp://hdl.handle.net/20.500.14038/34202
dc.description.abstractThe 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.
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12493736&dopt=Abstract">Link to article in PubMed</a>
dc.relation.urlhttp://dx.doi.org/10.1074/jbc.M211514200
dc.subjectAmino 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
dc.subjectLife Sciences
dc.subjectMedicine and Health Sciences
dc.titleDeterminants of Rab5 interaction with the N terminus of early endosome antigen 1
dc.typeJournal Article
dc.source.journaltitleThe Journal of biological chemistry
dc.source.volume278
dc.source.issue10
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_sp/859
dc.identifier.contextkey670705
html.description.abstract<p>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.</p>
dc.identifier.submissionpathgsbs_sp/859
dc.contributor.departmentProgram in Molecular Medicine
dc.contributor.departmentGraduate School of Biomedical Sciences
dc.source.pages8494-500


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