Mechanism for the selective interaction of C-terminal Eps15 Homology Domain Proteins With Specific Asn-Pro-Phe-containing partners

Fabien Kieken, Mahak Sharma, Marko Jović, Sai Srinivas Panapakkam Giridharan, Naava Naslavsky, Steve Caplan, Paul L. Sorgen

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

Epidermal growth factor receptor tyrosine kinase substrate 15 (Eps15) homology (EH)-domain proteins can be divided into two classes: those with an N-terminal EH-domain(s), and the C-terminal Eps15 homology domain-containing proteins (EHDs). Whereas many N-terminal EH-domain proteins regulate internalization events, the best characterized C-terminal EHD, EHD1, regulates endocytic recycling. Because EH-domains interact with the tripeptide Asn-Pro-Phe (NPF), it is of critical importance to elucidate the molecular mechanisms that allow EHD1 and its paralogs to interact selectively with a subset of the hundreds of NPF-containing proteins expressed in mammalian cells. Here, we capitalize on our findings that C-terminal EH-domains possess highly positively charged interaction surfaces and that many NPF-containing proteins that interact with C-terminal (but not N-terminal) EH-domains are followed by acidic residues. Using the recently identified EHD1 interaction partner molecule interacting with CasL (MICAL)-Like 1 (MICAL-L1) as a model, we have demonstrated that only the first of its two NPF motifs is required for EHD1 binding. Because only this first NPF is followed by acidic residues, we have utilized glutathione S-transferase pulldowns, two-hybrid analysis, and NMR to demonstrate that the flanking acidic residues "fine tune" the binding affinity to EHD1. Indeed, our NMR solution structure of the EHD1 EH-domain in complex with the MICAL-L1 NPFEEEEED peptide indicates that the first two flanking Glu residues lie in a position favorable to form salt bridges with Lys residues within the EH-domain. Our data provide a novel explanation for the selective interaction of C-terminal EH-domains with specific NPF-containing proteins and allow for the prediction of new interaction partners with C-terminal EHDs.

Original languageEnglish (US)
Pages (from-to)8687-8694
Number of pages8
JournalJournal of Biological Chemistry
Volume285
Issue number12
DOIs
StatePublished - Mar 19 2010
Externally publishedYes

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Electrohydrodynamics
Proteins
Glutathione Transferase
Epidermal Growth Factor Receptor
Protein-Tyrosine Kinases
Salts
Nuclear magnetic resonance
Peptides
Molecules
Protein Domains
Recycling
Cells
Substrates

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

Mechanism for the selective interaction of C-terminal Eps15 Homology Domain Proteins With Specific Asn-Pro-Phe-containing partners. / Kieken, Fabien; Sharma, Mahak; Jović, Marko; Giridharan, Sai Srinivas Panapakkam; Naslavsky, Naava; Caplan, Steve; Sorgen, Paul L.

In: Journal of Biological Chemistry, Vol. 285, No. 12, 19.03.2010, p. 8687-8694.

Research output: Contribution to journalArticle

Kieken, Fabien ; Sharma, Mahak ; Jović, Marko ; Giridharan, Sai Srinivas Panapakkam ; Naslavsky, Naava ; Caplan, Steve ; Sorgen, Paul L. / Mechanism for the selective interaction of C-terminal Eps15 Homology Domain Proteins With Specific Asn-Pro-Phe-containing partners. In: Journal of Biological Chemistry. 2010 ; Vol. 285, No. 12. pp. 8687-8694.
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AU - Giridharan, Sai Srinivas Panapakkam

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