Mechanism for the selective interaction of C-terminal Eps15 homology domain proteins with specific Asn-Pro-Phe-containing partners.
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.
Kieken
Fabien
F
Department of Biochemistry and Molecular Biology and Eppley Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska 68198-5870, USA.
Sharma
Mahak
M
Jovic
Marko
M
Giridharan
Sai Srinivas Panapakkam
SS
Naslavsky
Naava
N
Caplan
Steve
S
Sorgen
Paul L
PL
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry*; D011392:Proline / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; C486206:EPS15 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D010649:Phenylalanine; D001216:Asparagine; D011392:Proline; D005982:Glutathione Transferase; D008239:Lysine