Structure of the Eps15-stonin2 complex provides a molecular explanation for EH-domain ligand specificity.
Eps15 homology (EH) domain-containing proteins play a key regulatory role in intracellular membrane trafficking and cell signalling. EH domains serve as interaction platforms for short peptide motifs comprising the residues NPF within natively unstructured regions of accessory proteins. The EH-NPF interactions described thus far are of very low affinity and specificity. Here, we identify the presynaptic endocytic sorting adaptor stonin2 as a high-affinity ligand for the second EH domain (EH2) of the clathrin accessory protein Eps15. Calorimetric data indicate that both NPF motifs within stonin2 interact with EH2 simultaneously and with sub-micromolar affinity. The solution structure of this complex reveals that the first NPF motif binds to the conserved site on the EH domain, whereas the second motif inserts into a novel hydrophobic pocket. Our data show how combination of two EH-attachment sites provides a means for modulating specificity and allows discrimination from a large pool of potential binding partners containing NPF motifs.
Rumpf
Julia
J
Department of Biomolecular Mechanisms, Max-Planck-Institute for Medical Research, Heidelberg, Germany.
Simon
Bernd
B
Jung
Nadja
N
Maritzen
Tanja
T
Haucke
Volker
V
Sattler
Michael
M
Groemping
Yvonne
Y
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000378:metabolism
D002135:Calcium-Binding Proteins; D002966:Clathrin; C526966:Eps15 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008565:Membrane Proteins; D010750:Phosphoproteins; D033921:Vesicular Transport Proteins