Charge Effects in the Selection of NPF Motifs by the EH Domain of EHD1

The Eps15 homology (EH) domain is found in proteins associated with endocytosis and vesicle trafficking. EH domains bind to their target proteins through an asparagine-proline-phenylalanine (NPF) motif. We have measured the interaction energetics of the EH domain from EHD1 with peptides derived from two of its binding partners Rabenosyn-5 (Ac-GPSLNPFDEED-NH2) and Rab11-Fip2 (Ac-YESTNPFTAK-NH2). Heteronuclear single quantum coherence (HSQC) spectroscopy shows that both peptides bind in the canonical binding pocket of EHD1 EH and induce identical structural changes, yet the affinity of the negatively charged Ac-GPSLNPFDEED-NH2 (K-d = 8 x 10(5) M-1) is tighter by 2 orders of magnitude The thermodynamic profiles (Delta G, Delta H, Delta S) were measured for both peptides as a function of temperature The enthalpies of binding are essentially identical, and the difference in affinity is a consequence of the difference in entropic cost Ac-GPSLNPFDEED-NH2 binding is salt-dependent, demonstrating an electrostatic component to the interaction, whereas Ac-YESTNPFTAK-NH2 binding is independent of salt Successive replacement of acidic residues in Ac-GPSLNPFDEED-NH2 with neutral residues showed that all are important Lysine side chains in EH D I EH create a region of strong positive surface potential near the NIT binding pocket Contributions by lysine epsilon-ammo groups to complex formation with Ac-GPSLNPFDEED-NH2 was shown using direct-observe N-15 NMR spectroscopy. These experiments have enabled us to define a new extended interaction motif for EHD proteins, N-P-F-[DE]-[DE]-[DE], which we have used to predict new interaction partners and hence broaden the range of cellular activities involving the El-ID proteins