Solution structure of the Reps1 EH domain and characterization of its binding to NPF target sequences

The recently described EH domain recognizes proteins containing Asn-Pro-Phe (NPF) sequences. Using nuclear magnetic resonance (NMR) data, we determine d the solution structure of the EE-I domain from the Reps1 protein and char acterized its binding to linear and cyclic peptides derived from a navel ta rgeting protein. The structure calculation included 1143 distance restraint s and 122 angle restraints and resulted in structures with a root-mean-squa re deviation of 0.40 +/- 0.05 Angstrom for backbone atoms of superimposed s econdary structural elements. The structure comprises two helix-loop-helix motifs characteristic of EF-hand domains. Titration data with NPF-containin g peptides showed evidence of intermediate exchange on the NMR chemical shi ft time scale, which required an analysis that includes curve fitting to ob tain accurate equilibrium constants and dissociation rate constants. The cy clic and linear peptides bound with similar affinities (Kd = 65 +/- 17 and 46 +/- 14 muM, respectively) and Is the same hydrophobic pocket formed betw een helices B and C. The cyclic peptide formed a complex that dissociated m ore slowly (k(off) = 440 +/- 110 s(-1)) than the linear peptide (k(off) = 1 800 +/- 250 s(-1)), but had little change in affinity because of the slower rate of association of the cyclic peptide. In addition, we characterized b inding to a peptide containing a DPF sequence (Kd = 0.5 +/- 0.2 mM). The ch aracterization of binding between the Reps1 EH domain and its target protei ns provides information about their role in endocytosis.