Proteins containing EH domains and proteins containing Asn-Pro-Phe (NPF) sequences are ubiquitously involved in fundamental molecular sorting mechanisms. Reps1, an EH domain-containing protein, and FIP2, an NPF-containing protein, are downstream targets of Ras and Ral GTPases and the EGF receptor, suggesting that they are involved in both signal transduction and vesicle-mediated trafficking. Despite the importance of the EH domain:NPF interaction in building the protein networks responsible for vesicle-mediated transport and signal transduction, the factors that regulate their association are largely unknown. Knowing the basic principles that govern their regulation will lead to strategies to control these proteins and a better understanding of their biological roles. The EH domain of Reps1 binds directly to the NPF sequence of FIP2. Using Reps1:FIP2 as model system, the objective of this proposal is to evaluate the factors that modulate EH domain:NPF interaction. EH domains bind Ca2+ using the EF-hand fold. In Aim 1, EH domain affinity for Ca2+ and Mg2+ will be measured to test a new regulatory hypothesis operating in vesicle sorting. To examine the conformational changes that accompany metal ion binding, the structures of the apo and Mg2+-bound domains will be determined using NMR and compared to the Ca2+-bound structure. FIP2 and Reps1 both oligomerize through a coiled-coil domain. In Aim 2, we will determine the structure of the FIP2 coiled-coil domain and how oligomerization governs the strength of the interaction between these proteins. To understand the mechanism of binding, the structures from a series of unbound peptides as well as an EH domain-NPF complex will be determined in Aim 3. An inhibitory peptide, designed to be stable in cell-based assays, will also be optimized and tested as a tool to understand the roles of EH domains in vesicle-mediated internalization and secretion. Ultimately, the results of these experiments will provide insight into the vesicle sorting mechanisms important for diverse processes such as fertilization, neurotransmission, and cell growth. These investigations on the EH domain:NPF interaction are also expected to advance the application of NMR in understanding molecular recognition.