Title: Structural and biophysical studies of the Drosophila melanogaster Dpr and DIP families
Description: How neurons choose appropriate synaptic partners to form functional neural circuits is not well understood. Two subfamilies of Drosophila immunoglobulin superfamily (IgSF) cell surface proteins, Dprs (defective proboscis response) and DIPs (Dpr interacting proteins) are broadly expressed in the nervous system and involved in the development of neural circuits. A qualitative interactome developed from high-throughput experiments has shown that each DIP interacts with a unique set of Dpr proteins. Neurons with distinct synaptic specificities express distinct combinations of Dprs, while a subset of their synaptic partners express the complementary DIPs. These findings are consistent with the idea that the specificity of interactions between Dprs and DIPs help to define the synaptic connectivity of the neurons in which they are expressed. Thus, it is essential to fully understand interactions between members of these two protein families. Using surface plasmon resonance (SPR), we have generated a quantitative Dpr and DIP interactome, which contained several novel features.

We determined the binding affinities of the majority of Dpr-DIP interactions, revealing binding groups that span a range of affinities and reflect DIP and Dpr phylogeny. Crystal structures of Dpr-DIP heterocomplexes were determined and used to design site-specific mutants that, along with SPR experiments, reveal the major determinants of Dpr-DIP binding specificity. Using analytical ultracentrifugation (AUC), we show that some Dpr and DIP family members form homophilic dimers as well. Multiple crystal structures of DIP homodimers reveal the molecular determinants of homophilic binding and structure-guided mutants along with AUC experiments further validated their mechanism of interaction. The existence of DIP and Dpr homodimers suggests the possibility of still-unknown mechanisms of Dprs and DIPs in neural circuit formation. Based on information derived from our crystal structures and biophysical experiments, we designed, produced, and tested Dpr and DIP proteins with altered binding properties.

Many of the structural and biophysical studies described in this thesis were undertaken to produce tools to probe Dpr and DIP function in an in vivo setting. Parallel studies utilizing many of the mutant proteins described here (and other reagents that are not described here) are underway in the Zipursky lab, and are not described herein.