Abstract: There is a clear need to understand the molecular mechanism for interactions with the Wnt signaling pathway. In this project, we will use computational and experimental structural biology platforms to study interactions modulating Wnt signaling and also establish a focal point in the project for biophysical investigations in collaboration with other partners. Using approaches like that proposed here, we have already identified, and partly validated, a new mode of regulating Dishevelled molecules via the Kelch-like protein 12 (KLHL12), a regulator of the (BTB-CUL3-RBX1) E3 ubiquitin ligase complex, which is known to target Dishevelled for ubiquitin mediated degradation. A computational screen of the human interactome identified that the KLHL12/Dishevelled-3 interaction is likely mediated by a short, proline-rich, linear motif. Such motifs are known to play pivotal roles in signal transduction and most other biological processes. Initial tests with the two-hybrid system show that a minimal PGxP pattern (found in Dishevelleds and several other proteins) is responsible for recognition by KLHL12. Our first aim is to extend this finding using experimental biophysical approaches to assess the strength and nature of the interaction and thus better understand the role of KLHL12 in Wnt signaling. We will address how PGxP mediates the interaction and investigate the relationship between KLHL12 and Wnt signaling, particularly in regard to its other targeted molecules (e.g. Dopamine receptors). More generally, we will extend our established computational approach to rapidly model interactions by homology to study the entire range of interactions within the Wnt pathway, with an initial focus on Wnt/ receptor specificity. By exploiting the available crystal structures, known pathway members in multiple species, and interactions between them, we will predict, on the basis of the fit between each ligand/receptor pair to each known structure, the likelihood that the proteins can interact. Promising candidate interactions and interaction hotspots will then be passed to collaborators (in particular projects A4, A5 and A6) for additional investigations. For promising candidates, we will also express and purify interacting proteins or domains from using the already established protein production facility in order to better characterise the interaction specificity using biophysical and structural methods. Ultimately, we believe that these will form a set of proteins and interactions whereby manipulations can aid Synthetic and Systems Biology approaches to probe Wnt function. As an overarching aim in this project, we will establish a focal point for connecting computational and experimental investigations to dissect interaction mechanisms. This technology will be available to study any interactions of interest to or arising from members in this consortium.