| Summary: | Proteins containing the BTB domain conventionally homodimerise to perform a variety of critical biological processes and serve diverse roles such as transcriptional regulators, voltage-gated ion channels, and E3 ubiquitin ligase substrate adaptors. The molecular mechanisms for many of these proteins, particularly “atypical” BTB proteins forming higher order structures or containing tandem BTB domains, are poorly characterised. This thesis characterised the intramolecular and intermolecular protein interactions of atypical BTB proteins and how such interactions may determine function and disease, particularly of the craniofacial complex. Three complementary approaches were used to achieve the aims of this thesis and included 1) characterising the morphogenic role of the tandem BTB protein BTBD7 in 3D cell culture, 2) investigating the biophysical properties and molecular mechanisms of the tandem BTB protein class, and 3) characterising the structural, biophysical, and mechanistic alterations caused by mutations in the pentameric BTB protein KCTD15. Novel 3D cell culture models alongside several microscopy and proteomic assays revealed BTBD7 may contribute to <em>in vitro</em> cellular phenotypes consistent with tubular morphogenesis that were not observed <em>in vivo</em>, perhaps due to incomplete knockout of the gene. Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS), X-ray crystallography, and several other biophysical assays showed heterogeneity in tandem BTB domain oligomerisation and binding affinities for CUL3. Similar structural and biophysical approaches alongside analytical ultracentrifugation (AUC), differential scanning fluorimetry (DSF), and biolayer interferometry (BLI) showed that human mutations in the BTB domain of KCTD15 caused aberrant BTB domain oligomerisation and inhibited KCTD15 from binding its interaction partner transcription factor AP2. Collectively, this work enhances our understanding of molecular mechanisms of the atypical BTB proteins in normal development as well as pathobiology and provides clarity on how mutations in these proteins cause human disease.
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