Structural and functional studies of the hedgehog signalling pathway

Thesis

Hedgehog (Hh) morphogens play fundamental roles in development whilst dysregulation of Hh signalling leads to disease. Multiple receptors are involved in the modulation of Hh morphogens at the cell surface. Among these, the interactions of Hh ligands with glycosaminoglycan (GAG) (for example heparan or chondroitin sulphate) chains of proteoglycans in the extracellular matrix play a key role in shaping morphogen gradients and fulfil important functions in signal transduction. Several high resolution crystal structures of Sonic Hh (Shh)-GAG complexes have been determined. The interaction determinants, confirmed by binding studies and mutagenesis reveal a novel Hh site for GAG interactions, which appears to be common to all Hh proteins. This novel site is supported by a wealth of published functional data, and resides in a hot spot region previously found to be crucial for Hh receptor binding. Crystal packing analysis combined with analytical ultracentrifugation on Hh-GAG complexes suggest a potential mechanism for GAG-dependent multimerisation.

A key step in the Hh pathway is the transduction of the Hh signal into the receiving cell. The Hh signal transducer, Smoothened, is a key target drug target in the pathway with several modulators in clinical trials, despite an absence of structural data. Smoothened is required to activate all levels of Hh signalling. Recent evidence points to the conserved N-terminal ectodomain (ECD) in regulating Smo activity, from vertebrates to invertebrates. Despite the central importance of the ECD, its precise function remains elusive. A crystal structure of the ECD at 2.2 Å resolution is reported here. Structural analysis and biophysical experiments are discussed with reference to the potential function of this intriguing domain.

Authors: Whalen, D

• Publication date: 2012
• DOI: 10.5287/ora-xmgyyp2mo
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: developmental biology; smoothened; structure; protein chemistry; Hedgehog signalling; structural biology; smoothened ectodomain; macromolecular crystallography
• Subjects: High-Throughput Screening; Structural genomics; Biology (medical sciences); Developmental biology; Development (zoology); Molecular biophysics (biochemistry); Biochemistry; Crystallography; Chemical biology; Biophysics; Protein chemistry; Medical sciences; Glycobiology