Investigating the Role of Lipids and Small-Molecule Ligands in Hedgehog Signalling Using Molecular Dynamics Simulations

The Hedgehog (HH) signalling pathway orchestrates cellular patterning during embryonic development and adult stem cell programming. Sonic hedgehog (SHH) is the principle morphogenic ligand responsible for paracrine induction of HH signalling. Pathway dysregulation results in multiple cancer pathologies and ineffective tissue partitioning, leading to severe developmental diseases. SHH is directly conjugated to two lipids; a N-terminal palmitate and a C-terminal cholesterol moiety. Unique bilipidation of SHH occurs within the endoplasmic reticulum via autocatalytic cholesterylation and Hedgehog acyl-transferase (HHAT) catalysed palmitoylation steps, after which SHH is released from cells. SHH binds to and inhibits the tumour suppressor protein Patched1 (PTCH1) to relieve PTCH1 mediated inhibition of the Class F G-protein coupled receptor (GPCR) Smoothened (SMO). Small-molecule ligands, notably cholesterol and palmitate, are inextricably linked to HH signalling, inducing a perplexing range of signalling outputs. This thesis outlines a conveyor-belt tour of HH membrane proteins for SHH processing, engagement, and pathway initiation. Molecular dynamics (MD) simulations of PTCH1, SMO and HHAT are employed to rationalise the, often counter-inductive, roles of small-molecules in HH signalling. Simulations are coupled with structural and biochemical data, demonstrating how the SHH-cholesterol contributes to PTCH1 inhibition. Simulations and free energy calculations are used to provide energetic contextualisation of PTCH1 cholesterol transport models and predict ion-coupling stoichiometries. Additionally, we find simulation and biochemical evidence for cooperativity between cholesterol binding sites on SMO. Finally, simulations are used to assist HHAT structural interpretation, elucidating roles for residues in solvent gating, product exit and priming HHAT catalysis. Bioinformatics and simulations were also employed to assess structural divergence between HHAT and related homologues. Biological insights were assisted by concurrent development of computational methodologies for characterisation of protein-lipid interactions, their affinities and integration with experimental data. Thus, this thesis spotlights the diverse roles lipids and small-molecules play in HH pathway regulation - from activators to allosteric modulators and inhibitors, illuminating new avenues for therapeutic intervention.