AbstractThe cation-independent mannose 6-phosphate/ Insulin-like growth factor-2 receptor (CI-MPR/ IGF2R) is a ~300 kDa transmembrane glycoprotein that is critical for intracellular protein trafficking, lysosome biogenesis and regulation of cell growth. The extracellular region consists of fifteen domains homologous to one another including mannose 6-phosphate (M6P) binding domains (D) 3, 5, 9 and 15, and IGF2 binding domain 11. To date, high-resolution structures have been determined for human D1-5 and D11-14. Although low resolution cryoEM structures of bovine CI-MPR have recently been determined at pH 4.5 and 7.4, a structure of the full extracellular region of human CI-MPR has yet to be determined.
This thesis details structural studies on the central, uncharacterised region of human CI-MPR with particular focus on the elusive, specific and high-affinity M6P binding domain, D9. A modular approach has resulted in crystal structures of human CI-MPR D8, D9-10 and D7-11. D9-10 forms a rigid homodimer stabilised by a bridging N-linked glycan and maintained in D7-11, whereby two penta-domains intertwine to form a dimeric helical-type coil. Remarkably the D7-11 structure closely matches an IGF2 bound state of the receptor, suggesting this may be an intrinsically stable conformation at neutral pH. Interdomain clusters of histidine and proline residues at the D9-10 and D11-12 interfaces may impart receptor rigidity and play a role in cargo dissociation and structural rearrangement at low pH.
A parallel project took an iterative, structure-based approach to engineer a synthetic lectin. The hydrophobic IGF2 binding site of CI-MPR D11 was mutated by site-directed mutagenesis to resemble the positively charged M6P binding sites of D3 and 9. Following preparation, D11 mutants were screened by 1H-15N HSQC NMR for binding to monosaccharides. Although chemical shift perturbations were observed following addition of M6P, further work is required to validate these preliminary results.
|Date of Award||21 Jan 2021|
|Supervisor||Matthew P Crump (Supervisor)|