Structure and function studies of ciliary neurotrophic factor (CNTF) and its receptor complexes.

摘要

The binding of most signaling molecules to their receptors initiates a series of intracellular reactions that regulate virtually all aspects of cell behavior, including metabolism, proliferation, differentiation, or even programmed cell death (apoptosis). Ligand induced receptor oligomerization has been proposed as the first step and key mechanism of a cascade of signal transduction to be initiated for a family of helical cytokines. To study and elucidate the oligomerization mechanism, the extracellular portion of receptor molecules can be analyzed for their structures and interactions by structural biology techniques: NMR or X-ray crystallography.; Helical cytokines are a group of signal molecules that regulate the survival, growth and differentiation of cells in the immune, haematopoietic and neuropoietic system. This thesis predominately investigates one of the helical cytokine, Ciliary Neurotrophic Factor (CNTF) and the components of its receptor complex. Biophysical and biochemical techniques were employed. As the receptor proteins are highly modular in structure, this project took the approach of studying individual domains and examining the interactions of that domain with the ligand and/or other domain of the other receptor.; To examine the structure and function of CNTF and its modular receptor units, soluble protein or protein domain(s) have to be obtained. Different expression and purification systems have been extensively tried for CNTF and its different modular unit of CNTF a receptor (CNTFR) and gp130 beta receptor. With the suitable protein domain sample available, solution structure of C-terminal binding domain (BC) of cytokine binding domain (CBD) of CNTFR has been determined by NMR. Further investigation of its interaction with gp130 BC has also be elucidated by a combination of NMR and biochemical techniques.; NMR structure of CNTFR BC indicated that amino acid residue D269 forms a salt bridge with residue R220 and T268 forms a hydrogen bond with R221. These interaction help to stabilize the 3D structure of CNTFR BC. Mutations of D269A and T268A will destabilize the CNTFR BC structure, therefore mutants D269A and T268A of CNTFR BC bind more weakly with gp130 as shown in ELISA assay. The mutation of the two residues would also increase the net positive charge of the CNTFR BC domain in this region, possibly inducing electrostatic repulsion with gp130.; The technical breakthrough of using maltose binding protein as tagged to obtain soluble protein domain for protein crystallography and using sarkosyl to solubilze protein expressed normally in inclusion bodies can also extend the work in structural and functional studies of CNTF and its receptor complex.; The presented work has provided insights into the structure and receptor interactions of CNTF and help to explain part of the mechanism of CNTF receptor complex, more detailed information help to explain the interaction will be available with the structure determination done of another domain by crystallography from our collaborator.