In most parts of the body, nerves regenerate after injury. However, in the brain and spinal cord regeneration is very limited. One of the causes for this lack of regeneration is the presence of myelin proteins that inhibit outgrowth of neuronal projections. Three myelin-associated inhibitors of regeneration (MAIs) have been identified; myelin-associated glycoprotein (MAG), Nogo and oligodendrocyte myelin glycoprotein. These MAIs signal to neurons via receptor complexes on the neuronal cell surface. In this dissertation, molecular mechanisms of MAI signaling are described, focusing on the initial signaling events that happen at the plasma membrane. Three proteins were studied by structural biology techniques: the MAI ligand MAG, the neuronal Nogo Receptor (NgR) and an antagonist of MAG signaling via NgR; Olfactomedin-1 (Olfm1).
Apart from their role in MAI signaling, these proteins also serve various physiological functions in the nervous system. MAG is recognized as a cell adhesion molecule at the myelin-axon interface along the internode. MAG is involved in myelin formation and required for stability of myelin and axon. Moreover, it maintains the correct spacing between myelin and neuronal membranes and is engaged in axon-to-myelin signaling. NgR is a receptor for diverse plasticity-inhibiting ligands and functions in consolidating neuronal circuitry and memory. Olfm1 is a secreted protein vital for proper brain development and function.
The crystal structure of the extracellular segment of MAG is described. Structures of MAG-oligosaccharide complexes and biophysical techniques combined with site-directed mutagenesis reveal how MAG recognizes neuronal gangliosides (glycolipids). Dimers of MAG, formed by association between immunoglobulin domains four and five, were observed in different crystal forms. Site-directed mutagenesis and different solution-state techniques were used to validate this interface. Neurite outgrowth assays evaluated the role of MAG dimerization and ganglioside binding in regeneration-inhibiting signaling. Both dimerization and ganglioside binding were found to be required for the regeneration-inhibiting properties of MAG. The combination of MAG dimerization on the myelin membrane and ganglioside binding on the opposing neuronal membrane provides a mechanism for regulation of the myelin-axon spacing. As a monomer, the rod-like MAG extracellular segment has angular freedom with respect to the myelin membrane. However, it is locked in a specific orientation by dimerization and ganglioside binding, maintaining the myelin-axon spacing like a molecular leaf spring.
Crystal structures of the leucine-rich repeat (LRR) domain of NgR were solved, for the first time with the correct disulfide structure. An extra C-terminal loop is formed, which is important for co-receptor binding, but appears mostly flexible in the absence of stabilizing interactions. An extensive dimerization interface was observed in different crystal forms, which we argue represents a previously-reported NgR self-interaction on the neuronal cell surface.
Olfm1 is shown to form disulfide-linked tetramers. Limited proteolysis was combined with X-ray crystallography to determine the structure of a double olfactomedin (Olf) domain and coiled coil. We show that this Olf domain is stabilized by calcium and use a combination of techniques to provide the architecture of the full-length tetramer. The V-shaped dimer-of-dimers architecture suggests a role for Olfm1 in receptor clustering at cell surfaces.