Decoding receptor selection and specificity in Class 3 Semaphorins

Thesis

Robust control over axon navigation is crucial for the development of a healthy, functional nervous system, and is partially afforded by Semaphorins. Their jurisdiction is not confined to axon guidance either, with diverse contributions to synaptogenesis, dendritic spine regulation, and even vascular repair depending on the given receptor. Precise selection of receptor complexes enables targeted activation of desired signalling pathways and firm control of downstream biological outcomes, however the source of Sema receptor specificity is elusive. As such, the determinants of Sema3 recognition of Neuropilin, Plexin and auxiliary L1-IgCAM receptors were sought, through the lens of Sema3G, Nrp2 and PlxnA4/D1, in the following thesis.

Structural and biophysical examination of Sema3G homo-dimerisation and assembly with Nrp2 culminated in an unprecedented hetero-oligomeric array rich in atypical contacts and compelling insights into Sema3 receptor recognition. Further, mutagenesis of a targeted Sema loop revealed a short motif exclusive to Sema3G vital for associating with Nrp2, but not Nrp1. Manifold efforts to optimise Sema3G complexation with Nrp2/Plxn holoreceptor complexes enabled the preparation of pure samples for structural studies, but were ultimately unable to capture the elusive Plexin. Crystal structures of the first four and first six NrCAM Ig domains revealed Ig1-4 ‘horseshoe’ architectures and homo-dimerisation modes evocative of related L1-IgCAMs, however attempts to situate NrCAM in Sema3/Nrp signalling were foiled by unsuccessful Sema3F/Nrp2 and NrCAM/Nrp2 complexation, poor expression of the cognate PlxnA3 receptor and dubious structural predictions. Nonetheless, these persistent bids built the foundations for impending studies targeting co-receptor assembly with Plxns and IgCAMs.

Enriching the current understanding of class 3 Semaphorin receptor complex assembly, the following thesis demonstrates a novel mechanism of Sema-Nrp oligomerisation and bestows notable advances in decoding Nrp specificity.

Authors: Rowland, E

• DOI: 10.5287/ora-gazqb12nw
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: structural biology; neuropilin; neuroscience; cryoEM; protein crystallography; semaphorin; plexin
• Subjects: Protein-protein interactions; Biochemistry