Studies on structures, Dynamics and Interactions with small molecules of CNS regeneration Inhibitory components associated with Nogo-A and EphA4

Abstract

The re-growth of injured neurons in CNS (central nervous system) is largely inhibited by the non-permissive environment around, and indeed several growth inhibitors have been identified so far. My thesis is aimed to study structures, dynamics and protein-protein interactions, as well as protein-small molecule interactions for two CNS regeneration inhibitors: Nogo-A and EphA4 receptor. Intracellular Nogo-A protein level is believed to correlate with stroke, as well as other neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer?s disease. Thus, it is of great interest to understand the mechanism of how Nogo-A protein level is regulated in vivo. An E3 ubiquitin ligase WWP1 was identified to be a novel interacting partner for Nogo-A both in vitro and in vivo, and down-regulated Nogo-A protein level by initiating the ubiquitination of Nogo-A. By using CD, ITC, and NMR, we have further conducted extensive studies on all four WWP1 WW domains and their interactions with a Nogo-A peptide carrying the only PPxY motif. Moreover, the solution structure of the best-folded WW4 domain is determined, and the binding-perturbed residues were derived for both WW4 and Nogo-A (650-666) by NMR HSQC titrations. On the basis of the NMR data, the complex model is constructed by HADDOCK 2.0. This study provides rationales as well as a template for further design of molecules to intervene in the WWP1-Nogo-A interaction which may regulate the Nogo-A protein level by controlling its ubiquitination. EphA4 was proved to play key roles in the inhibition of the regeneration of injured axons, synaptic plasticity, platelet aggregation, and so on. In addition, EphA4 has unique ability to bind all ephrins including 6 A-ephrins and 3 B-ephrins. Therefore, studies of EphA4 structure, dynamics, and its interaction with ephrin ligands and small molecules will be critical in understanding mechanisms underlying the binding between Eph receptor and ephrin ligands as well as molecule design targeting disease-involved Eph receptors. Both crystal and NMR structures of free EphA4 LBD were resolved, revealing the highly dynamic property of loops that comprising the classical binding pocket. Dynamics study shows that the whole EphA4 ligand binding domain undergoes dramatic conformational exchanges on ?s-ms time scales. These results may have crucial implications in understanding why EphA4 owns a unique ability to bind all 9 ephrins. The results with EphA4 dynamics may also help to design and optimize small molecule agonists and antagonists with high affinity and specificity for EphA4. The crystal structure of the EphA4-ephrin-B2 complex was also determined and an additional interaction surface was identified which will enhance the affinity and specificity of the interclass binding. These findings contribute to our understanding of the distinctive binding determinants that characterize selectivity versus promiscuity of Eph receptor-ephrin interactions and suggest that diverse strategies may be needed to design antagonists for effectively disrupting different Eph-ephrin complexes. The first two small molecules which antagonize ephrin-induced effects on EphA4-expressing cells were also presented in our work. Their binding with EphA4 LBD were studied by ITC, NMR and computer docking. Our results demonstrate that the high-affinity ephrin-binding pocket of the Eph receptors is amenable to targeting with small molecule antagonists and suggest avenues for further optimization.