NMR study of the human NCK2 SH3 domains structure determination, binding diversity, folding and amyloidogenesis 7

164 Chapter 7 Conclusions and future work 7.1 Conclusions Nck2 is an adaptor protein responsible for transmitting signals from transmembrane receptors including EGF receptor and ephrin

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Chapter 7 Conclusions and future work

7.1 Conclusions

Nck2 is an adaptor protein responsible for transmitting signals from transmembrane receptors including EGF receptor and ephrin-B to downstream effectors controlling cytoskeletonal dynamics and remodeling Nck2 contains three SH3 domains which are responsible for recruiting various effector proteins The main objectives of the project are to determine structures, dynamics, binding specificities, folding properties and amyloidogenesis of all three human Nck2 SH3 domains by use of molecular biology techniques, NMR spectroscopy and other biophysical methods Firstly, the NMR structures of two SH3 domains were successfully determined and the molecular mechanism for their binding to a large set of peptide ligands was defined Secondly, for the first time, the NMR evidence was obtained to show that a non-native helical conformation was highly-populated in a partially-folded SH3 domain Moreover, a 4-residue region was identified to be critical for transforming the helical state to all-β native state Thirdly, the mechanism for amyloidogenesis of SH3 mutants was investigated and finally, Apllp, a protein for establishing long-term memory, was demonstrated to be intrinsically unstructured but fully active This study not only sheds light on the structural basis for Nck2-involved signaling pathways, but also contributes to our fundamental understanding of protein folding and amyloidogenesis underlying human neurodegenerative diseases

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7.2 Future work

1) Structure determinations and study of binding diversity are carried out for both SH3-2 and SH3-3 domain in this project However, whether the hydrogen bond network is involved in the binding between SH3 domains and proline-rich peptides is still need to be answered Specific NMR experiments can be done to investigate the hydrogen bond connections in future

2) Complex dynamics study should also be carried our either for complex system in which peptide is connected by poly-GS link region with N/C terminal of SH3 domain

or the one in which no covalent bond exists between SH3 and proline-rich peptides The dynamic information of loops and 310-helix in the complex system will give us more functional implication

3) If the solubility and stability problems of whole hNck2 protein can be overcome, it

is necessary to cocrystalize hNck2 with proteins carrying proline-rich peptides

4) Urea-induced denaturation of hNck2 SH3-1 should also be done in future