| Split PH domain is a unique subtype of PH domain, which is split into two halves by other protein modules. It occurs in several proteins during evolution, including scaffold protein alpha-Syntrophin, phospholipase C gamma (PLC-gamma), endosome transport machinary ESCRTII component Vps 36, neuronal PI(3)K enhancer GTPase PIKE (also named GGAP and AGAP), Rho kinase ROCK and unconventional myosin motor Myosin X. However, little is known about split PH domain prior to this thesis study. The central theme of this thesis work has been focusing on structural characterization of individual split PH domain and elucidation of the functional roles of split PH domain mediated regulation of protein localization and activities.;Chapter 1 provides an overall introduction about the background of PH domain and PH domain mediated lipid or protein interaction. From Chapter 2 to Chapter 5, I systematically characterized four split PH domains and their potential functions. In chapter 2, I characterized the structure of split PH domain from alpha-Syntrophin. We reported for the first time split PH domain adopts a canonical PH domain fold. We also showed two halves of PH domain can stably exist in solution and they undergo de novo folding when encountering each other. We also reported for the first time PDZ domain inserted into split PH domain is a weak lipid binder and it works synergistically with the split PH domain in lipid binding.;In chapter 3, I moved on to the split PH domain in Phospholipase C gamma. Again, this split PH domain also adopts a canonical PH domain fold. However, it does not contain the conventional lipid binding pocket and thus it does not serve as a lipid binding module. Our biochemical and structural data proved that the two halves of split PH domain form a tight and stable beta barrel and it is less possible to be opened. We further showed that the split PH domain directly inhibits lipase activity. In chapter 4, I presented the structure of split PH domains from PIKE, and showed the split PH domain works together with inserted nuclear localization signal (NLS) and regulate the proteins localization. In chapter 5, structure of the Myosin-X PH12 tandem was solved. Unlike other split PH domain, this split PH domain packs together with its inserted PH domain in a neck to neck pattern, leaving the lipid binding pockets on the same side and thus results in enhanced lipid binding avidity. |
