Structural characterization of proteins involved in cellular signaling and trafficking

The endomembrane system is the characteristic symbol for eukaryotic cells, which provides a much more precise and high-speed performance of various cellular events. The functional integrity of individual organelle relies on specific protein trafficking to these compartments, as well as the tight regulation of local lipid turnover. The former is highly dependent on the collaborative actions of biomoelcues in the membrane trafficking nano-machinery, whereas the latter is always sensible to different extracellular stimulations and subsequently triggers the signal transduction inside the cell. The main focus of this dissertation is to unveil the biological functions and underlying molecular mechanisms of selected signaling and trafficking proteins that are involved in membrane binding and/or membrane trafficking. A unique subclass of signaling proteins called split PH domain proteins have been systematically studied in this dissertation. In the mammalian genome, there are only six split PH domain proteins, which are characterized by insertion of one or more autonomously folded protein modules. I have determined the three dimensional structure of the PHN-PDZ-PHC tandem of α-syntrophin using NMR spectroscopy. For the first time, I showed that two halves of the split PH domain fold into a canonical PH domain conformation intra- and inter-molecularly, and the PDZ insertion in the PH domain does not change the structure of the PH domain. Although completely unstructured in their isolated form, de novo folding could be observed by simply mixing of two complementary split PH domain fragments. Further biochemical studies demonstrated that the site specific insertion of PDZ domain in the middle of the PH domain creates a supramodule with distinct lipid binding properties. Structure studies of other split PH domains from phospholipase C-γ1 (PLC-γ1), PI 3-kinase enhancer (PIKE), and Rho-kinases (ROCK) further demonstrated that two halves of split PH domain fragments interact with each other to fold into canonical PH domain structure, and the domain insertion may occur in different loop regions of the domain. In addition, the supramodular nature of split PH domain together with its inserted sequence is not an exclusive property for the PH-PDZ tandem of syntrophin, but also extended to the PH-insertion tandems from PLC-γ1, PIKE, and ROCK. For PIKE and ROCK, the domain insertions also modify the lipid binding properties of their split PH domains. Whereas for PLC-γ1, whose split PH domain cannot bind to phospholipid, the split PH domain controls the orientation of the inserted SH2-SH2-SH3 tandem toward the catalytic X-Y boxes, and thus regulates the lipase activity of this enzyme. The systematic structural and functional studies of split PH domains indicate that these special tandem-arranged protein-protein interaction modules are not just simple attachments of “beads on a string”, but often represent functional supramodules with distinct structures and biological functions to satisfy the diverse requirements of an organism. This dissertation also describes regulatory mechanism of Dynein Light Chain 1 (DLC1), which functions as a dimer and plays a critical role in the molecular motor dynein-mediated protein trafficking process. With a combination of gel filtration chromatography, NMR spectroscopy, and fluorescence spectroscopy, I showed that substitution of Ser88 (which is buried in the dimer interface) with phosphorylation mimicking Glu disrupts DLC1 dimer formation, and consequently impairs its target binding property. This structural insight was further demonstrated by our cellular data showing that DLC1 phosphorylation on Ser88 by p21-activated kinase 1, promotes mammalian cell survival by regulating its interaction with Bim and Bim’s cellular stability