The Structural And Functional Research Of Eukaryotic AU-rich Element-binding Proteins HuR And GAPDH3

Part I. The structural and functional research of human mRNA binding protein HuRIn mammalian cells, mRNA decay requires exquisite regulation of specific cis-regulatiory sequences and trans-acting factors. Adenylate-and uridylate-rich elements (ARES) which are located in3’-untranslated regions (UTR) are the most well studied cis-regulatory element responsible for mRNA degration. The significant feature of ARE is the presence of AUUUA pentamer. Among scores of ARE-binding proteins, Hu antigen R (HuR) is the best characterizaed mRNA decay factor. HuR, one member of Hu family (the other ones are HuB, HuC and HuD), is a ubiquitously expressed-36kDa protein. It contains three RNA recognition motifs (RRM). The N-terminal tandem RRM domains (RRM1/2) recognize ARE with high affinity while the third RRM (RRM3) interacts with poly (A) and other protein ligands. HuR has been implicated as a key posttranscriptional regulator. Previous small-angle x-ray scattering (SAXS) studies on HuR suggest that the conformation of HuR RRM1/2changes when binding to RNA. However the structural details are still unclear since the lack of comparable crystal structures of HuR RRM1/2. Therefore, it is important to investigate the structure-function relationship of HuR RRM1/2.The thesis presents the structural basis for ARE recognition by HuR RRM1/2. We determined the crystal structure of HuR RRM1/2in RNA-free form, revealing an open conformation with no interactions between two RRM domains. Mutagenesis analysis showed that HuR RRM1/2binds to mRNA through its β-sheets. We also solved the crystal structure of HuR RRM1/2complexed with11-base mRNA (5’-AUUUUUAUUUU-3’). This structure presents a closed state compared to the RNA-free form. HuR RRM1/2binds this target RNA via its positively charged cleft. Fluorescence polarization assays (FPA) indicate that RRM1is the primary ARE-binding domain in HuR. Combined with the results of free energy analysis, we speculate that HuR RRM1/2undergoes dramatical conformational changes during RNA binding process. And this conformational changes induce subsequent contacts between RRM2and inter-domain linker with RNA, increasing the RNA binding affinity of HuR greatly. Part Ⅱ. The structural and functional research of glyceraldehyde-3-phosphate dehydrogenase3(GAPDH3) from Saccharomyces cerevisiaeThe enzyme glyceraldehydes-3-phosphate dehydrogenase (GAPDH, EC:1.2.1.12) is one of the essential enzyme in the glycolytic pathway which is the primary pathway in the cells responsible for the production of ATP. GAPDH catalyzes the sixth step reaction which is the oxidative phosphorylation of glyceraldehydes-3-phosphate (GAP) to1,3-bisphosphoglycerate (BPG) using the cofactor NAD+. GAPDH is a ubiquitous enzyme of～37kDa that is located prominently in the cytoplasm and can shuttle between other compartments. Although GAPDH is originally thought of as a housekeeping enzyme and an important enzyme for glycolysis specially, numerous studies have described a range of new functions for this protein. These diverse roles include DNA repair, cell apoptosis, membrane fusion, tRNA export and mRNA stability regulation. A lot of GAPDH structures from different specises have been solved, however, the RN A recognition mechanism of GAPDH remains unclear. There are three isoforms of GAPDH in Saccharomyces cerevisiae:GAPDH1, GAPDH2and GAPDH3. Previous studies reported that only the most basic isoform of yeast GAPDH (GAPDH1) could bind poly (U). However, we showed that a less basic one (GAPDH3) also possesses poly (U) binding capacity using fluorescence polarization assays (FPA).To explore the recognition mechanism how GAPDH3binds to RNA, we determined the crystal structure of yeast GAPDH3. The overall structure of GAPDH3is similar to GAPDHs of other species and is composed of two domains:the NAD+binding domain (residues1-149) and the catalytic domain (residues150-332). GAPDH3presents as a homotetramer in solution. Enzymatic activity studies of GAPDH3showed that the apparentKNAD+is about682μM which is a little higher than the values reported for the homolgous GAPDHs. The results of FPA showed that GAPDH3can bind to mRNA (5’-AUUUAUUUAUUUA-3’) with a Kd value about18μM. FPA assays also indicated that the RNA binding activity of GAPDH3is inhibited by NAD+in a concentration-dependent manner. Further studies on the RNA recognition mechanism is currently in progress.