Structural And Functional Study Of Prp20p From Saccharomyces Cerevisiae And Human DESR1

In this thesis, we will present the 1.9 ? crystal structure of Prp20p from Saccharomyces cerevisiae by X-ray crystallography and the solution structure of human DESR1, a CSL zinc-binding protein by multidimensional NMR spectroscopy.The main part of this thesis will be divided into three chapters.Chapter 1 is a review of nucleocytoplasmic transport which is an important cellular physiological process that Prp20p participates in and plays key roles in it. In eukaryotic cells, the different composition of the nucleus and cytoplasm is maintained by the transport of macromolecules through the nuclear pore complex (NPC). Nuclear proteins are imported into the nucleus by importins, while most of the RNAs are exported into the cytoplasm by exportins. The directionality of the transport is determined by the RanGTP gradient across the nuclear envelope. Ran in the nucleus is mainly in the GTP-bound form at high concentrations, and mostly in the GDP-bound form in the cytoplasm. Importins bind their cargo in the cytoplasm, with or without the assistance of adaptor proteins, and the cargo will be released by the binding of RanGTP to importins. On the contrary, complexes are formed in the nucleus by exportins and RanGTP and cargo, and then the complexes are dissociated in the cytosol after the hydrolysis of GTP to GDP. GTP hydrolysis requires a RanGAP (Ran GTPase-activating protein) in cytosol, while the exchange of GTP to GDP is catalyzed by a guanine nucleotide exchange factor (GEF, RCC1 in mammals and Prp20p in baker's yeast) in nuclear.In chapter 2, we will focus on the introduction of Prp20p and the experimental methods that we used and the subsequent results we obtained. Prp20p, also terms as Srm1, is the functional homolog of mammalian RCC1 (Regulator of Chromosome Condensation 1) in Saccharomyces cerevisiae, and acts as the guanine nucleotide exchange factor (GEF) for Gsp1p (Ran in mammals). Prp20p/RCC1 plays important roles throughout the whole cell cycle, particularly in nucleocytoplasmic transport and mitosis regulation. Prp20p is composed of a NLS (nuclear localization signal)-containing N-terminal tail and a typical RCC1-like domain (RLD) that contains seven RCC1 repeats. Prp20p also functions as a linker between nuclear pore complex (NPC) and chromatin and play important role in the NPC-mediated boundary activity (BA) in the gene regulation. Here we present the 1.9 ? crystal structure of Prp20p, which appears as a classical seven bladesβ-propeller as expected. From this structure, we found an additionalβ-wedge and it was proved to be an important element in the interaction between Prp20p and Gsp1p. We then built the complex model of Prp20p and Gsp1p and optimized it by molecular dynamics (MD) simulations. Besides, we surprisingly discovered that the histone binding properties of Prp20p are different to that of RCC1.Our work on the solution structure of human DESR1 will present in chapter 3. DESR1, also designated Dph3, is required for the first step in the posttranslational modification of translation elongation factor 2 (eEF-2) at H715 (H699 in yeast) that yields diphthamide, the target site for ADP ribosylation by DT and ETA. Succeeding investigation had attested to the physiological importance of Dph3 in mouse embryonic and placental development. Then we compared the 3D structure of DESR1 with its homologs in mouse (mDESR1) and yeast (KTI11), which showed the conservation and minor difference. Lastly, we discussed the classification of these CSL zinc finger-containing proteins.