Structural And Functional Research Of Eukaryotic Translation Initiation Factor 5A And Structural Insights Into The Substrate Tunnel Of Saccharomyces Cerevisiae Carbonic Anhydrase Nce103

(I) The protein eIF-5A is a highly conserved eukaryotic translation initiation factor (eIF) which has been found in eukaryotes and archaea. Biochemical and molecular studies revealed that eIF-5A is the sole protein which has been identified to contain the modified amino acid residue hypusine (Nε-(4-amino-2-hydroxybutyl) lysine). eIF-5A was first purified and identified from immature red blood cells. However, different from the classic translation initiation factors, eIF-5A is not essential for the global protein synthesis but might be involved in mRNA translocation across the nuclear envelope. The hypusinated yeast eIF-5A was recently found to promote translation elongation. Moreover, hypusine of the yeast eIF-5A has been found to be required for the sequence-specific interaction with RNA. To help clarify these diverse and even somewhat controversial functions, seven structures of eIF-5A from various organisms have been solved. They all share a similar overall structure of two domains, both of which resemble the nucleic acid binding fold.The plant Arabidopsis thaliana genome encodes three isoforms of eIF-5A: AteIF-5A1, A2 and A3. As the best investigated one, AteIF-5A2 has been found to play a crucial role in plant growth and development by controlling cell proliferation and senescence. Here we report the crystal structure of AteIF-5A2 from Arabidopsis thaliana at 2.3 A resolution, with two subunits in an asymmetry unit, each of which contains two domains linked by a flexible hinge. Distinct from the previous dimeric homolgs, the two molecules of AteIF-5A2 stand in parallel to form a dimer with a two-fold rotation axis, resulting in an enlarged positively charged groove around the hypusination site, which may facilitate the specific binding of nucleic acids. Moreover, the highly conserved residues at the N-terminal interface suggest a unique dimerization pattern of plant eIF5As.The hypusination modification of eIF-5A is made by two sequential reactions catalyzed by deoxyhypusine synthase (EC 1.1.1.249) and deoxyhypusine hydroxylase (EC 1.14.99.29). Deoxyhypusine synthase (DHS) catalyzes the first reaction, which converts lysine to deoxyhypusine [Nε-(4-aminobutyl) lysine] by a NAD-dependent transfer of the butylamine moiety of spermidine. In order to decipher the molecular mechanism of this reaction, four structures of DHS have been solved. In all structures, DHS forms a homotetramer, with the substrate binding sites at the interface of two monomers, and the N-terminal motif as a guard to control the enzymatic activity.We report the crystal structure of DHS in the budding yeast Sachomyces cerevisae at 2.8 A resolution, with two subunits and two cofactors NAD+in an asymmetry unit. Moreover, based on the crystal structures of Dysl and Hyp2, we propose an interface between the two proteins, by generating a putative three-dimensional model of Dys1 in complex with Hyp2 with the program HADDOCK. (Ⅱ) The carbonic anhydrases (CAs) are involved in inorganic carbon utilization. They have been classified into six families from the evolutionary and structural points of view:α-,β-,γ-,δ-,ε-,ζ-CAs. P-CAs presents in higher plants, algae and prokaryotes. The yeast Saccharomyces cerevisiae encodes a single copy ofβ-CA Nce103/YNL036W.We determined the crystal structure of Nce103 in complex with a substrate analog at 2.04 A resolution. It assembles as a homodimer, with the active site located at the interface between two monomers. At the bottom of the substrate pocket, a zinc ion is coordinated by the three highly conserved residues Cys57, His 112 and Cys 115 in addition to a water molecule. Residues Asp59, Arg61, Glyl11, Leu 102, Val80, Phe75 and Phe97 form a tunnel to the bottom of the active site which is occupied by a molecule of the substrate analog acetate. The quaternary structure of Nce 103 resembles the typical plant type P-CAs of known structure, with an N-terminal arm indispensable for the enzymatic activity. Comparative structure analysis enables us to draw a possible tunnel for the substrate to access the active site which is located at the bottom of a funnel-shaped substrate pocket. Furthermore, activity assays of the full length and two truncated versions of Nce103 indicated that the N-terminal arm is indispensable.