| Hdal is a typical class ? histone deacetylase from Saccharomyces cerevisiae.Two related protein Hda2 and Hda3 are essential for the activity of Hdal. Together,they form the yeast Hdal HDAC complex, which specifically deacetylates H2B and H3, with Hdal homo-dimer as the catalytic subunit. Hdal contains a N-terminal catalytic HDAC domain and a C-terminal ARB2 domain. In regard to the HDAC domain, there are a large number of structural and functional studies, with the catalytic mechanism clearly elucidated. The ARB2 domain is defined by sequence homology to the Arb2 protein, which is a subunit of the recently found Argonaute complex from Schizosaccharomyces pombe and participates in the RNA-induced transcriptional silencing. Therefore, structural and functional studies on the ARB2 domain contribute to the further understanding of the deacylation process of Hdal and the recognition of the Arb2 protein family as well.In this study, we determine the crystal structure of the Hdal-ARB2 domain at a resolution of 2.86 A. The ARB2 domain is composed of a core ?/? sandwich architecture and a protruding arm region. Although there is one ARB2 domain molecule observed in each asymmetric unit, further examination of symmetry-related molecules indicates that they can form a homodimer by the protruding arm region. Two ARB2 domain molecules assemble as an inverse "V"shape via the hydrophobic interaction among the residues located in the protruding arm region, which is demonstrated by mutant experiment and size exclusion chromatography assay. The ARB2 domain shows structural resemblance to the ?/?fold hydrolases. However, the protruding arm regions are totally different between these enzymes, and the catalytic triplets for the hydrolysis are missing in ARB2 domain. Thus, ARB2 domain does not possess the hydrolytic activity. The pull-down and ITC results reveal that the ARB2 domain possesses the histone binding ability,recognizing both the H2A/H2B and H3/H4. Perturbation of the dimer interface abolishes the histone binding ability of the ARB2 domain, indicating that the unique dimer architecture of the ARB2 domain coincides with the function for anchoring to histone. The mutant experiment and ITC results show that the groove formed by ARB2 dimer is employed to bind to histone.Glyceraldehyde-3-phosphate dehydrogenase is an essential enzyme in the glycolytic pathway. It catalyses the oxidative phosphorylation of glyceraldehyde-3-phosphate (GAP) to 1,3-bisphosphoglycerate (BPG) in the presence of the cofactor nicotinamide adenine dinucleotide (NAD+). A number of studies have indicated that GAPDH is not merely a simple glycolytic protein but rather has multiple roles in DNA replication and repair, apoptosis initiation,localization at the cell surface, binding to cellular molecules, membrane fusion and transport, tRNA export, and mRNA stability regulation. The ability to regulate mRNA stability comes from the binding to AU-rich element (ARE). However, the RNA recognition mechanism of GAPDH is still poorly understood.In this study, we demonstrate that Saccharomyces cerevisiae GAPDH3 can bind to an mRNA sequence containing three iterations of AUUUA, with the similar binding ability to the same length polyA, by fluorescence polarization assay (FPA).However, it can not bind to the same length polyU and polyC. The crystal structure of apo-form GAPDH3 is refined at 2.49 A resolution, with one molecule in each asymmetric unit. GAPDH3 exists as a tetramer in solution, indicated by size exclusion chromatography assay. By comparing to homologous structures and crystallographic symmetry operation, the tetrameric state of GAPDH3 is indeed obtained. By analyzing the surface electrostatic potential we find that there are two consecutive positively charged regions located on each side of the tetramer, and each region contains one NAD+ binding site. FPA result reveals that NAD+ inhibit the RNA binding ability of GAPDH3 to RNA. Furthermore, via structural analysis of GAPDH3 complex with NAD+, we find that the NAD+ molecule inserts the pyrimidine ring into a narrow hydrophobic binding pocket on the positively charged surface of GAPDH3. Additionally, adenosine contains the same adenine head with NAD+. Collectively, we speculate that the sequence-specific recognition of GAPDH3 comes from the tight binding of adenosine to the two hydrophobic pyrimidine ring binding pocket on the positively charged grooves, while the other regions on the positively charged groove only provide a nonspecific binding surface to RNA substrate.Staphylococcus aureus (S. aureus) can produce a variety of virulence factors,which can cause a wide range of diseases. The expression of virulence factors is tightly regulated by a series of regulatory elements. Recently, a novel nucleoside phosphatase SA1684 was found to contribute to S. aureus virulence. Nucleoside phosphatases are widespread from prokaryote to eucaryon. SA1684 is totally different from the other reported nucleoside phosphatases based on the protein sequence alignment, which indicates that SA1684 could be a promising anti-S.aureus target. Therefore, it is worth studying the mechanism of substrate recognition and catalysis of SA1684 protein.In this study, we solve the structures of SA1684 protein in apo form, complex with ATPyS and calcium, complex with GDP?S and magnesium and complex with GTP?S and calcium, with one molecule in each asymmetric unit. Structure comparisons indicate that there are two base binding site, located in the substrate binding pocket, to accommodate nucleoside diphosphates (NDPs) and nucleoside triphosphates (NTPs) respectively. There are two metal ions in each of these complex structures, and a water, located in the middle of the two metal ions, is rather conserved. Comparing the complex structures of SA1684 with its homology structure suggests that this water could participate in the hydrolysis by nucleophilic attacking the phosphorus in the substrate. |
PDF Full Text Request