The Structural Biology Research Of Synaptotagmin C2Domain And Mannonate Dehydratase

Synaptotagmins constitute a family of multifunctional integral membrane proteins found predominantly on vesicles in neural and endocrine tissues.17isoforms of synaptotagmin family in mammalians have been identified,7isoforms among them are known to be able to bind Ca2+via their C2domains. This study reports the crystal structure of synaptotagmin5C2A domain associating with three Ca2+ions and an acetate ion at1.90A resolution. The acetate binding site might mimic the binding site of phospholipid phosphate. The Ca2+-binding pocket of synaptotagmin5C2A domain was compared with other synaptotagmins and key residues leading to variable conformations of Ca2+-binding pockets of these C2domains were analyzed. We deduced that the conformation of Ca2+-binding pocket could influence the electrostatic repulsion potential energy between SYT and phospholipid phosphate during Ca2+-dependent synaptotagmin-phospholipid bilayer disassembly. Synaptotagmin5C2A domain has a folding pattern similar to C2domains of other synaptotagmins, but the conformation of its Ca+-binding pocket is intermediately close between synaptotagmins, which is consistent with the experimental observations that disassembly rate constant of synaptotagmin5cytosolic region-phospholipid membrane complex is intermediate among synaptotagmins capable of Ca2+binding. Thus, we deduced that variation of conformation of Ca2+-binding pocket is one of the most important reasons of synaptotagmins sharing high sequence and overall structure similarity are able to regulate various types of vesicle-membrane fusion processes with different Ca2+response rates.Mannonate dehydratase (ManD; EC4.2.1.8) catalyzes the dehydration of D-mannonate to2-keto-3-deoxygluconate. It is the third enzyme in the pathway for dissimilation of D-glucuronate to2-keto-3-deoxygluconate involving in the Entner-Doudoroff pathway in certain bacterial and archaeal species. ManD from Gram negative bacteria has an insert sequence as compared to those from Gram positives revealed by sequence analysis. To evaluate the impact of this insert sequence on the catalytic efficiency, we solved the crystal structures of ManD from Escherichia coli strain K12and its complex with D-mannonate, which reveal that this insert sequence forms two a helices locating above the active site. The two insert a helices introduce a loop that forms a cap covering the substrate binding pocket, which restricts the tunnels of substrate entering and product releasing from the active site. Site-directed mutations and enzymatic activity assays confirm that the catalytic rate is decreased by this loop. These features are conserved among Gram negative bacteria. Thus, the insert sequence of ManD from Gram negative bacteria acts as a common inducer to decrease the catalytic rate and consequently the glucuronate metabolic rate as compared to those from Gram positives. Moreover, residues essential for substrate to enter the active site were characterized via structural analysis and enzymatic activity assays.In Saccharomyces cerevisiae, TRM6and TRM61compose a tRNA methyltransferase, which catalyzes methylation of the N1of adenine at position58(m’A58) in tRNAs, especially initiator methionine tRNA. TRM61is the subunit that binds S-adenosyl-L-methionine, and both the two subunits contribute to target tRNA binding. Expression, purification, crystallization and X-ray diffraction analysis of the complex of TRM6-TRM61were preformed in this study. The crystals diffracted to a maximum resolution of2.80A, which provides a foundation for elucidating the catalytic mechanism of TRM6-TRM61and the interaction mode between the two subunits in the future.In bacteria and eukaryotes, the last two steps of de novo purine biosynthesis are catalyzed by bifunctional purine biosynthesis protein (PurH), which is composed of two functional independent domains linked by a flexible region. The N-terminal domain posseses the activity of IMP cyclohydrolase and the C-terminal domain posseses the activity of aminoimidazole-4-carboxamide ribonucleotide transformylase. Bacterial PurH possesses several insert and delete sequences with compared to eukaryotic and archaeal PurH. These sequence variation could cause the inter domain position of bacterial PurH be different from those of PurH from other species. This study reports the expression, purification, crystallization and preliminary X-ray crystallographic analysis of PurH from Escherichia coli K12strain (EcPurH). The crystals diffracted to a maximum resolution of3.05A, which provides a foundation for exploring the spatial relationship of the two active sites in bacterial PurH and its impact to catalytic efficiency in the future.