SynopsisThe first X-ray structure of the catalytic nucleotide-binding subunit A of the A 1 -ATPase has been determined at 2.55 Å resolution.Abstract H + -transporting ATP synthase is a multi-subunit enzyme involved in the production of ATP, which is essential molecule for living organisms as a source of energy. Archaeal A-type ATPase (A-ATPase) is thought to act as a functional ATP synthase in Archaea and is thought to have chimeric properties of F-ATPase and V-ATPase.From the previous structural studies of F-ATPase, it is indicated that the major nucleotide-binding subunits α and β consist of three domains. The catalytic nucleotide-binding subunit A of V/A-ATPase contains an insertion of about 90 residues, which is absent from the F 1 -β subunit. Here we describe the first X-ray structure of the catalytic nucleotide-binding subunit A of the A 1 -ATPase determined at 2.55 Å resolution. A 1 -ATPase subunit A from Pyrococcus horikoshii consists of four domains. A novel domain, including a part of this insertion, corresponds to the "knob-like structure" observed in electron microscopy of A 1 -ATPase. Based on the structure, it is highly likely that this inserted domain is related to the peripheral stalk common to the A-and V-ATPases. The arrangement of this inserted domain suggests that this region plays an important role in A-ATPase as well as in V-ATPase.

Macrophage migration inhibitory factor (MIF) is an immunoregulatory cytokine involved in both acquired and innate immunity. MIF also has many functions outside the immune system, such as isomerase and oxidoreductase activities and control of cell proliferation. Considering the involvement of MIF in various intraand extracellular events, we expected that MIF might also be important in vertebrate development. To elucidate the possible role of MIF in developmental processes, we knocked down MIF in embryos of the African clawed frog Xenopus laevis, using MIF-specific morpholino oligomers (MOs). For the synthesis of the MOs, we cloned a cDNA for a Xenopus homolog of MIF. Sequence analysis, determination of the isomerase activity, and x-ray crystallographic analysis revealed that the protein encoded by the cDNA was the ortholog of mammalian MIF. We carried out whole mount in situ hybridization of MIF mRNA and found that MIF was expressed at high levels in the neural tissues of normal embryos. Although early embryogenesis of MO-injected embryos proceeded normally until the gastrula stage, their neurulation was completely inhibited. At the tailbud stage, the MO-injected embryos lacked neural and mesodermal tissues, and also showed severe defects in their head and tail structures. Thus, MIF was found to be essential for axis formation and neural development of Xenopus embryos.

Hepatocyte nuclear factor-6 (HNF-6), a liver-enriched transcription factor, controls the development of various tissues, such as the pancreas and liver, and regulates the expression of several hepatic genes. This protein belongs to the ONECUT class of homeodomain proteins and contains a bipartite DNA-binding domain composed of a single cut domain and a characteristic homeodomain. This transcription factor has two distinct modes of DNA binding and transcriptional activation that use different coactivators depending on the target gene. The crystal structure of the bipartite DNA-binding domain of HNF-6alpha complexed with the HNF-6-binding site of the TTR promoter revealed the DNA recognition mechanism of this protein. Comparing our structure with the DNA-free structure of HNF-6 or the structure of Oct-1, we discuss characteristic features associated with DNA binding and the structural basis for the dual mode of action of this protein, and we suggest a strategy for variability of transcriptional activation of the target gene.

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Trypsin, an important enzymes belonging to the large and diverse family of serine proteases, has been found in numerous higher mammals, fish, plants, insects and bacteria. Several studies on the isolation and characterization of trypsins from cold-adapted fishes, such as Atlantic salmon (Salmo salar), 1,2) chum salmon (Oncorhynchus keta), [3][4][5] Atlantic cod (Gadus morhua), 6) Greenland cod (Gadus ogac), 7,8) Antarctic fish (Paraotohhthenia magellanica), 9) Japanese anchovy (Engraulis japonicus), 10) and anchovy (Engraulis encrasicholus) 11) have been reported. These fish trypsins resemble mammalian trypsins in many respects, but are also different in some respects. For example, most of the fish trypsins studied are more unstable than those from mammals at acidic pH and high temperatures. 2,4,6,9,11) Moreover, trypsins from cold-adapted fish have higher catalytic efficiencies (measured by the k cat /K m ratio) than those from bovine and porcine. 2,[5][6][7][9][10][11] Furthermore multiple isoforms of trypsin have been found in many fish species, 2-6,10,11) while one cationic and one anionic form of trypsin have been found in humans, 12) bovines, 13) porcine, 14) and dogs. 15)Trypsin from chum salmon (Oncorhynchus keta) was first reported by Uchida et al. 3,4) Chum salmon trypsin is composed of seven anionic trypsins and one cationic trypsin, and they were separated by ion-exchange resin. Their functional properties were similar to those of bovine cationic trypsin; however, the detailed catalytic properties have not been reported. Thus, in our previous study, we isolated an anionic trypsin from chum salmon and measured the catalytic properties toward some substrates. The catalytic efficiency (k cat /K m ) of an anionic chum salmon trypsin for benzoyl-Larginine-p-nitroanilide (BAPA) hydrolysis is 22-fold higher than that of bovine trypsin at 5°C. 5) To improve our understanding of the higher level of catalytic efficiency at a lower temperature of enzymes from cold-adapted fish compared to mammalian enzymes, we also examined the X-ray crystallographic structure of an anionic chum salmon trypsin.16) The result suggested that a higher level of catalytic efficiency of anionic chum salmon trypsin might be achieved from the lower electrostatic potential of the S1-binding pocket. Studies on related enzymes from organisms adapted to living in a wide range of environments have revealed general patterns for how variations in sequence and structure allow enzymes to perform similar catalytic functions under different environmental conditions. These general patterns suggest mechanisms for how specific changes in structure may result in changes in enzymatic functions. Our studies are directed at the synthesis of non-covalent inhibitors of trypsin or trypsin-like enzymes (e.g. thrombin, kallikrein and urokinase) that may be used as scaffolds for the development of more specific drugs 17,18) ; therefore we are interested in the catalytic properties and three-dimensional structures of other isoforms of chum salmon trypsin. The...