Hydrogen bonds of DsrD protein revealed by neutron crystallography

Hydrogen bonds of DNA-binding protein DsrD have been determined by neutron diffraction. In terms of proton donors and acceptors, DsrD protein shows striking differences from other proteins

Structural fluctuation observed in Z-DNA d(CGCGCG)2 in the absence of divalent metal cations and polyamines.

In the present study, Z-DNA d(CGCGCG)2 was crystallized from a DNA solution in the absence of divalent metal cations and polyamines, and its X-ray structure was determined at 0.98 Å resolution. Comparison of this structure and previously reported Z-DNA structures, containing Mg(2+) cations and/or polyamines, demonstrated that Z-DNA can have structural fluctuations with respect to phosphate groups and hydration in the minor groove. At the GpC steps, a two-state structural equilibrium between the ZI and ZII conformations was frequently observed. In contrast, at the CpG steps, the phosphate groups exhibited rotational fluctuation, which could induce distortion of sugar puckering. In addition, alternative positions of water molecules were found in the middle of the minor groove of the Z-DNA. These structural fluctuations were likely observable because of the absence of Mg(2+) cations and polyamines. The results related to these phenomena were supported by those of other experimental methods, suggesting the possibility of these fluctuations occurring in biological conditions

DNA conformational transitions inferred from reevaluation of m|Fo|-D|Fc| electron density maps

Conformational flexibility of DNA plays important roles in biological processes such as transcriptional regulation and DNA packaging etc. To understand the mechanisms of these processes, it is important to analyse when, where and how DNA shows conformational variations. Recent analyses have indicated that conventional refinement methods do not always provide accurate models of crystallographic heterogeneities and that some information on polymorphism has been overlooked in previous crystallographic studies. In the present study, the m|Fo| D|Fc| electron-density maps of double-helical DNA crystal structures were calculated at a resolution equal to or better than 1.5 A ˚ and potential conformational transitions were found in 27% of DNA phosphates. Detailed analyses of the m|Fo| D|Fc| peaks indicated that some of these unassigned densities correspond to ZI $ ZII or A/B ! BI conformational transitions. A relationship was also found between ZI/ZII transitions and metal coordination in Z-DNA from the detected peaks. The present study highlights that frequent transitions of phosphate backbones occur even in crystals and that some of these transitions are affected by the local molecular environment

Recent structural insights into the mechanism of lysozyme hydrolysis

Lysozyme hydrolyzes the glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycans located in the bacterial cell wall. The mechanism of the hydrolysis reaction of lysozyme was first studied more than 50 years ago; however, it has not yet been fully elucidated and various mechanisms are still being investigated. One reaction system that has commonly been proposed is that the lysozyme intermediate undergoes covalent ligand binding during hydrolysis. However, these findings resulted from experiments performed under laboratory conditions using fluorine-based ligands, which facilitate the formation of covalent bonds between the ligands and the catalytic side chain of lysozyme. More recently, high-resolution X-ray structural analysis was used to study the complex of lysozyme with an N-acetylglucosamine tetramer. As a result, the carboxyl group of Asp52 was found to form a relatively strong hydrogen-bond network and had difficulty binding covalently to C1 of the carbohydrate ring. To confirm this hydrogen-bond network, neutron test measurements were successfully performed to a resolution of better than 1.9 Å

X-ray structure determination and deuteration of nattokinase.

Nattokinase (NK) is a strong fibrinolytic enzyme, which is produced in abundance by Bacillus subtilis natto. Although NK is a member of the subtilisin family, it displays different substrate specificity when compared with other subtilisins. The results of molecular simulations predict that hydrogen arrangements around Ser221 at the active site probably account for the substrate specificity of NK. Therefore, neutron crystallographic analysis should provide valuable information that reveals the enzymatic mechanism of NK. In this report, the X-ray structure of the non-hydrogen form of undeuterated NK was determined, and the preparation of deuterated NK was successfully achieved. The non-hydrogen NK structure was determined at 1.74 Å resolution. The three-dimensional structures of NK and subtilisin E from Bacillus subtilis DB104 are near identical. Deuteration of NK was carried out by cultivating Bacillus subtilis natto in deuterated medium. The D2O resistant strain of Bacillus subtilis natto was obtained by successive cultivation rounds, in which the concentration of D2O in the medium was gradually increased. NK was purified from the culture medium and its activity was confirmed by the fibrin plate method. The results lay the framework for neutron protein crystallography analysis