Neutron Crystallographic Analysis Research Articles

Neutron crystallographic analysis of the nucleotide-binding domain of Hsp72 in complex with ADP

The 70 kDa heat-shock proteins (Hsp70s) are ATP-dependent molecular chaperones that contain an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain. Hsp70s bind to misfolded/unfolded proteins and thereby prevent their aggregation. The ATP hydrolysis reaction in the NBD plays a key role in allosteric control of the binding of substrate proteins. In the present study, the neutron crystal structure of the NBD of Hsp72, a heat-inducible Hsp70 family member, was solved in complex with ADP in order to study the structure–function relationship with a focus on hydrogens. ADP bound to Hsp72 was fully deprotonated, and the catalytically important residues, including Asp10, Asp199 and Asp206, are also deprotonated. Neutron analysis also enabled the characterization of the water clusters in the NBD. Enzymatic assays and X-ray crystallographic analysis revealed that the Y149A mutation exhibited a higher ATPase activity and caused disruption of the water cluster and incorporation of an additional magnesium ion. Tyr149 was suggested to contribute to the low intrinsic ATPase activity and to stabilize the water cluster. Collectively, these structural studies will help to elucidate the molecular basis of the function of Hsp72.

Large-scale crystallization and neutron crystallographic analysis of HSP70 in complex with ADP.

HSP70 belongs to the heat-shock protein family and binds to unfolded proteins, driven by ATP hydrolysis, in order to prevent aggregation. Previous X-ray crystallographic analyses of HSP70 have shown that HSP70 binds to ADP with internal water molecules. In order to elucidate the role of the water molecules, including their H/D atoms, a neutron diffraction study of the human HSP70 ATPase domain was initiated. Deuterated large crystals of the HSP-ADP complex (1.2-1.8 mm3) were successfully grown by large-scale crystallization, and a neutron diffraction experiment at BIODIFF resulted in diffraction to a maximum resolution of 2.2 Å. After data reduction, the overall completeness, Rmeas and average I/σ(I) were 90.4%, 11.7% and 8.1, respectively, indicating that the data set was sufficient to visualize H and D atoms.

Neutron crystallography of photoactive yellow protein reveals unusual protonation state of Arg52 in the crystal

Because of its high pKa, arginine (Arg) is believed to be protonated even in the hydrophobic environment of the protein interior. However, our neutron crystallographic structure of photoactive yellow protein, a light sensor, demonstrated that Arg52 adopts an electrically neutral form. We also showed that the hydrogen bond between the chromophore and Glu46 is a so-called low barrier hydrogen bond (LBHB). Because both the neutral Arg and LBHB are unusual in proteins, these observations remain controversial. To validate our findings, we carried out neutron crystallographic analysis of the E46Q mutant of PYP. The resultant structure revealed that the proportion of the cationic form is higher in E46Q than in WT, although the cationic and neutral forms of Arg52 coexist in E46Q. These observations were confirmed by the occupancy of the deuterium atom bound to the Nη1 atom combined with an alternative conformation of the N(η2)D2 group comprising sp2 hybridisation. Based on these results, we propose that the formation of the LBHB decreases the proton affinity of Arg52, stabilizing the neutral form in the crystal.

Neutron crystallographic analysis of human α-thrombin using iBIX at J-PARC

α-Thrombin is a serine protease, which plays the central role in the coagulation system. Investigating the protonation states of the enzyme is useful to reveal the reaction mechanism and to design anticoagulant drugs. We have studied the protonation states of the α-Thrombin-bivalirudin complex using neutron diffraction method. The complex is regarded as the enzyme-product complex, because the hydrolyzed bivalirudin fragments keep staying in the binding sites in the crystal. Previously we had performed a neutron crystallographic analysis of α-Thrombin-bivalirudin complex at pD 5.0 (space group P21212) at 2.8 Å resolution.[1] To observe the protonation states of the active site more clearly, we carried out time-of-flight neutron diffraction experiments for a different crystal form of this complex (space group C2 at pD 7.9) using IBARAKI biological crystal diffractometer, iBIX, installed in J-PARC. Using improved 30 neutron detectors with high-efficiency, we have succeeded in collecting the reflections at around 2.0 Å resolution. XN-joint refinements were performed using PHENIX program. The neutron scattering length OMIT map showed a density on the hydroxyl group of serine 195, which could be a deuterium. Since the density was not observed for P21212 crystal at pD 5.0 and the position was too far from an acceptor atom to form a stiff hydrogen bond, currently we are confirming the result. In this presentation, details of the neutron crystallographic analysis and the comparison between the structures, especially, the protonation states of amino acid residues in the active site of the complex at pD 5.0 and pD 7.9 will be given.

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.

Solvent penetration in photoactive yellow protein R52Q mutant: A theoretical study

An R52Q mutation of photoactive yellow protein creates a cavity mimicking an intermediate state in the wild-type. This mutation, of a positively charged arginine to a neutral glutamine, does not affect the spectral tuning of photoactive yellow protein. Contradicting explanations for this phenomenon suggest either water or hydronium occupation in the cavity. To solve this controversy, we have performed 3D-RISM calculations on the mutant structure examining the solvation structure. Our results show that while there is a high probability of hydronium in the cavity compared to bulk, there is still a relatively low occupation when considering realistic concentrations. Thus our findings suggest that while the cavity is clearly accessible by hydronium, it is mostly occupied by water molecules. We expect high-resolution neutron crystallographic analysis of the mutant to confirm our prediction.

Growth of Protein Crystals by Syringe-Type Top-Seeded Solution Growth

Neutron crystallography is an experimental method for visualizing the hydrogen atoms of proteins, thereby elucidating the mechanism of the target proteins. The technique requires large crystals of proteins to overcome the low flux of the available neutron beam. We previously reported that top-seeded solution growth (TSSG) is an efficient strategy for rapidly growing large protein crystals, but obstacles still remain. Here we report a syringe-type TSSG for growing protein crystals. In this method, a seed crystal is hung in the syringe. The method permits small protein crystals to be attached with the desired crystal orientation under an optical microscope. Similarly to previous results of TSSG, the shape of the lysozyme and superoxide dismutase crystals were strongly influenced by the orientation of the seed crystal. This new approach is expected to expand the scope of target proteins for neutron crystallographic analysis.

Metal Ion Roles and the Movement of Hydrogen during Reaction Catalyzed by D-Xylose Isomerase: A Joint X-Ray and Neutron Diffraction Study

Conversion of aldo to keto sugars by the metalloenzyme D-xylose isomerase (XI) is a multistep reaction that involves hydrogen transfer. We have determined the structure of this enzyme by neutron diffraction in order to locate H atoms (or their isotope D). Two studies are presented, one of XI containing cadmium and cyclic D-glucose (before sugar ring opening has occurred), and the other containing nickel and linear D-glucose (after ring opening has occurred but before isomerization). Previously we reported the neutron structures of ligand-free enzyme and enzyme with bound product. The data show that His54 is doubly protonated on the ring N in all four structures. Lys289 is neutral before ring opening and gains a proton after this; the catalytic metal-bound water is deprotonated to hydroxyl during isomerization and O5 is deprotonated. These results lead to new suggestions as to how changes might take place over the course of the reaction.

2P343 フェレドキシン依存性ビリン還元酵素PcyAの中性子構造解析に向けた結晶成長(結晶成長・結晶化技術,第48回日本生物物理学会年会)

2P343 フェレドキシン依存性ビリン還元酵素PcyAの中性子構造解析に向けた結晶成長(結晶成長・結晶化技術,第48回日本生物物理学会年会)

A neutron crystallographic analysis of T6 porcine insulin at 2.1 A resolution.

Neutron diffraction data for T(6) porcine insulin were collected to 2.1 A resolution from a single crystal partly deuterated by exchange of mother liquor. A maximum-likelihood structure refinement was undertaken using the neutron data and the structure was refined to a residual of 0.179. The hydrogen-bonding network of the central core of the hexamer was observed and the charge balance between positively charged Zn ions and their surrounding structure was interpreted by considering the protonation and/or deprotonation states and interactions of HisB10, water and GluB13. The observed double conformation of GluB13 was essential to interpreting the charge balance and could be compared with the structure of a dried crystal of T(6) human insulin at 100 K. Differences in the dynamic behaviour of the water molecules coordinating the upper and lower Zn ions were observed and interpreted. The hydrogen bonds in the insulin molecules, as well as those involving HisB10 and GluB13, are discussed. The hydrogen/deuterium (H/D) exchange ratios of the amide H atoms of T(6) porcine insulin in crystals were obtained and showed that regions highly protected from H/D exchange are concentrated in the centre of a helical region of the B chains. From the viewpoint of soaking time versus H/D-exchange ratios, the amide H atoms can be classified into three categories.

A neutron crystallographic analysis of phosphate-free ribonuclease A at 1.7 Å resolution

A neutron crystallographic analysis of phosphate-free bovine pancreatic RNase A has been carried out at 1.7 A resolution using the BIX-4 single-crystal diffractometer at the JRR-3 reactor of the Japan Atomic Energy Agency. The high-resolution structural model allowed us to determine that His12 acts mainly as a general base in the catalytic process of RNase A. Numerous other distinctive structural features such as the hydrogen positions of methyl groups, hydroxyl groups, prolines, asparagines and glutamines were also determined at 1.7 A resolution. The protonation and deprotonation states of all of the charged amino-acid residues allowed us to provide a definitive description of the hydrogen-bonding network around the active site and the H atoms of the key His48 residue. Differences in hydrogen-bond strengths for the alpha-helices and beta-sheets were inferred from determination of the hydrogen-bond lengths and the H/D-exchange ratios of the backbone amide H atoms. The correlation between the B factors and hydrogen-bond lengths of the hydration water molecules was also determined.

Generalized X-ray and neutron crystallographic analysis: more accurate and complete structures for biological macromolecules

X-ray and neutron crystallographic techniques provide complementary information on the structure and function of biological macromolecules. X-ray and neutron (XN) crystallographic data have been combined in a joint structure-refinement procedure that has been developed using recent advances in modern computational methodologies, including cross-validated maximum-likelihood target functions with gradient-based optimization and simulated annealing. The XN approach for complete (including hydrogen) macromolecular structure analysis provides more accurate and complete structures, as demonstrated for diisopropyl fluorophosphatase, photoactive yellow protein and human aldose reductase. Furthermore, this method has several practical advantages, including the easier determination of the orientation of water molecules, hydroxyl groups and some amino-acid side chains.

A preliminary neutron crystallographic study of an A-DNA crystal.

The LADI-III diffractometer at the Institut Laue-Langevin has been used to carry out a preliminary neutron crystallographic study of the self-complementary DNA oligonucleotide d(AGGGGCCCCT)(2) in the A conformation. The results demonstrate the viability of a full neutron crystallographic analysis with the aim of providing enhanced information on the ion-water networks that are known to be important in stabilizing A-DNA. This is the first account of a single-crystal neutron diffraction study of A-DNA. The study was carried out with the smallest crystal used to date for a neutron crystallographic study of a biological macromolecule.

An abnormal pKa value of internal histidine of the insulin molecule revealed by neutron crystallographic analysis

Insulin is stored in pancreatic β-cell as hexameric form with Zn2+ ions, while the hormonally active form is monomer. The hexamer requires the coordination of Zn2+ ions to the HisB10. In order to reveal the mechanism of the hexamerization of insulin, we investigated the Zn2+ free insulin at pD6.6 and pD9 by neutron crystallographic analyses. HisB10 is doubly protonated not only at pD6.6 but also at pD9, indicating an abnormal pKa of this histidine. It is suggested that HisB10 acts on a strong cation capture and contributes to the high stability of the hexameric form in pancreas.

A preliminary neutron crystallographic study of thaumatin.

A preliminary neutron crystallographic study of the sweet protein thaumatin is presented. Large hydrogenated crystals were prepared in deuterated crystallization buffer using the gel-acupuncture method. Data were collected to a resolution of 2 A on the LADI-III diffractometer at the Institut Laue Langevin (ILL). The results demonstrate the feasibility of a full neutron crystallographic analysis of this structure aimed at providing relevant information on the location of H atoms, the distribution of charge on the protein surface and localized water in the structure. This information will be of interest for understanding the specificity of thaumatin-receptor interactions and will contribute to further understanding of the molecular mechanisms underlying the perception of taste.

Biophysical reactions affect of the protonation state of amino acids in protein

Protonation is the key to elucidate a function of protein.Neutron crystallographic analysis is a powerful method that can observe the protonation states of amino acid residues in a protein molecule. However the neutron crystallographic technique was very difficult method, because of low flux neutron source mainly. Recently, a neutron imaging plate and an elastically bent perfect Si monochromator enabled neutron experiment. As the result, neutron structural biology has been progressed greatly in these ten years. In this report, we explain the various biological reactions affected by the protonation state on protein molecules by our studies on neutron crystallographic analysis. Furthermore, we also introduce our recent study on the protonation of cubic insulin molecule.

Ｘ線および中性子線結晶学によるＤＮＡの水素および水和の構造生物学的研究

It is well known that hydrogen atoms in DNA play important roles for folding, stability, enzymatic function, and recognition via hydrogen bonds. A combination of X-ray and neutron diffraction study is a powerful technique to reveal hydrogen and hydration structures of DNA. Here, my present study of high resolution neutron crystallographic analysis of Z-DNA is described together with my previous X-ray analysis of chemically damaged DNA, in which hydrogen bonding pattern was converted so as to cause mutagenesis. These results illustrate complicated and artistic features of DNA structures.

DsrD蛋白質の中性子結晶構造解析

DsrD蛋白質の中性子結晶構造解析