Hydration in proteins observed by high-resolution neutron crystallography

It is well known that water molecules surrounding a protein play important roles in maintaining its structural stability. Water molecules are known to participate in several physiological processes through the formation of hydrogen bonds. However, the hydration structures of most proteins are not known well at an atomic level at present because X-ray protein crystallography has difficulties to localize hydrogen atoms. In contrast, neutron crystallography has no problem in determining the position of hydrogens with high accuracy.1 In this article, the hydration structures of three proteins are described- myoglobin, wild-type rubredoxin, and a mutant rubredoxin-the structures of which were solved at 1.5- or 1.6-A resolution by neutron structure determination. These hydration patterns show fascinating features and the water molecules adopt a variety of shapes in the neutron Fourier maps, revealing details of intermolecular hydrogen bond formation and dynamics of hydration. Our results further show that there are strong relationships between these shapes and the water environments.     

Production and characterization of recombinant perdeuterated cholesterol oxidase

Cholesterol oxidase (CO) is a FAD (flavin adenine dinucleotide) containing enzyme that catalyzes the oxidization and isomerization of cholesterol. Studies directed toward elucidating the catalytic mechanism of CO will provide an important general understanding of Flavin-assisted redox catalysis. Hydrogen atoms play an important role in enzyme catalysis; however, they are not readily visualized in protein X-ray diffraction structures. Neutron crystallography is an ideal method for directly visualizing hydrogen positions at moderate resolutions because hydrogen and deuterium have comparable neutron scattering lengths to other heavy atoms present in proteins. The negative coherent and large incoherent scattering lengths of hydrogen atoms in neutron diffraction experiments can be circumvented by replacing hydrogen atoms with its isotope, deuterium. The perdeuterated form of CO was successfully expressed from minimal medium, purified, and crystallized. X-ray crystallographic structures of the enzyme in the perdeuterated and hydrogenated states confirm that there are no apparent structural differences between the two enzyme forms. Kinetic assays demonstrate that perdeuterated and hydrogenated enzymes are functionally identical. Together, structural and functional studies indicate that the perdeuterated protein is suitable for structural studies by neutron crystallography directed at understanding the role of hydrogen atoms in enzyme catalysis.     

Hydrogen and hydration in proteins

Neutron diffraction provides an experimental method of directly locating hydrogen atoms in proteins. High-resolution neutron diffractometers dedicated to biological macromolecules (BIX-type diffractometer) have been constructed at the Japan Atomic Energy Research Institute and they have been used in the 1.5A-resolution crystal structure analyses of several proteins. Interesting topics relevant to hydrogen and hydration in proteins, such as (1) the detailed geometry of hydrogen bonds; (2) information regarding hydrogen/deuterium exchange behavior; (3) the acidities of certain H atoms; (4) the role of hydrogen atoms in enzyme mechanisms and thermostability; (5) the location methyl hydrogen atoms; and (6) dynamical behavior of hydration structures that include H positions have been extracted from these structural results. In addition, a method for the systematic growth of large single crystals based on phase diagrams has been introduced and will be briefly described in this article.     

Neutrons expand the field of structural biology.

Neutron protein crystallography aids the identification of all the hydrogen atoms in biological macromolecules and has helped to establish hydration patterns in proteins. Recent technical innovations, such as the development of the neutron imaging plate, have made it possible to shorten the prohibitively long amount of time required to collect a full diffraction data set. These instrumental advances have been applied to Laue diffractometry, as well as to more conventional data collection techniques, such as those using monochromatized neutron beams.     

Neutron protein crystallography: current status and a brighter future

Hydrogen atoms are rarely seen in X-ray protein crystal structures, but are readily visualized by neutron crystallography, even at typical (1.5-2.5A) resolutions. Recent advances in neutron beamlines and deuterium labeling technologies have dramatically extended the scale and range of structures studied. High-quality neutron data can be collected to near atomic resolution ( approximately 1.5-2.5A) for proteins of 50-175kDa molecular weight, from perdeuterated samples, from crystals with volumes of 0.1mm(3) and at cryogenic temperatures (15K). These structures are providing unique and complementary insights into hydrogen-bonding interactions, protonation states, catalytic mechanisms and hydration states of biological structures that are not available from X-ray analysis alone. The new generation of spallation neutron sources promises further 10-50-fold gains in performance.     

Hydration Structures of the Human Protein Kinase CK2α Clarified by Joint Neutron and X-ray Crystallography

Casein kinase 2 (CK2) has broad phosphorylation activity against various regulatory proteins, which are important survival factors in eukaryotic cells. To clarify the hydration structure and catalytic mechanism of CK2, we determined the crystal structure of the alpha subunit of human CK2 containing hydrogen and deuterium atoms using joint neutron (1.9 Å resolution) and X-ray (1.1 Å resolution) crystallography. The analysis revealed the structure of conserved water molecules at the active site and a long potential hydrogen bonding network originating from the catalytic Asp156 that is well known to enhance the nucleophilicity of the substrate OH group to the γ-phospho group of ATP by proton elimination. His148 and Asp214 conserved in the protein kinase family are located in the middle of the network. The water molecule forming a hydrogen bond with Asp214 appears to be deformed. In addition, mutational analysis of His148 in CK2 showed significant reductions by 40%–75% in the catalytic efficiency with similar affinity for ATP. Likewise, remarkable reductions to less than 5% were shown by corresponding mutations on His131 in death-associated protein kinase 1, which belongs to a group different from that of CK2. These findings shed new light on the catalytic mechanism of protein kinases in which the hydrogen bond network through the C-terminal domain may assist the general base catalyst to extract a proton with a link to the bulk solvent via intermediates of a pair of residues.     

Characterization of perdeuterated high-potential iron-sulfur protein with high-resolution X-ray crystallography

Perdeuteration in neutron crystallography is an effective method for determining the positions of hydrogen atoms in proteins. However, there is shortage of evidence that the high-resolution details of perdeuterated proteins are consistent with those of the nondeuterated proteins. In this study, we determined the X-ray structure of perdeuterated high-potential iron-sulfur protein (HiPIP) at a high resolution of 0.85 å resolution. The comparison of the nondeuterated and perdeuterated structures of HiPIP revealed slight differences between the two structures. The spectroscopic and spectroelectrochemical studies also showed that perdeuterated HiPIP has approximately the same characteristics as nondeuterated HiPIP. These results further emphasize the suitability of using perdeuterated proteins in the high-resolution neutron crystallography.     

Neutron Laue macromolecular crystallography

Recent progress in neutron protein crystallography such as the use of the Laue technique and improved neutron optics and detector technologies have dramatically improved the speed and precision with which neutron protein structures can now be determined. These studies are providing unique and complementary insights on hydrogen and hydration in protein crystal structures that are not available from X-ray structures alone. Parallel improvements in modern molecular biology now allow fully (per)deuterated protein samples to be produced for neutron scattering that essentially eradicate the large-and ultimately limiting-hydrogen incoherent scattering background that has hampered such studies in the past. High quality neutron data can now be collected to near atomic resolution (approximately 2.0 A) for proteins of up to approximately 50 kDa molecular weight using crystals of volume approximately 0.1 mm3 on the Laue diffractometer at ILL. The ability to flash-cool and collect high resolution neutron data from protein crystals at cryogenic temperature (15 K) has opened the way for kinetic crystallography on freeze trapped systems. Current instrument developments now promise to reduce crystal volume requirements by a further order of magnitude, making neutron protein crystallography a more accessible and routine technique.     

A quasi-Laue neutron crystallographic study of d-xylose isomerase

The location of hydrogen atoms in enzyme structures can bring critical understanding of catalytic mechanism. However, whilst it is often difficult to determine the position of hydrogen atoms using X-ray crystallography even with subatomic (<1.0 A) resolution data available, neutron crystallography provides an experimental tool to directly localize hydrogen/deuterium atoms in biological macromolecules at resolution of 1.5-2.0 A. D-Xylose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5) is a 43 kDa enzyme that catalyses the first reaction in the catabolism of D-xylose. Linearization and isomerization of D-xylose at the active site of D-xylose isomerase rely upon a complex hydrogen transfer. Neutron quasi-Laue data at 2.2 A resolution were collected at room temperature on a partially deuterated Streptomyces rubiginosus D-xylose isomerase crystal using the LADI instrument at ILL with the objective to provide insight into the enzymatic mechanism. The neutron structure shows unambiguously that residue His 53 is doubly protonated at the active site of the enzyme. This suggests that the reaction proceeds through an acid catalyzed opening of the sugar ring, which is in accord with the mechanism suggested by Fenn et al. (Biochemistry 43(21): 6464-6474, 2004). This is the first report of direct observation of double protonation of His 53 and the first validation of the ring opening mechanism at the active site of D-xylose isomerase.     

Water-protein interactions from high-resolution protein crystallography

To understand the role of water in life at molecular and atomic levels, structures and interactions at the protein-water interface have been investigated by cryogenic X-ray crystallography. The method enabled a much clearer visualization of definite hydration sites on the protein surface than at ambient temperature. Using the structural models of proteins, including several hydration water molecules, the characteristics in hydration structures were systematically analysed for the amount, the interaction geometries between water molecules and proteins, and the local and global distribution of water molecules on the surface of proteins. The tetrahedral hydrogen-bond geometry of water molecules in bulk solvent was retained at the interface and enabled the extension of a three-dimensional chain connection of a hydrogen-bond network among hydration water molecules and polar protein atoms over the entire surface of proteins. Networks of hydrogen bonds were quite flexible to accommodate and/or to regulate the conformational changes of proteins such as domain motions. The present experimental results may have profound implications in the understanding of the physico-chemical principles governing the dynamics of proteins in an aqueous environment and a discussion of why water is essential to life at a molecular level.     

Neutron crystallographic studies of T4 lysozyme at cryogenic temperature

Bacteriophage T4 lysozyme (T4L) has been used as a paradigm for seminal biophysical studies on protein structure, dynamics, and stability. Approximately 700 mutants of this protein and their respective complexes have been characterized by X‐ray crystallography; however, despite the high resolution diffraction limits attained in several studies, no hydrogen atoms were reported being visualized in the electron density maps. To address this, a 2.2 Å‐resolution neutron data set was collected at 80 K from a crystal of perdeuterated T4L pseudo‐wild type. We describe a near complete atomic structure of T4L, which includes the positions of 1737 hydrogen atoms determined by neutron crystallography. The cryogenic neutron model reveals explicit detail of the hydrogen bonding interactions in the protein, in addition to the protonation states of several important residues.     

Structural characterizations of nonpeptidic thiadiazole inhibitors of matrix metalloproteinases reveal the basis for stromelysin selectivity.

The binding of two 5-substituted-1,3,4-thiadiazole-2-thione inhibitors to the matrix metalloproteinase stromelysin (MMP-3) have been characterized by protein crystallography. Both inhibitors coordinate to the catalytic zinc cation via an exocyclic sulfur and lay in an unusual position across the unprimed (P1-P3) side of the proteinase active site. Nitrogen atoms in the thiadiazole moiety make specific hydrogen bond interactions with enzyme structural elements that are conserved across all enzymes in the matrix metalloproteinase class. Strong hydrophobic interactions between the inhibitors and the side chain of tyrosine-155 appear to be responsible for the very high selectivity of these inhibitors for stromelysin. In these enzyme/inhibitor complexes, the S1'' enzyme subsite is unoccupied. A conformational rearrangement of the catalytic domain occurs that reveals an inherent flexibility of the substrate binding region leading to speculation about a possible mechanism for modulation of stromelysin activity and selectivity.     

Polar hydrogen positions in proteins: Empirical energy placement and neutron diffraction comparison

A method for the prediction of hydrogen positions in proteins is presented. The method is based on the knowledge of the heavy atom positions obtained, for instance, from X-ray crystallography. It employs an energy minimization limited to the environment of the hydrogen atoms bound to a common heavy atom or to a single water molecule. The method is not restricted to proteins and can be applied without modification to nonpolar hydrogens and to nucleic acids. The method has been applied to the neutron diffraction structures of trypsin ribonuclease A, and bovine pancreatic trypsin inhibitor. A comparison of the constructed and the observed hydrogen positions shows few deviations except in situations in which several energetically similar conformations are possible. Analysis of the potential energy of rotation of Lys amino and Ser, Thr, Tyr hydroxyl groups reveals that the conformations of lowest intrinsic torsion energies are statistically favored in both the crystal and the constructed structures.     

Positive contribution of hydration structure on the surface of human lysozyme to the conformational stability

Water molecules make a hydration structure with the network of hydrogen bonds, covering on the surface of proteins. To quantitatively estimate the contribution of the hydration structure to protein stability, a series of hydrophilic mutant human lysozymes (Val to Ser, Tyr, Asp, Asn, and Arg) modified at three different positions on the surface, which are located in the alpha-helix (Val-110), the beta-sheet (Val-2), and the loop (Val-74), were constructed. Their thermodynamic parameters of denaturation and crystal structures were examined by calorimetry and by x-ray crystallography at 100 K, respectively. The introduced polar residues made hydrogen bonds with protein atoms and/or water molecules, sometimes changing the hydration structure around the mutation site. Changes in the stability of the mutant proteins can be evaluated by a unique equation that considers the conformational changes resulting from the substitutions. Using this analysis, the relationship between the changes in the stabilities and the hydration structures for mutant human lysozymes substituted on the surface could be quantitatively estimated. The analysis indicated that the hydration structure on protein surface plays an important role in determining the conformational stability of the protein.     

Protein-water interactions in ribonuclease A and angiogenin: a molecular dynamics study

It is known that water molecules play an important role in the biological functioning of proteins. The members of the ribonuclease A (RNase A) family of proteins, which are sequentially and structurally similar, are known to carry out the obligatory function of cleaving RNA and individually perform other diverse biological functions. Our focus is on elucidating whether the sequence and structural similarity lead to common hydration patterns, what the common hydration sites are and what the differences are. Extensive molecular dynamics simulations followed by a detailed analysis of protein-water interactions have been carried out on two members of the ribonuclease A superfamily-RNase A and angiogenin. The water residence times are analyzed and their relationship with the characteristic properties of the protein polar atoms, such as their accessible surface area and mean hydration, is studied. The capacity of the polar atoms to form hydrogen bonds with water molecules and participate in protein-water networks are investigated. The locations of such networks are identified for both proteins.     

Cryo neutron crystallography demonstrates influence of RNA 2′-OH orientation on conformation,sugar pucker and water structure

The ribose 2′-hydroxyl is the key chemical difference between RNA and DNA and primary source of their divergent structural and functional characteristics. Macromolecular X-ray diffraction experiments typically do not reveal the positions of hydrogen atoms. Thus, standard crystallography cannot determine 2′-OH orientation (H2′-C2′-O2′-HO2′ torsion angle) and its potential roles in sculpting the RNA backbone and the expansive fold space. Here, we report the first neutron crystal structure of an RNA, the Escherichia coli rRNA Sarcin-Ricin Loop (SRL). 2′-OD orientations were established for all 27 residues and revealed O-D bonds pointing toward backbone (O3′, 13 observations), nucleobase (11) or sugar (3). Most riboses in the SRL stem region show a 2′-OD backbone-orientation. GAGA-tetraloop riboses display a 2′-OD base-orientation. An atypical C2′-endo sugar pucker is strictly correlated with a 2′-OD sugar-orientation. Neutrons reveal the strong preference of the 2′-OH to donate in H-bonds and that 2′-OH orientation affects both backbone geometry and ribose pucker. We discuss 2′-OH and water molecule orientations in the SRL neutron structure and compare with results from a solution phase 10 μs MD simulation. We demonstrate that joint cryo-neutron/X-ray crystallography offers an all-in-one approach to determine the complete structural properties of RNA, i.e. geometry, conformation, protonation state and hydration structure.     

Large-scale networks of hydration water molecules around proteins investigated by cryogenic X-ray crystallography.

The structures at protein-water interface, i.e. the hydration structure of proteins, have been investigated by cryogenic X-ray crystal structure analyses. Hydration structures appeared far clearer at cryogenic temperature than at ambient temperature, presumably because the motions of hydration water molecules were quenched by cooling. Based on the structural models obtained, the hydration structures were systematically analyzed with respect to the amount of water molecules, the interaction modes between water molecules and proteins, the local and the global distribution of them on the surface of proteins. The standard tetrahedral interaction geometry of water in bulk retained at the interface and enabled the three-dimensional chain connection of hydrogen bonds between hydration water molecules and polar protein atoms. Large-scale networks of hydrogen bonds covering the entire surface of proteins were quite flexible to accommodate to the large-scale conformational changes of proteins and seemed to have great influences on the dynamics and function of proteins. The present observation may provide a new concept for discussing the dynamics of proteins in aqueous solution.     

A systematic method for analysing the protein hydration structure of T4 lysozyme

A systematic method for the analysis of the hydration structure of proteins is demonstrated on the case study of lysozyme. The method utilises multiple structural data of the same protein deposited in the protein data bank. Clusters of high water occupancy are localised and characterised in terms of their interaction with protein. It is shown that they constitute a network of interconnected hydrogen bonds anchored to the protein molecule. The high occupancy of the clusters does not directly correlate with water–protein interaction energy as was originally hypothesised. The highly occupied clusters rather correspond to the nodes of the hydration network that have the maximum number of hydrogen bonds including both the protein atoms and the surrounding water clusters. Copyright © 2013 John Wiley & Sons, Ltd.     

A neutron Laue diffraction study of endothiapepsin: implications for the aspartic proteinase mechanism.

Current proposals for the catalytic mechanism of aspartic proteinases are largely based on X-ray structures of bound oligopeptide inhibitors possessing nonhydrolyzable analogues of the scissile peptide bond. However, the positions of protons on the catalytic aspartates and the ligand in these complexes have not been determined with certainty. Thus, our objective was to locate crucial protons at the active site of an inhibitor complex since this will have major implications for a detailed understanding of the mechanism of action. We have demonstrated that high-resolution neutron diffraction data can be collected from crystals of the fungal aspartic proteinase endothiapepsin bound to a transition state analogue (H261). The neutron structure of the complex has been refined at a resolution of 2.1 A to an R-factor of 23.5% and an R(free) of 27.4%. This work represents the largest protein structure studied to date by neutron crystallography at high resolution. The neutron data demonstrate that 49% of the main chain nitrogens have exchanged their hydrogen atoms with D2O in the mother liquor. The majority of residues resisting exchange are buried within core beta-sheet regions of the molecule. The neutron maps confirm that the protein has a number of buried ionized carboxylate groups which are likely to give the molecule a net negative charge even at very low pH, thereby accounting for its low pI. The functional groups at the catalytic center have clearly undergone H-D exchange despite being buried by the inhibitor occupying the active site cleft. Most importantly, the data provide convincing evidence that Asp 215 is protonated and that Asp 32 is the negatively charged residue in the transition state complex. This has an important bearing on mechanistic proposals for this class of proteinase.     

Hydration-coupled dynamics in proteins studied by neutron scattering and NMR: the case of the typical EF-hand calcium-binding parvalbumin

The influence of hydration on the internal dynamics of a typical EF-hand calciprotein, parvalbumin, was investigated by incoherent quasi-elastic neutron scattering (IQNS) and solid-state 13C-NMR spectroscopy using the powdered protein at different hydration levels. Both approaches establish an increase in protein dynamics upon progressive hydration above a threshold that only corresponds to partial coverage of the protein surface by the water molecules. Selective motions are apparent by NMR in the 10-ns time scale at the level of the polar lysyl side chains (externally located), as well as of more internally located side chains (from Ala and Ile), whereas IQNS monitors diffusive motions of hydrogen atoms in the protein at time scales up to 20 ps. Hydration-induced dynamics at the level of the abundant lysyl residues mainly involve the ammonium extremity of the side chain, as shown by NMR. The combined results suggest that peripheral water-protein interactions influence the protein dynamics in a global manner. There is a progressive induction of mobility at increasing hydration from the periphery toward the protein interior. This study gives a microscopic view of the structural and dynamic events following the hydration of a globular protein.