The hydration shell of myoglobin

The space in the unit cell of a metmyoglobin crystal not occupied by myoglobin atoms was filled with water using Monte Carlo calculations. Independent calculations with different amounts of water have been performed. Structure factors were calculated using the water coordinates thus obtained and the known coordinates of the myoglobin atoms. A comparison with experimental structure factors showed that both the low and the high resolution regime could be well reproduced with 814 Monte Carlo water molecules per unit cell with a B-value of 50 Ų. The Monte Carlo water molecules yield a smaller standard R-value (0.166) than using a homogeneous electron density for the simulation of the crystal water (R = 0.212). A reciprocal space refinement of the water and the protein coordinates has been performed. Monte Carlo calculations can be used to obtain information for crystallographically invisible parts of the unit cell and yield better coordinates for the visible part in the refinement. Correspondence to: F. Parak     

Conservation of solvent-binding sites in 10 crystal forms of T4 lysozyme.

Solvent-binding sites were compared in 10 different crystal forms of phage T4 lysozyme that were refined using data from 2.6 A to 1.7 A resolution. The sample included 18 crystallographically independent lysozyme molecules. Despite different crystallization conditions, variable crystal contacts, changes due to mutation, and varying attention to solvent during crystallographic refinement, 62% of the 20 most frequently occupied sites were conserved. Allowing for potential steric interference from neighboring molecules in the crystal lattice, this fraction increased to 79% of the sites. There was, however, no solvent-binding site that was occupied in all 18 lysozyme molecules. A buried double site was occupied in 17 instances and 2 other internal sites were occupied 15 times. Apart from these buried sites, the most frequently occupied sites were often at the amino-termini of alpha-helices. Solvent molecules at the most conserved sites tended to have crystallographic thermal factors lower than average, but atoms with low B-factors were not restricted to these sites. Although superficial inspection may suggest that only 50-60% (or less) of solvent-binding sites are conserved in different crystal forms of a protein, it appears that many sites appear to be empty either because of steric interference or because the apparent occupancy of a given site can vary from crystal to crystal. The X-ray method of identifying sites is somewhat subjective and tends to result in specification only of those solvent molecules that are well ordered and bound with high occupancy, even though there is clear evidence for solvent bound at many additional sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystalline beta-cyclodextrin.12H2O reversibly dehydrates to beta-cyclodextrin.10.5 H2O under ambient conditions.

In contact with mother liquor, crystalline beta-cyclodextrin (beta-CD) hydrate has composition approximately beta-CD.12H2O. If crystals are dried at ambient conditions (18 degrees C, approximately 50% humidity), the unit cell volume diminishes approximately 30 to 50 A3. X-ray structure analysis of a dry crystal (0.89 A resolution, 4617 data, R = 0.059) showed the composition beta-CD.10.5 H2O, with approximately 5.5 water molecules in the beta-CD cavity (7 partially and 2 fully occupied sites) and approximately 5.0 between the beta-CD molecules. The positions of the beta-CD host and of most of the hydration waters are conserved during dehydration, but the occupancies of the waters in the beta-CD cavity diminish. Dry crystals put into solvent re-hydrate to the original form. The mechanism of de- and re-hydration is not evident.     

Buried water molecules contribute to the conformational stability of a protein

This study sought to attain a better understanding of the contribution of buried water molecules to protein stability. The 3SS human lysozyme lacks one disulfide bond between Cys77 and Cys95 and is significantly destabilized compared with the wild-type human lysozyme (4SS). We examined the structure and stability of the I59A-3SS mutant human lysozyme, in which a cavity is created at the mutation site. The crystal structure of I59A-3SS indicated that there were ordered new water molecules in the cavity created. The stability of I59A-3SS is 5.5 kJ/mol less than that of 3SS. The decreased stability of I59A-3SS (5.5 kJ/mol) is similar to that of Ile to Ala mutants with newly introduced water molecules in other globular proteins (6.3 +/- 2.1 kJ/mol), but is less than that of Ile/Leu to Ala mutants with empty cavities (13.7 +/- 3.1 kJ/mol). This indicates that water molecules partially compensate for the destabilization by decreasing hydrophobic and van der Waals interactions. These results provide further evidence that buried water molecules contribute to protein stability.     

Molecular replacement study on form-B monoclinic crystal of insulin

The form-B monodinic insulin crystal was obtained from the sodium citrate buffer with 1% zinc chloride, keeping phenolic content between 0.76% and 1.25%. Its space group is P21, cell constants are: a = 4.924nm, b=6.094nm, c=4.818nm, β=95.8°. There are 6 insulin molecules which form a hexamer. The initial phase was obtained by using rotation function program of X-PLOR program package and molecular packing program of our laboratory. The molecular model was chosen from 4 zinc bovine insulin hexamer. After the preliminary refinement by using the rnacromolecular rigid body refinement technique, the molecular model was further refined and adjusted by using the energy-minimizing stereochemically restrained least-squared refinement on the difference Fourier maps. The finial R-factor is 214% at 0.3nm resolution, the r.m.s. deviations from standard bond length and bond angle are 0.0022nm and 4.7°, respectively.     

A compact RNA tertiary structure contains a buried backbone-K+ complex

The structure of a 58 nucleotide ribosomal RNA fragment buries several phosphate groups of a hairpin loop within a large tertiary core. During refinement of an X-ray crystal structure containing this RNA, a potassium ion was found to be contacted by six oxygen atoms from the buried phosphate groups; the ion is contained completely within the solvent-accessible surface of the RNA. The electrostatic potential at the ion chelation site is unusually large, and more than compensates for the substantial energetic penalties associated with partial dehydration of the ion and displacement of delocalized ions. The very large predicted binding free energy, approximately -30 kcal/mol, implies that the site must be occupied for the RNA to fold. These findings agree with previous studies of the ion-dependent folding of tertiary structure in this RNA, which concluded that a monovalent ion was bound in a partially dehydrated environment where Mg2+ could not easily compete for binding. By compensating the unfavorable free energy of buried phosphate groups with a chelated ion, the RNA is able to create a larger and more complex tertiary fold than would be possible otherwise.     

Crystal structure determination of basic phospholipase A_2 from venom of Agkistrodon halys pallas by molecular replacement method

Basic phospholipase A2 (BPLA2) from the venom of Agkistrodon halys pallas has a strong ability to hemolyze erythrocytes. The asymmetrical unit of P212121 crystal of BPLA2 contains two molecules. Self-rotation function was used to study the orientation relationship of these two molecules. Cross-rotation and translation functions were then used to determine the orientations and positions of the two molecules in the unit cell. The model building and preliminary structure refinement were carried out. The result shows that the two molecules in the asymmetrical unit of orthorhombic crystal are related by a non-crystallographic 2-fold symmetry axis.     

Accessibility and order of water sites in and around proteins: A crystallographic time-averaging study.

Water plays an essential role in most biological processes. Water molecules solvating biomolecules are generally in fast exchange with the environment. Nevertheless, well-defined electron density is seen for water associated with proteins whose crystal structure is determined to high resolution. The relative accessibility of these water sites is likely to be relevant to their biological role but is difficult to assess. A time-averaging crystallographic refinement simulation on basic pancreatic trypsin inhibitor successfully characterizes the relative accessibility of the crystallographic water sites. In such a refinement simulation water diffuses through the crystal lattice in a manner that is consistent with the crystallographic data. This refinement discovers that internal crystallographic waters in this particular protein are bridged to the outside protein surface via a series of progressively more accessible water sites. On the surface of the protein, water molecules exchange quickly between crystallographic water sites. Time-averaging crystallographic refinement provides a view based on experimental data of the relative accessibility of water sites in and around a protein in a crystalline environment. Proteins 1999;36:501-511.     

Crystallographic refinement and atomic models of two different forms of citrate synthase at 2·7 and 1·7 Å resolution

The structures of pig heart and chicken heart citrate synthase have been determined by multiple isomorphous replacement and restrained crystallographic refinement for two crystal forms, a tetragonal form at 2·7 Å and a monoclinic form at 1·7 Å resolution, with crystallographic R-values of 0·199 and 0·192, respectively. The structure determination involved a novel application of restrained crystallographic refinement, in that the refinement of incomplete models was necessary in order to completely determine the course of the polypeptide chain. The recently determined amino acid sequence (Bloxham et al., 1981) has been fitted to the models. The molecule has substantially different conformations in the two crystal forms, and there is evidence that a conformational change is required for enzymatic activity.The molecule is a dimer of identical subunits with 437 amino acid residues each. The conformation is all α-helix, with 40 helices per dimer packing tightly to form a globular molecule. Many of the helices are kinked in various ways or bent smoothly over a large angle. Several of the helices show an unusual antiparallel packing.Each subunit is clearly divided into a large and a small domain. The two crystal forms differ by the relative arrangement of the two domains. The tetragonal form represents an open configuration with a deep cleft between the two domains, the monoclinic form is closed. The structural change from the open to the closed form can be described by an 18 ° rotation of the small domain relative to the large domain.Crystallographic analyses were performed with the product citrate bound in both crystal forms, with coenzyme A (CoA) and a citryl-CoA analogue bound to the monoclinic form. These studies establish the CoA and the citrate binding sites, and the conformations of the two product molecules in atomic detail. The subunits are extensively interdigitated, with one subunit making significant contributions to both the citrate and the CoA binding sites of the other subunit. The adenine moiety of CoA is bound to the small domain, and the pantothenic arm is bound to the large domain. The citrate molecule is bound in a cleft between the large domain. The citrate molecule is bound in a cleft between the large and small domain, with the si carboxymethylene group facing the SH arm of coenzyme A. In the monoclinic form, the cysteamine part of CoA shields the bound citrate completely from the solution. Partial reaction of CoA-SH and aspartate 375 to form aspartyl-CoA, and citrate to form citryl-CoA may occur in the crystals. The conformation of CoA is compact, characterized by an internal hydrogen bond O-52 … N-7 and a tightlybound water molecule O-51 … HOH … O-20.     

Molecular dynamics simulation of replacement of methane hydrate with carbon dioxide

Molecular dynamics simulation was performed to analyse the phenomena of replacement of methane hydrate with carbon dioxide (CO₂) at 270 K and 5.0 MPa for 5300 ps. The methane hydrate phase was constructed with 16 unit cells of hydrate. Every cage in the hydrate was occupied by one methane molecule. The methane hydrate phase was sandwiched between two CO₂ phases. During the simulation the hydrate partially melted and liquid water phase appeared, and CO₂ dissolved in the liquid water phase. The replacements were observed three times at the hydrate–liquid water interface during the simulation. In the first case, the replacement occurred at a S-cage without changing the structure. In the second case, an M-cage of methane hydrate partially collapsed, and methane and CO₂ molecules exchanged. After the exchange, the cage occupied by CO₂ remained in the M-cage structure. In the third case, a S-cage of methane hydrate partially collapsed, and methane and CO₂ molecules exchanged. After the exchange, the cage occupied by CO₂ changed to an M-cage-like structure.     

Metal ions bound at the active site of the junction-resolving enzyme T7 endonuclease I

T7 endonuclease I is a nuclease that is selective for the structure of the four-way DNA junction. The active site is similar to those of a number of restriction enzymes. We have solved the crystal structure of endonuclease I with a wild-type active site. Diffusion of manganese ions into the crystal revealed two peaks of electron density per active site, defining two metal ion-binding sites. Site 1 is fully occupied, and the manganese ion is coordinated by the carboxylate groups of Asp55 and Glu65, and the main chain carbonyl of Thr66. Site 2 is partially occupied, and the metal ion has a single protein ligand, the remaining carboxylate oxygen atom of Asp55. Isothermal titration calorimetry showed the sequential exothermic binding of two manganese ions in solution, with dissociation constants of 0.58 +/- 0.019 and 14 +/- 1.5 mM. These results are consistent with a two metal ion mechanism for the cleavage reaction, in which the hydrolytic water molecule is contained in the first coordination sphere of the site 1-bound metal ion.     

Molecular and crystal structure of the regenerated form of (1----3)-alpha-D-mannan.

The crystal structure of the hydrated form of (1----3)-alpha-D-mannan, obtained by solid-state deacetylation of the partially O-acetylated mannan, was analyzed by combined X-ray diffraction and stereochemical-model refinement techniques. The structure crystallizes in a four-chain, monoclinic unit cell with parameters a = 11.33 A, b = 18.36 A, c (fiber repeat) = 8.25 A, and gamma = 101.75 degrees, and the most probable space group is P2(1). In the most probable structure the chain-backbone conformation is a two-fold helix, but with all four O-6 rotational positions nonequivalent. The chains pack with antiparallel polarity and are connected by pairs of intermolecular hydrogen bonds that form an infinite, zig-zag sheet. There are 16 water molecules in the unit cell, generally embedded between the sheets in crystallographic positions, providing additional hydrogen bonding and establishing a three-dimensional hydrogen-bond network in the crystal structure. The reliability of the structure analysis is indicated by the X-ray residual R" = 0.281, based on 98 hkl reflection intensities.     

The crystal structure of the 1:1 inclusion complex of beta-cyclodextrin with squaric acid.

The crystal and molecular structure of the 1:1 inclusion complex of beta-cyclodextrin (cyclomaltoheptaose) with squaric acid (3,4-dihydroxycyclobutene-1,2-dione) was determined by X-ray diffraction. The complex crystallizes in the monoclinic P2(1) space group and belongs to the monomeric cage-type, characterized by a herringbone-like packing motif. Co-crystallized water molecules are present on seven sites, of which six are fully occupied. The guest molecule is placed inside the beta-cyclodextrin cavity, perpendicular to the plane defined by the glycosidic O-4n atoms, and held in place by direct and water-mediated hydrogen bonds mainly involving symmetry-related beta-cyclodextrin molecules. The accommodation of the planar guest molecule into the beta-cyclodextrin cavity determines a significant distortion of the latter from the sevenfold symmetry.     

Crystallographic structure refinement of Chromatium high potential iron protein at two Angstroms resolution.

The structure of Chromatium high potential iron protein (HiPIP) has been refined by semiautomatic Fo-Fc (observed minus calculated structure amplitude Fourier methods to a convential R index, R=sum of the absolute value of Fo-Fc divided by the sum of Fo, of 24.7% for a model in which bond distances and angles are constrained to standard values. Bond length and angle constraints were applied only intermittenly during the computations. At a late stage of the refinement, atomic parameters for only the Fe4S4 cluster plus the 4 associated cystein S-gamma atoms were adjusted by least squares methods and kept fixed during the rest of the refinement. The refined model consists of 625 of the 632 nonhydrogen atoms in the protein plus 75 water molecules. Seven side chain atoms could not be located in the final electron density map. A computer program rather than visual inspection was used wherever possible in the refinement: for locating water molecules, for removing water molecules that too closely approach other atoms, for deleting atoms that lay in regions of low electron density, and for evaluating the progress of refinement. Fo-Fc Fourier refinement is sufficiently economical to be applied routinely in protein crystal structure determinations. The complete HiPIP refinement required approximately 12 hours of CDC 3600 computer time and cost less than $3000 starting from a "trial structure," based upon multipe isomorphoous replacement phases, which gave an R of 43%...     

On the mechanism of biological methane formation: structural evidence for conformational changes in methyl-coenzyme M reductase upon substrate binding

Methyl-coenzyme M reductase (MCR) catalyzes the final reaction of the energy conserving pathway of methanogenic archaea in which methylcoenzyme M and coenzyme B are converted to methane and the heterodisulfide CoM-S-S-CoB. It operates under strictly anaerobic conditions and contains the nickel porphinoid F430 which is present in the nickel (I) oxidation state in the active enzyme. The known crystal structures of the inactive nickel (II) enzyme in complex with coenzyme M and coenzyme B (MCR-ox1-silent) and in complex with the heterodisulfide CoM-S-S-CoB (MCR-silent) were now refined at 1.16 A and 1.8 A resolution, respectively. The atomic resolution structure of MCR-ox1-silent describes the exact geometry of the cofactor F430, of the active site residues and of the modified amino acid residues. Moreover, the observation of 18 Mg2+ and 9 Na+ ions at the protein surface of the 300 kDa enzyme specifies typical constituents of binding sites for either ion. The MCR-silent and MCR-ox1-silent structures differed in the occupancy of bound water molecules near the active site indicating that a water chain is involved in the replenishment of the active site with water molecules. The structure of the novel enzyme state MCR-red1-silent at 1.8 A resolution revealed an active site only partially occupied by coenzyme M and coenzyme B. Increased flexibility and distinct alternate conformations were observed near the active site and the substrate channel. The electron density of the MCR-red1-silent state aerobically co-crystallized with coenzyme M displayed a fully occupied coenzyme M-binding site with no alternate conformations. Therefore, the structure was very similar to the MCR-ox1-silent state. As a consequence, the binding of coenzyme M induced specific conformational changes that postulate a molecular mechanism by which the enzyme ensures that methylcoenzyme M enters the substrate channel prior to coenzyme B as required by the active-site geometry. The three different enzymatically inactive enzyme states are discussed with respect to their enzymatically active precursors and with respect to the catalytic mechanism.     

Buried waters and internal cavities in monomeric proteins

We have analyzed the buried water molecules and internal cavities in a set of 75 high-resolution, nonhomologous, monomeric protein structures. The number of hydrogen bonds formed between each water molecule and the protein varies from 0 to 4, with 3 being most common. Nearly half of the water molecules are found in pairs or larger clusters. Approximately 90% are shown to be associated with large cavities within the protein, as determined by a novel program, PRO_ACT. The total volume of a protein's large cavities is proportional to its molecular weight and is not dependent on structural class. The largest cavities in proteins are generally elongated rather than globular. There are many more empty cavities than hydrated cavities. The likelihood of a cavity being occupied by a water molecule increases with cavity size and the number of available hydrogen bond partners, with each additional partner typically stabilizing the occupied state by 0.6 kcal/mol.     

Calcium binding to thermitase. Crystallographic studies of thermitase at 0, 5, and 100 mM calcium

The three-dimensional crystal structure of thermitase complexed with eglin-c in the presence of 100 mM calcium has been determined and refined at 2.0-A resolution to a R-factor of 16.8%. This crystal structure is compared with previously determined structures of thermitase at 0 and 5 mM calcium concentration. In the presence of 100 mM calcium all three calcium binding sites in thermitase are fully occupied. At 100 mM CaCl2 the "weak" calcium binding is occupied by a calcium ion, which is chelated by three protein ligands and four water molecules in a pentagonal bipyramid geometry. Thermitase has, apparently, a monovalent and divalent cation binding position at 2.5-A distance from each other at this site. At low calcium concentrations the monovalent-ion position is occupied by a sodium or potassium ion. The "medium strength" binding site shows in the presence of 100 mM CaCl2 a square antiprism arrangement with eight ligands, of which seven are donated by the protein. At low calcium concentrations we observe a distorted pentagonal bipyramid coordination at this site. The largest difference between these two conformations is observed for ligand Asp-60, which has two conformations with 0.8-A difference in C alpha positions. The "strong" calcium binding site has a pentagonal bipyramid coordination and is fully occupied in all three structures. Structural changes on binding calcium to the weak and "medium strength" calcium binding sites of thermitase are limited to the direct surroundings of these sites. Thermitase resembles in this respect subtilisin BPN' and does not exhibit long-range shifts as have been reported for proteinase K.     

Correlation between occupancy and B factor of water molecules in protein crystal structures

An empirical relationship between occupancy and the atomic displacement parameter of water molecules in protein crystal structures has been found by comparing a set of well refined sperm whale myoglobin crystal structures. The relationship agrees with a series of independent structural features whose impact on water occupancy can easily be predicted as well as with other known data and is independent of the protein fold. The estimation of the water occupancy in protein crystal structures may help in understanding the physico-chemical properties of the protein-solvent interface and can allow the monitoring of the accuracy of the protein crystal structure refinement.     

Insights into carbohydrate recognition by Narcissus pseudonarcissus lectin: the crystal structure at 2 A resolution in complex with alpha1-3 mannobiose.

Carbohydrate recognition by monocot mannose-binding lectins was studied via the crystal structure determination of daffodil (Narcissus pseudonarcissus) lectin. The lectin was extracted from daffodil bulbs, and crystallised in the presence of alpha-1,3 mannobiose. Molecular replacement methods were used to solve the structure using the partially refined model of Hippeastrum hybrid agglutinin as a search model. The structure was refined at 2.0 A resolution to a final R -factor of 18.7 %, and Rfreeof 26.7 %.The main feature of the daffodil lectin structure is the presence of three fully occupied binding pockets per monomer, arranged around the faces of a triangular beta-prism motif. The pockets have identical topology, and can bind mono-, di- or oligosaccharides. Strand exchange forms tightly bound dimers, and higher aggregation states are achieved through hydrophobic patches on the surface, completing a tetramer with internal 222-symmetry. There are therefore 12 fully occupied binding pockets per tetrameric cluster. The tetramer persists in solution, as shown with small-angle X-ray solution scattering. Extensive sideways and out-of-plane interactions between tetramers, some mediated via the ligand, make up the bulk of the lattice contacts.A fourth binding site was also observed. This is unique and has not been observed in similar structures. The site is only partially occupied by a ligand molecule due to the much lower binding affinity. A comparison with the Galanthus nivalis agglutinin/mannopentaose complex suggests an involvement of this site in the recognition mechanism for naturally occurring glycans.     

Comparison of crystal structures of human type 3 3alpha-hydroxysteroid dehydrogenase reveals an "induced-fit" mechanism and a conserved basic motif involved in the binding of androgen

The aldo-keto reductase (AKR) human type 3 3alpha-hydroxysteroid dehydrogenase (h3alpha-HSD3, AKR1C2) plays a crucial role in the regulation of the intracellular concentrations of testosterone and 5alpha-dihydrotestosterone (5alpha-DHT), two steroids directly linked to the etiology and the progression of many prostate diseases and cancer. This enzyme also binds many structurally different molecules such as 4-hydroxynonenal, polycyclic aromatic hydrocarbons, and indanone. To understand the mechanism underlying the plasticity of its substrate-binding site, we solved the binary complex structure of h3alpha-HSD3-NADP(H) at 1.9 A resolution. During the refinement process, we found acetate and citrate molecules deeply engulfed in the steroid-binding cavity. Superimposition of this structure with the h3alpha-HSD3-NADP(H)-testosterone/acetate ternary complex structure reveals that one of the mobile loops forming the binding cavity operates a slight contraction movement against the citrate molecule while the side chains of many residues undergo numerous conformational changes, probably to create an optimal binding site for the citrate. These structural changes, which altogether cause a reduction of the substrate-binding cavity volume (from 776 A(3) in the presence of testosterone/acetate to 704 A(3) in the acetate/citrate complex), are reminiscent of the "induced-fit" mechanism previously proposed for the aldose reductase, another member of the AKR superfamily. We also found that the replacement of residues Arg(301) and Arg(304), localized near the steroid-binding cavity, significantly affects the 3alpha-HSD activity of this enzyme toward 5alpha-DHT and completely abolishes its 17beta-HSD activity on 4-dione. All these results have thus been used to reevaluate the binding mode of this enzyme for androgens.