Crystal Soaking Research Articles

The Three-dimensional Structure of Mammalian Ribonucleotide Reductase Protein R2 Reveals a More-accessible Iron-radical Site than Escherichia coliR2

The three-dimensional structure of mouse ribonucleotide reductase R2 has been determined at 2.3 Å resolution using molecular replacement and refined to an R-value of 19.1% ( R free=25%) with good stereo-chemistry. The overall tertiary structure architecture of mouse R2 is similar to that from Escherichia coliR2. However, several important structural differences are observed. Unlike the E. coliprotein, the mouse dimer is completely devoid of β-strands. The sequences differ significantly between the mouse and E. coliR2s, but there is high sequence identity among the eukaryotic R2 proteins, and the identities are localized over the whole sequence. Therefore, the three-dimensional structures of other mammalian ribonucle otide reductase R2 proteins are expected to be very similar to that of the mouse enzyme. In mouse R2 a narrow hydrophobic channel leads to the proposed binding site for molecular oxygen near to the iron-radical site in the interior of the protein. In E. coliR2 this channel is blocked by the phenyl ring of a tyrosine residue, which in mouse R2 is a serine. These structural variations may explain the observed differences in sensitivity to radical scavengers. The structure determination is based on diffraction data from crystals grown at pH 4.7. Unexpectedly, the protein is not iron-free, but contains one iron ion bound at one of the dinuclear iron sites. This ferric ion is bound with partial occupancy and is coordinated by three glutamic acids (one bidentate) and one histidine in a bipyramidal coordination that has a free apical coordination position. Soaking of crystals in a solution of ferrous salt at pH 4.7 increased the occupancy on the already occupied site, but without any detectable binding at the second site.

Preparation and Crystallization of a Complex between Human Adenovirus Serotype 2 Proteinase and Its 11-Amino-Acid Cofactor pVIc

Crystals have been obtained of the recombinant human adenovirus serotype 2 proteinase (AVP) in a complex with its 11-amino-acid cofactor pVIc. AVP–pVIc complexes were formed by the incubation of AVP with a 1.2-fold molar excess of pVIc prior to the crystallization trials. Diffraction-quality crystals were obtained at 18°C by the vapor-diffusion method with 5.6 mg/ml AVP–pVIc in 1.4Msodium acetate and 0.1MHepes, pH 7.5. Diffraction data (99% complete to 2.6 Å resolution withRmergeof 0.077) were collected from native crystals at room temperature at beamline X12-C at the National Synchrotron Light Source. The crystals belong to space group P61with unit cell dimensionsa=b= 114.2 Å,c= 50.1 Å; α = β = 90°, γ = 120°. The unit cell dimensions and likely mass of the molecular species in the crystals were consistent with there being one 25000-Da complex (1:1) per asymmetric unit. Additionally, one heavy-atom derivative, obtained by the soaking of preformed crystals, was isomorphous to the native crystal. Diffraction data obtained on these crystals were 95% complete to 3.0 Å resolution with anRmergeof 0.076. Difference-Patterson analysis indicates three heavy atom sites in the derivative asymmetric unit.

Characterization of the metal ion binding helix-hairpin-helix motifs in human DNA polymerase beta by X-ray structural analysis.

X-ray crystallographic studies have shown that DNA binding by human polymerase beta (pol beta) occurs primarily through two structurally and sequentially homologous helix-hairpin-helix (HhH) motifs, one in the fingers subdomain and the other in the 8-kDa domain [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996a) Biochemistry 35, 12742-12761]. In that DNA binding by each HhH motif is facilitated by a metal ion, we set out to determine the identity of the metal ion that most likely binds to the HhH motif in vivo. Crystal soaking experiments were performed on human pol beta-DNA cocrystals with Mg2+, Ca2+, Na+, and K+, the four most prevalent metal ions in the cell, and in each case a data set was collected and the resulting structure was refined. Under the conditions tested, the HhH motifs of pol beta have an affinity for these biologically prevalent metal ions in the order Mg2+ < Ca2+ < Na+ < K+, with K+ displaying the strongest binding. Crystals soaked in the presence of Tl+, a commonly used spectroscopic probe for K+, were too X-ray-sensitive to establish the binding behavior of Tl+, but soaking experiments with Ba2+ and Cs+ resulted in relatively stable crystals that gave evidence of metal ion binding in both HhH motifs, confirming that larger monovalent and divalent metal ions are capable of binding to the HhH metal sites. Although Mn2+, which has been categorized as a potent polymerase mutagen, binds to the HhH motifs with a greater affinity than Mg2+, Mn2+ does not bind to the HhH motifs in the presence of equimolar concentrations of Na+. These results suggest that in vivo, where Mn2+ is present only in trace amounts, Mn2+ probably does not have a large effect on DNA binding and may instead manifest a mutagenic effect on pol beta primarily by distorting nucleotide binding or by directly affecting the catalytic step [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996b) Biochemistry 35, 12762-12777]. Crystal soaking experiments with 31-kDa apoenzyme crystals show that, in the absence of DNA, the HhH motif in the fingers subdomain binds metal ions with either much lower occupancy or not at all, indicating that metal ion binding is dependent on the presence of the DNA substrate.

The application of crystal soaking technique to study the effect of zinc and cresol on insulinotropin crystals grown from a saline solution.

The purpose of this study is to investigate the effect of zinc and cresol on the structure of insulinotropin crystals. Insulinotropin crystals grown from a saline solution were treated with zinc and/or m-cresol using a crystal soaking technique. The effects of these additives on the crystal structure were investigated with powder X-ray diffraction, photomicrography, and differential scanning calorimetry. The molecular interaction between insulinotropin and m-trifluorocresol in solution was also studied by 19F NMR: The data suggest that the original crystals grown from a saline solution have relatively weak lattice forces. After the addition of m-cresol to the suspension of the insulinotropin crystals, the crystals were immediately rendered amorphous. The m-cresol molecules which diffused into the crystals through solvent channels may have disturbed the lattice interactions that maintain the integrity of the crystal. In contrast, the zinc added to the suspension stabilized the crystal lattice so that the subsequent addition of m-cresol did not alter the integrity of the crystals. A marked increase in melting point (206 degrees versus 184 degrees) and heat of fusion (24.6 J/g versus 1.4 J/g) of the crystals was observed after the treatment with zinc. The solubility of the zinc treated crystals in a pH 7.1 phosphate buffered saline was 1/20 of that of the original crystals. When the insulinotropin crystals were treated with the additives using a crystal soaking method, the crystals underwent structural changes. Zinc stabilized the crystal lattice, and reduced the solubility of the peptide.

Recipes for the protein crystallographer

Practical Protein Crystallography by Duncan E McRee Academic Press 1993, 386 pages. £35/$70 hardback (ISBN 0-12-486050-8).The area of protein crystallography has not been flooded with textbooks. After the classical Blundell and Johnson text appeared in 1976, not a single crystallography textbook focussing on protein crystallography has been published. Interested students have to consult more general texts on X-ray crystallography which devote not more than one or two chapters to proteins.Duncan McRee has done an excellent job in presenting his personal experience in this field. He writes in a casual style which makes reading easy. The title of this book truly covers its content. It is exclusively devoted to practical problems in protein X-ray crystallography. Chapter 1, on Laboratory Techniques, gives details about protein sample preparation and crystal growth, and storing and soaking of crystals. In chapter 2, data collection techniques are discussedin extenso, including crystal mounting, X-ray sources, strategies and data reduction. Chapter 3 is equally useful. It presents data processing and the solution of the phase problem, including the necessary elementary terminology, such as R-factors, space groups, and resolution. After the more general chapters 1–3, the book becomes a real cookbook. In 60 pages the author discusses eight specific problems from his own work and from the work of his colleagues at the Scripps Research Institute. Four of the examples concern multiple isomorphous replacement (40 pages) with only one example (3 pages) of molecular replacement, which is somewhat surprising in view of the extensive application of this technique for structure determination. The remaining three examples are on mutant studies and substrate-analog binding. These examples can be illuminating for the student who is struggling with his/her own structure. In the remaining 80 pages of the book, software is discussed. This is of only limited interest to most protein crystallographers because it focuses on the specific package (Xtal View) used in the author's laboratory.The book is not free of errors. Apart from an occasional printing error, it also contains some technical errors (for example, on page 33; in the X-ray tube drawing the tube current is indicated as the filament current, and in the legend to this figure it is stated that the filament is 40 kV above the target (anode), whereas in fact it is below). It is also sloppy to write: “soaking in an inhibitor or heavy atom”. One easily forgives the author these imperfections, however, because of the pleasant way in which he addresses the reader. The question is: what sort of reader? The experienced protein crystallographer will recognize the problems discussed and may have the same or different solutions for them. The absolute beginner and those with hardly any background in crystallography will have great difficulty, because the most elementary concepts (such as Ewald sphere and reciprocal lattice) are used but not explained. If there is an explanation it is in a dictionary-like style. Theory is not dealt with at all, and readers needing to understand diffraction and structure determination are referred to the literature (e.g. Blundell and Johnson, and Methods in Enzymology, Vol. 114 and 115). Therefore, this book is not a textbook for the beginner but a manual for the laboratory. The book will be appreciated most by the student who has already made a start in protein crystallography and who knows and understands what he/she is doing. Such students will find it instructive to learn from the author's extensive experience and to find out that the standard techniques in their own laboratory are not the only ones in the world. As a manual the book is highly useful and should be on the shelf in every protein crystallography laboratory.

NADH binding site and catalysis of NADH peroxidase.

The structure of the complex between cofactor NADH and the enzyme NADH peroxidase from Streptococcus faecalis 10C1 (Enterococcus faecalis) has been determined by crystal soaking, X-ray data collection, model building of NADH and refinement at 0.24-nm resolution based on the known enzyme structure [Stehle, T., Ahmed, S. A., Claiborne, A. & Schulz, G. E. (1991) J. Mol. Biol. 221, 1325-1344]. Apart from NADH, the catalytic center of the enzyme contains FAD and a cysteine that shuttles between thiolate and sulfenic acid states. Unfortunately, this cysteine was irreversibly oxidized to a cysteine sulfonic acid in the established enzyme structure. Based on the geometry of the catalytic center, we discuss the stabilization of the oxidation-sensitive sulfenic acid and propose a reaction mechanism.

Soaking of crystals for macromolecular crystallography in a capillary

Soaking of crystals for macromolecular crystallography in a capillary

Refined crystal structures of “aged” and “non-aged” organophosphoryl conjugates of γ-chymotrypsin

“Aged” organophosphoryl conjugates of serine hydrolases differ from the corresponding “non-aged” conjugates in their striking resistance to nucleophilic reactivation. The refined X-ray structures of “aged” and “non-aged” organophosphoryl conjugates of γ-chymotrypsin were compared in order to understand the molecular basis for this resistance of “aged” conjugates. “Aged” and “non-aged” crystalline organophosphoryl-γ-chymotrypsin conjugates were obtained by prolonged soaking of native γ-chymotrypsin crystals with appropriate organophosphates. Thus, a representative “non-aged” conjugate, diethylphosphoryl-γ-chymotrypsin, was obtained by soaking native crystals with paraoxon (diethyl- p-nitrophenyl phosphate), and a closely related “aged” conjugate, monoisopropyl-γ-chymotrypsin, was obtained by soaking with diisopropylphosphorofluoridate. In both crystalline conjugates, the refined structures clearly reveal a high occupancy of the active site by the appropriate organophosphoryl moiety within covalent bonding distance of Ser195 O γ. Whereas in the “non-aged” conjugate both ethyl groups can be visualized clearly, in the putative “aged” conjugate, as expected, only one isopropyl group is present. There is virtually no difference between the “aged” and “non-aged” conjugates either with respect to the conformation of the polypeptide backbone as a whole or with respect to the positioning of the side-chains within the active site. In the “aged” conjugate, however, close proximity (2·6 Å) of the negatively charged phosphate oxygen atom of the dealkylated organophosphoryl group to His57 N ε2 indicates the presence of a salt bridge between these two moieties. In contrast, in the “non-aged” conjugate the DEP moiety retains its two alkyl groups; thus, lacking a negative oxygen atom, it does not enter into such a charge-charge interaction and its nearest oxygen atom is 3·6 Å away from His57 N ε2. It is suggested that steric constraints imposed by the salt bridge in the “aged” conjugate lie at the basis of its resistance to reactivation.

Crystal structure of Penicillium citrinum P1 nuclease at 2.8 A resolution.

P1 nuclease from Penicillium citrinum is a zinc dependent glyco-enzyme consisting of 270 amino acid residues which cleaves single-stranded RNA and DNA into 5'-mononucleotides. The X-ray structure of a tetragonal crystal form of the enzyme with two molecules per asymmetric unit has been solved at 3.3 and refined at 2.8 A resolution to a crystallographic R-factor of 21.6%. The current model consists of 269 amino acid residues, three Zn ions and two N-acetyl glucosamines per subunit. The enzyme is folded very similarly to phospholipase C from Bacillus cereus, with 56% of the structure displaying an alpha-helical conformation. The three Zn ions are located at the bottom of a cleft and appear to be rather inaccessible for any phosphate group in double-stranded RNA or DNA substrates. A crystal soaking experiment with a dinucleotide gives clear evidence for two mononucleotide binding sites separated by approximately 20 A. One site shows binding of the phosphate group to one of the zinc ions. At both sites there is a hydrophobic binding pocket for the base, but no direct interaction between the protein and the deoxyribose. A cleavage mechanism is proposed involving nucleophilic attack by a Zn activated water molecule.

X-ray Diffraction Analysis on Single Crystals of Recombinant Escherichia coli Ornithine Transcarbamoylase

Single crystals of recombinant Escherichia coli ornithine transcarbamoylase suitable for x-ray analysis have been grown from polyethylene glycol and 2-methyl-2,4-pentanediol. The space group has been determined as P3(1) or P3(2), with one protein trimer of three identical 36.8-kDa subunits in the asymmetric unit. The unit cell dimensions are a = b = 105.1 A and c = 87.8 A. The crystals diffract well to 3-A resolution and are quite resistant to radiation damage. Single crystals have also been grown of a genetically engineered site-specific mutant for which the replacement of an arginine (Arg-57) to a glycine has been shown to not only drastically affect the enzyme activity but also its kinetic mechanism (Kuo, L. C., Miller, A. W., Lee, S., and Kozuma, C. (1988) Biochemistry 27, 8823-8832). The crystals of the Arg-57----Gly mutant protein are isomorphous to those of the wild type. Crystal soaking experiments using both wild-type and Arg-57----Gly crystals in the presence of various ligands have provided evidence of specific conformational changes upon substrate binding which supports our previous kinetic and spectroscopic observations.

X-ray crystallographic investigation of substrate binding to carboxypeptidase A at subzero temperature.

A high-resolution x-ray crystallographic investigation of the complex between carboxypeptidase A (CPA; peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) and the slowly hydrolyzed substrate glycyl-L-tyrosine was done at -9 degrees C. Although this enzyme-substrate complex has been the subject of earlier crystallographic investigation, a higher resolution electron-density map of the complex with greater occupancy of the substrate was desired. All crystal chemistry (i.e., crystal soaking and x-ray data collection) was performed on a diffractometer-mounted flow cell, in which the crystal was immobilized. The x-ray data to 1.6-A resolution have yielded a well-resolved structure in which the zinc ion of the active site is five-coordinate: three enzyme residues (glutamate-72, histidine-69, and histidine-196) and the carbonyl oxygen and amino terminus of glycyl-L-tyrosine complete the coordination polyhedron of the metal. These results confirm that this substrate may be bound in a nonproductive manner, because the hydrolytically important zinc-bound water has been displaced and excluded from the active site. It is likely that all dipeptide substrates of carboxypeptidase A that carry an unprotected amino terminus are poor substrates because of such favorable bidentate coordination to the metal ion of the active site.

Crystallographic studies of protein denaturation and renaturation. 1. Effects of denaturants on volume and X-ray pattern of cross-linked triclinic lysozyme crystals.

Triclinic crystals of hen egg-white lysozyme cross-linked with glutaraldehyde have been treated with various denaturants and found to be susceptible to x-ray structure analysis even after major conformational changes in the protein. Cross-linked crystals were isomorphous with the native form, and electron density difference maps indicated the locations of intermolecular corss-links, but showed no appreciable differences in the protein conformation. Soaking of the cross-linked crystals in danaturant solutions of increasing concentrations caused corresponding increases in crystal volume and decreases in minimum observable x-ray spacings. These changes proved partly reversible on diluting the solutions, and measurements of crystal volume and minimums x-ray spacing were used to follow denaturation and renaturation as a function of concentration for several denaturants. Some of these, including bromoethanol and sodium dodecyl sulfate, had little effect on the crystals below critical concentrations at which there was a sharp volume increase and loss of x-ray pattern, which could, however, be regenerated to about 3.2-A resolution. Others, including KCNS and urea, caused more gradual changes, but with a smaller degree of recovery. It is suggested that at least two different denaturation mechanisms are involved with detergent-like reagents disrupting the hydrophobic interactions joining the two wings of the lysozyme molecule and hydrophilic denaturants interacting primarily with polar groups on the molecular surface.