Preliminary x-ray data on crystals of mandelate racemase

The recent development of a high-yield expression system and purification scheme for mandelate racemase has enabled us to produce sufficiently large quantities of pure enzyme to pursue x-ray crystallographic study. Large, single crystals of mandelate racemase have been grown from buffered polyethylene glycol (pH 8.0) in the presence of 10 mM magnesium chloride. The crystals grow in several habits, and we have identified two distinct tetragonal space groups in preliminary x-ray diffraction analysis. Crystals shaped as rectangular plates demonstrate 4/mmm Laue symmetry and systematic absences consistent with the space group I422. They have cell dimensions of a = b = 153 A and c = 181 A. Octahedrally shaped crystals of mandelate racemase display 4/m Laue symmetry and systematic absences consistent with the space group 14. Cell dimensions for these crystals are a = b = 113 A and c = 124 A. Based on estimates of Vm and on the measured density of the 1422 form, we suggest that two subunits of mandelate racemase (38,570 daltons/subunit) occupy the asymmetric unit in both crystal forms. Crystals of both forms diffract to beyond 3.0-A resolution. We are currently screening for isomorphous heavy-atom derivatives.     

Preliminary X-ray crystallographic studies of pig kidney fructose-1,6-bisphosphatase

Preliminary x-ray data have been obtained from large single crystals of pig kidney fructose-1,6-bisphosphatase, grown from polyethylene glycol. The crystals have the symmetry of space group P3(1)21 or its enantiomorph P3(2)21, contain two subunits of the 146,000-dalton tetramer/asymmetric unit, and diffract to 2.9-A resolution on still photographs. The unit cell dimensions are a = b = 132.5 A and c = 68.0 A. Small single crystals have been grown in the presence of the inhibitor fructose 2,6-bisphosphate, with and without the allosteric effector AMP added. Crystals grown in the presence of both ligands are isomorphous with native crystals and generate diffraction patterns that show significant intensity changes.     

Crystallization of human neutrophil elastase

Human neutrophil elastase was inactivated by methoxysuccinyl-L-Ala-L-Ala-L-Pro-L-Ala-chloromethane. The modified enzyme was crystallized from 40 mM ammonium phosphate, pH 7.0 in the hexagonal space group P6(3) with unit cell parameters a = 74.53 A, b = 74.53 A, c = 70.88 A, alpha = beta = 90 degrees, gamma = 120 degrees. These crystals were resistant to radiation damage and diffracted beyond 1.84-A resolution. The asymmetric unit contained one 25,000-dalton monomer of human neutrophil elastase. Crystals were also grown from the enzyme modified with the analogous iodinated inactivator, p-iodoanilinosuccinyl-L-Ala-L-Ala-L-Pro-L-Ala-chloromethane. These crystals proved to be isomorphous with those of methoxysuccinyl-L-Ala-L-Ala-L-Pro-L-Ala-chloromethane-modified human neutrophil elastase, and served as a single-site, heavy atom derivative for solving the tertiary structure of the enzyme.     

Preliminary X-ray diffraction studies of the putative catalytic domain of gamma delta resolvase from Escherichia coli

Two crystal forms of the putative catalytic domain (residues 1-140) of gamma delta resolvase from Escherichia coli have been obtained. Type I is isomorphous with crystals of the intact protein, and type II is suitable for high resolution structure analysis. Type II crystals belong to the orthorhombic space group C222(1), with a = 76.8 A, b = 191.3 A, and c = 63.4 A. They contain two molecules (15,500 daltons each)/asymmetric unit and show diffraction beyond 2.7-A resolution. Calculation of a rotation function using 7-A data shows the orientation of the noncrystallographic axes.     

Crystallization and preliminary X-ray analysis of the cAMP-dependent protein kinase catalytic subunit from Saccharomyces cerevisiae

A truncated variant of TPK1, the yeast cAMP-dependent protein kinase catalytic subunit, was overexpressed in an engineered strain of Saccharomyces cerevisiae, purified by liquid chromatography, and crystallized from solutions of 2-propanol and magnesium at alkaline pH. The crystals are hexagonal dipyramids, space group P6(1)22 (P6(5)22), with unit-cell parameters a = b = 61 A, c = 320 A. Large single crystals suitable for diffraction analysis are obtainable by microseeding, and diffract beyond 2.8-A resolution. Crystal density measurements reveal 12 kinase monomers per unit cell with a single kinase monomer per asymmetric unit.     

Beta-bungarotoxin. Preparation and characterization of crystals suitable for structural analysis

Phospholipases in some snake venoms are potent neurotoxins that target their enzymatic action to the synaptic membrane. One of these is the heterodimeric neurotoxin, beta-bungarotoxin, which binds with a protease inhibitor-like subunit to a presynaptic potassium channel and then blocks neurotransmission with a second subunit that has phospholipase A2 activity. We have prepared and characterized well ordered crystals of the most toxic beta-bungarotoxin isoform, beta 1-bungarotoxin. The crystals are monoclinic, space group C2, with unit cell parameters: a = 176.5 A, b = 39.3 A, c = 92.7 A, and beta = 114.8 degrees. Rotation-function analysis of the Patterson function, as calculated from a 2.3-A data set, reveals an asymmetric unit composed of four heterodimers. These heterodimers appear to be associated as two crystallographically distinct (AB)4 tetramers, each having dihedral D2 symmetry. The two are positioned with equivalent molecular 2-fold axes coincident with crystallographic dyads, but rotated by 55 degrees relative to one another. X-ray analysis of these crystals will permit direct visualization of the specific structural motifs and chemical features that underlie phospholipase neurotoxicity.     

Crystallization and preliminary x-ray investigation of the regulatory subunit of cAMP-dependent protein kinase

Crystals of type I cAMP-dependent protein kinase regulatory subunit have been grown from solutions of ammonium sulfate. The crystals are square bipyramids, space group P4(1)2(1)2 (P4(3)2(1)2), with a = b = 106.9 +/- 0.6 A and c = 212.4 +/- 1.0 A. There are two dimers of the regulatory subunit/crystallographic asymmetric unit. The crystals are stable for 3-4 days in the x-ray beam and diffract to at least 3.5-A resolution.     

X-ray grade crystals of the enzymatic fragment of diphtheria toxin

The enzymatic fragment of diphtheria toxin, fragment A (Mr = 21,167), complexed to the dinucleotide adenosine 3',5'-uridine (ApU), has been crystallized at two different values of pH by hanging drop vapor diffusion. Crystals grown at a pH value of 5.0 (from I) belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a = 71.2 A, b = 73.0 A, c = 139.8 A and four protomers in the asymmetric unit. Crystals grown at a pH value of 8.1 (form II) belong to the monoclinic space group C2, with unit cell parameters a = 65.2 A, b = 85.6 A, c = 34.6 A, beta = 103.0 degrees and one protomer in the asymmetric unit. Both crystal forms diffract to 2.5 A resolution. The molecular structures of fragment A obtained from these two crystal forms may illuminate the pH-dependent transition of diphtheria toxin during membrane translocation.     

Crystallization and preliminary X-ray studies of pyridoxal kinase from sheep brain

Crystals of pyridoxal kinase from sheep brain have been grown from solutions of ammonium sulfate or Na+/K+ phosphate. The crystals are trigonal, space group P3(1)21 with axes a = b = 102.2 A and c = 58.5 A. The crystals are quite stable to x-rays and diffract at 2.2-A resolution. This macromolecule is a 80,000-kDa dimer and utilizes the 2-fold symmetry of the space group, resulting in one subunit/asymmetric unit.     

Expression, crystallization, and preliminary X-ray analysis of a sialic acid-binding fragment of sialoadhesin in the presence and absence of ligand.

Sialoadhesin is a macrophage-restricted cell surface receptor, consisting of 17 immunoglobulin domains, which mediates cell adhesion via the recognition of specific sialylated glycoconjugates. A functional fragment of sialoadhesin, comprising the N-terminal immunoglobulin domain, has been expressed in Chinese hamster ovary cells as both native (SnD1) and selenomethionyl (Se-SnD1) stop protein. The successful production of 86% selenomethionine-incorporated protein represents a rare example of production of selenium-labeled protein in mammalian cells. SnD1 and Se-SnD1 have been crystallized in the absence of ligand, and SnD1 has also been crystallized in the presence of its ligand 2,3 sialyllactose. The ligand-free crystals of SnD1 and Se-SnD1 were isomorphous, of space group P3(1)21 or P3(2)21, with unit cell dimensions a = b 38.9 A,c = 152.6 A, alpha = beta = 90 degrees, gamma = 120 degrees, and diffracted to a maximum resolution of 2.6 A. Cocrystals containing 2,3 sialyllactose diffracted to 1.85 A at a synchrotron source and belong to space group P2(1)2(1)2(1), with unit cell dimensions a = 40.9 A, b = 97.6 A,c = 101.6 A, alpha = beta = gamma = 90 degrees.     

Three-dimensional structure of catalase from Micrococcus lysodeikticus at 1.5 A resolution.

The three-dimensional crystal structure of catalase from Micrococcus lysodeikticus has been solved by multiple isomorphous replacement and refined at 1.5 A resolution. The subunit of the tetrameric molecule of 222 symmetry consists of a single polypeptide chain of about 500 amino acid residues and one haem group. The crystals belong to space group P4(2)2(1)2 with unit cell parameters a = b = 106.7 A, c = 106.3 A, and there is one subunit of the tetramer per asymmetric unit. The amino acid sequence has been tentatively determined by computer graphics model building and comparison with the known three-dimensional structure of beef liver catalase and sequences of several other catalases. The atomic model has been refined by Hendrickson and Konnert's least-squares minimisation against 94,315 reflections between 8 A and 1.5 A. The final model consists of 3,977 non-hydrogen atoms of the protein and haem group, 426 water molecules and one sulphate ion. The secondary and tertiary structures of the bacterial catalase have been analyzed and a comparison with the structure of beef liver catalase has been made.     

Crystals and a low resolution structure of interleukin-2

Recombinant derived human interleukin-2 and an analog in which cysteine 125 has been replaced with alanine have been crystallized in a form suitable for x-ray diffraction. The crystals are triclinic, space group P1, with two protein molecules in the unit cell; unit cell parameters are a = 55.8 A, b = 40.1 A, c = 33.7 A, alpha = 90.0 degrees, beta = 109.3 degrees, gamma = 93.2 degrees. The interleukin-2 structure has been solved to 5.5 A resolution using heavy atom isomorphous replacement methods. The resultant low resolution model reveals a significant fraction of alpha helical secondary structure and outlines the overall tertiary structure of the molecule.     

Crystallization and preliminary X-ray diffraction studies of HutP protein: an RNA-binding protein that regulates the transcription of hut operon in Bacillus subtilis

HutP is an RNA-binding protein and regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis by binding to cis-acting regulatory sequences on hut mRNA. HutP and its mutant, which has increased affinity for the regulatory sequences, were purified and crystallized by the hanging-drop vapor diffusion method. The space group was P2(1)3 with unit cell dimensions a=b=c=95.6A for HutP and a=b=c=96.8A for the mutant. Complete data sets of 3.0-A resolution for wild-type HutP and of 2.70-A resolution for the mutant HutP were collected.     

Dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia exhibits activity against a substrate containing a 4-hydroxyproline residue

The crystal structure of dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia was determined at 2.8-A resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space group P4(3)2(1)2, with unit cell parameters a = b = 105.9 A and c = 161.9 A. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal beta-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes. Stenotrophomonas dipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In the Stenotrophomonas enzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.     

Crystallization of isoelectrically homogeneous cholera toxin

Past difficulty in growing good crystals of cholera toxin has prevented the study of the crystal structure of this important protein. We have determined that failure of cholera toxin to crystallize well has been due to its heterogeneity. We have now succeeded in overcoming the problem by isolating a single isoelectric variant of this oligomeric protein (one A subunit and five B subunits). Cholera toxin purified by our procedure readily forms large single crystals. The crystal form (space group P2(1), a = 73.0 A, b = 92.2 A, c = 60.6 A, beta = 106.4 degrees, one molecule in the asymmetric unit) has been described previously [Sigler et al. (1977) Science (Washington, D.C.) 197, 1277-1278]. We have recorded data from native crystals of cholera toxin to 3.0-A resolution with our electronic area detectors. With these data, we have found the orientation of a 5-fold symmetry axis within these crystals, perpendicular to the screw dyad of the crystal. We are now determining the crystal structure of cholera toxin by a combination of multiple heavy-atom isomorphous replacement and density modification techniques, making use of rotational 5-fold averaging of the B subunits.     

Nonintercalative binding of proflavin to Z-DNA: structure of a complex between d(5BrC-G-5BrC-G) and proflavin

The crystal structure of a disordered 1:1 complex between the tetradeoxyoligomer d(5BrC-G-5BrC-G) and proflavin has been determined and refined to an R factor of 26.9% for 474 reflections initially in space group P6(5) and to an R factor of 22.2% for 475 reflections in space group P2(1), both at 2-A resolution with Fobsd greater than or equal to 4.0. The unit cell constants are a = b = 17.9 A, c = 44.5 A, and gamma = 120 degrees. The final models are essentially the same in the two space groups with greater disorder in space group P6(5). In space group P2(1), the asymmetric unit is a tetranucleotide duplex, two sandwiched proflavin molecules, and four "outside-bound" proflavins. The tetranucleotide duplex is in the Z conformation and is located at the origin of the unit cell with a pair of proflavins sandwiched between the tetranucleotides. Thus, the tetranucleotides and proflavin dimers stack alternatively forming a quasi-continuous helix with the helix axis coincident with the c axis. The structure analysis revealed the presence of outside-bound proflavins as well. It is interesting that one type of outside-bound proflavins occupies a similar environment as the cobalt hexaammines in their complex with the decadeoxyoligomer d(CGTACGTACG) [Brennan, R. G., Westhof, E., & Sundaralingam, M. (1986) J. Biomol. Struct. Dyn. 3, 649]. Crystals of the latter are isomorphous to the present complex. The outside-bound proflavins penetrate the deep minor groove, thereby closing it off, and provide a visualization of a quasi-internal mode of binding of proflavin to a nucleic acid.     

Preliminary X-ray diffraction studies of acyl carrier protein from Escherichia coli

Crystals of the acyl carrier protein of Escherichia coli have been grown and analyzed by X-ray diffraction. The crystals grow in space group C2 with unit cell dimensions a = 46.8 A, b = 52.1 A, c = 47.3 A and beta = 93.2 degrees. An isomorphous derivative, HgCl2, has been identified and characterized.     

Structure determination of histidine decarboxylase from Lactobacillus 30a at 3.0 A resolution

The crystal structure of histidine decarboxylase from Lactobacillus 30a has been determined by X-ray diffraction methods to a resolution of 3.0 A. This protein is a pyruvoyl-dependent enzyme that is formed by an unusual self-activation process. The structure was determined from an electron density map calculated using multiple isomorphous replacement phases from two heavy-atom derivatives and included contributions from anomalous scattering measurements. The final mean figure of merit was 0.79, based on 28,805 independent reflections. The molecule has an (alpha beta)6 subunit composition and crystallizes in the space group 14122 with a = b = 221.7 A and c = 107.1 A. There is one (alpha beta)3 half molecule per asymmetric unit. The (alpha beta)6 particle is dumbbell-shaped, with each (alpha beta)3 unit being approximately spherical, with a diameter of about 65 A. There is a large central cavity approximately 30 A deep around the molecular 3-fold axis of the (alpha beta)3 unit. The 3-fold related active site pockets are located around the bottom of this cavity and are separated from each other by a distance of approximately 23 A. The inner portion of each (alpha beta) unit, which lies near the interface between the two (alpha beta)3 particles, consists mainly of random coil with several small helical and sheet regions. The outer region of each (alpha beta) unit has an unusual structure consisting of two overlapping, predominantly antiparallel beta-pleated sheets, lined on each side by an alpha-helix. The walls of the central cavity are formed by the 3-fold repeat of two strands from this beta-sandwich structure and one of the helices.     

The molecular structure of the high potential iron-sulfur protein isolated from Ectothiorhodospira halophila determined at 2.5-A resolution

The molecular structure of a high potential iron-sulfur protein (HiPIP) isolated from the purple photosynthetic bacterium, Ectothiorhodospira halophila strain BN9626, has been solved by x-ray diffraction analysis to a nominal resolution of 2.5 A and refined to a crystallographic R value of 18.4% including all measured x-ray data from 30.0- to 2.5-A resolution. Crystals used in the investigation contained two molecules/asymmetric unit and belonged to the space group P21 with unit cell dimensions of a = 60.00 A, b = 31.94 A, c = 40.27 A, and beta = 100.5 degrees. An interpretable electron density map, obtained by combining x-ray data from one isomorphous heavy atom derivative with non-crystallographic symmetry averaging and solvent flattening, clearly showed that this high potential iron-sulfur protein contains 71 amino acid residues, rather than 70 as originally reported. As in other bacterial ferredoxins, the [4Fe-4S] cluster adopts a cubane-like conformation and is ligated to the protein via four cysteinyl sulfur ligands. The overall secondary structure of the E. halophila HiPIP is characterized by a series of Type I and Type II turns allowing the polypeptide chain to wrap around the [4Fe-4S] prosthetic group. The hydrogen bonding pattern around the cluster is nearly identical to that originally observed in the 85-amino acid residue Chromatium vinosum HiPIP and consequently, the 240 mV difference in redox potentials between these two proteins cannot be simply attributed to hydrogen bonding patterns alone.     

Single and twinned crystals of ribulose-1,5-bisphosphate carboxylase-oxygenase from Alcaligenes eutrophus

Ribulose-1,5-bisphosphate carboxylase-oxygenase (L8S8) from Alcaligenes eutrophus has been crystallized by equilibrium vapor diffusion techniques with ammonium sulfate as precipitant. Crystals thus obtained either as the ternary complex with CO2 and Mg2+ or as the quaternary complex with CO2, Mg2+, and 2-carboxyarabinitol 1,5-bisphosphate, a transition state analogue, diffract at least to 2.8-A resolution. Both are essentially isomorphous to each other, having orthorhombic space group C222(1) with cell dimensions a = 159 A, b = 159 A, and c = 200 A, and there is half a molecule in the asymmetric unit. The crystals of the ternary complex are sometimes twinned about the c axis so that the space group appears to be tetragonal. In this light, our earlier report (Bowien, B., Mayer, F., Spiess, E., P?hler, A., Englisch, U., and Saenger, W. (1982) Eur. J. Biochem. 106, 405-410) on a tetragonal space group P4(2)2(1)2 with crystals obtained from the same enzyme with Mg2+ and CO2 but without 2-carboxyarabinitol 1,5-bisphosphate might be incorrect.