Marvin L. Hackert

The Structural and Functional Analysis of the Hemoglobin D Component from Chicken

Oxygen binding by chicken blood shows enhanced cooperativity at high levels of oxygen saturation. This implies that deoxy hemoglobin tetramers self-associate. The crystal structure of an R-state form of chicken hemoglobin D has been solved to 2.3-Å resolution using molecular replacement phases derived from human oxyhemoglobin. The model consists of an α2β2 tetramer in the asymmetric unit and has been refined to a R-factor of 0.222 (R-free = 0.257) for 29,702 reflections between 10.0- and 2.3-Å resolution. Chicken Hb D differs most from human oxyhemoglobin in the AB and GH corners of the α subunits and the EF corner of the β subunits. Reanalysis of published oxygen binding data for chicken Hbs shows that both chicken Hb A and Hb D possess enhanced cooperativity in vitro when inositol hexaphosphate is present. The electrostatic surface potential for a calculated model of chicken deoxy-Hb D tetramers shows a pronounced hydrophobic patch that involves parts of the D and E helices of the β subunits. This hydrophobic patch is a promising candidate for a tetramer-tetramer interface that could regulate oxygen binding via the distal histidine.

Crystallization of a dihydrolipoyl transacetylase-dihydrolipoyl dehydrogenase subcomplex and its implications regarding the subunit structure of the pyruvate dehydrogenase complex from Escherichia coli

Abstract A subcomplex consisting of dihydrolipoyl transacetylase and dihydrolipoyl dehydrogenase, two of the three enzymes comprising the Escherichia coli pyruvate dehydrogenase complex, has been crystallized. X-ray diffraction data establish that the space group is P 2 1 3 with unit cell dimension a=211 .5 A . The unit cell contains four molecules of the subcomplex, each possessing 3-fold crystallographic and molecular symmetry. This finding, together with biochemical and electron microscopic data reported elsewhere, establish unequivocally that dihydrolipoyl transacetylase, the core enzyme of the pyruvate dehydrogenase complex, consists of 24 identical subunits with octahedral (432) symmetry. In the case presented here, the 432 symmetry of the transacetylase is reduced to 3-fold symmetry in the subcomplex by the addition of dihydrolipoyl dehydrogenase subunits. Crystal density measurements indicate that the dihydrolipoyl transacetylase present in these crystals is considerably smaller than the core mass generally reported for intact transacetylase. The implications of these findings are discussed with respect to the subunit stoichiometry and structure of the E. coli pyruvate dehydrogenase complex.

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.

Crystallization of C-phycocyanin from the marine blue-green alga Agmenellum quadruplicatum

The biliprotein photoreceptor, C-phycocyanin, from the blue-green alga Agmenellum quadruplicatum, was crystallized from ammonium sulfate solutions using microdiffusion cells. The prismatic crystals belong to the trigonal space group P321 with unit cell dimensions, a=b=184·5 A, c=60·5 A and γ=120°. Density measurements of these crystals indicate that the unit cell contains 36 (αβ) units. A sublattice feature associated with low-order hk0 X-ray reflections and the intensity distribution along the h00 lattice row suggests that these units are probably aggregated into three (αβ)12 entities per unit cell. The symmetry and size of the unit cell suggest that these C-phycocyanin oligomers possess 32 molecular symmetry and restrict their approximate dimensions to a disk 106·5 A in diameter and 60°5 A in thickness. There is a striking resemblance between the resulting crystal packing of these C-phycocyanin units and the association of similar units proposed in models of the phycobilisomes.

The quaternary structure of a unique phycobiliprotein: B-phycoerythrin from Porphyridium cruentum

B-phycoerythrin, from the unicellular red alga Porphyridium cruentum, was crystallized in the rhombohedral space group R3 with a=111.0A and α=116.8° or A=B=189.1A and C=60.1A and γ=120°. Density measurements on the crystals indicate that the hexagonal unit cell can acconmodate three cylindrical molecules, 109A in diameter and 60A in height, each of approximately 275,000 daltons. The crystallographic symmetry of the unit cell requires at least 3-fold symmetry for the particle. However, the particle stoichiometry has been reported as (αβ)6γ and this composition is also supported by SDS gel electrophoresis on the crystalline protein. These results are discussed in light of preliminary model calculations on the quaternary structure of B-phycoerythrin.

Three-dimensional structure of the Gly121Tyr dimeric form of ornithine decarboxylase from Lactobacillus 30a.

Ornithine decarboxylases catalyze the conversion of ornithine to putrescine at the beginning of the polyamine pathway. Ornithine decarboxylase (ODC) from Lactobacillus 30a is a 990612 Da dodecamer composed of six homodimers. A single point mutation (Gly121Tyr) was found to prevent association of dimers into dodecamers. The dimeric protein has been crystallized at pH 7.0 in the presence of guanosine triphosphate (GTP). Crystals belong to space group P3(2)21, with unit-cell parameters a = 111.8, c = 135.9 A and one monomer in the asymmetric unit. The structure was determined by molecular replacement and refined using simulated annealing to R = 0.211 at 2. 7 A resolution. The GTP-binding site was analyzed in detail. The protein exhibits a novel binding mode for GTP which is different from that seen in most G-proteins or GTPases. Central to this binding scheme appear to be three lysines, Lys190, Lys374 and Lys382, which form salt bridges with the three phosphates, and Thr191, which hydrogen bonds with the guanine base. Furthermore, the structure suggests that there is some flexibility in the wing domain, which can change its orientation as the protein adapts to its environment. The active site is similar to that of the native enzyme, consistent with the observation that the enzyme activity does not depend on its dodecameric state.

Coordination chemistry of alkali and alkaline earth cations: Synthesis and X-ray crystal structure of cesium (picrate) (benzo-15-crown-5) Cs+C6H2N3O7 -(C14H20O5)

Crystals of the Cs+ Pic− (B15C5) complex (Pic=picrate; B15C5=benzo-15-crown-5) (Mr=629.3) are yellow prisms which belong to the triclinic space groupPī witha=7.377(4),b=11.372(2),c=14.806(2) Å, α=90.31(1), β=91.06(2), γ=108.32(2)0,Z=2,Dx=1.77, andDm=1.77 g cm−3. FinalR=0.055 for 3575 observed reflections out of a total of 4004 measured reflections. Cesium forms a 1:1 anion paired complex with B15C5 like sodium rather than a charge separated sandwich structure as found for potassium and expected for cesium in view of the ion-cavity radius concept. The Cs cation is 9-coordinate involving the five crown oxygens (Cs...O, 3.00(1) to 3.24(1) Å), the phenoxide (Cs...O−, 3.03(1) Å) and anortho nitro group oxygen (Cs...O, 3.01(1) Å) of the picrate counteranion and, uniquely, with two additional oxygens (Cs...O, 3.17(1) and 3.40(1) Å) from apara nitro group of the picrate belonging to the adjacent molecule in the lattice. The Cs+ ion lies 2.07 Å out of the mean plane formed by the crown oxygens. This system provides the first structural evidence that the interaction stoichiometry of an alkali cation with a cyclic multidentate ligand is not a function of the ion and cavity size alone but also of its Lewis acid strength as modified by the charge neutralizing anion.

Crystal structures of native and inactivated cis-3-chloroacrylic acid dehalogenase: Implications for the catalytic and inactivation mechanisms.

The isomeric mixture of cis- and trans-1,3-dichloropropene constitutes the active component of a widely used nematocide known as Telone II®. The mixture is processed by various soil bacteria to acetaldehyde through the 1,3-dichloropropene catabolic pathway. The pathway relies on an isomer-specific hydrolytic dehalogenation reaction catalyzed by cis- or trans-3-chloroacrylic acid dehalogenase, known respectively as cis-CaaD and CaaD. Previous sequence analysis and crystallographic studies of the native and covalently modified enzymes identified Pro-1, His-28, Arg-70, Arg-73, Tyr-103, and Glu-114 as key binding and catalytic residues in cis-CaaD. Mutagenesis of these residues confirmed their importance to the dehalogenation reaction. Crystal structures of the native enzyme (2.01Å resolution) and the enzyme covalently modified at the Pro-1 nitrogen by 2-hydroxypropanoate (1.65Å resolution) are reported here. Both structures are at a resolution higher than previously reported (2.75Å and 2.1Å resolution, respectively). The conformation of the covalent adduct is strikingly different from that previously reported due to its interaction with a 7-residue loop (Thr-32 to Leu-38). The participation of another active site residue, Arg-117, in catalysis and inactivation was also examined. The implications of the combined findings for the mechanisms of catalysis and inactivation are discussed.

Three-dimensional structure of a hemichrome hemoglobin from Caudina arenicola

The structure of a monomeric hemichrome form of an invertebrate hemoglobin, Hb-C chain, from Caudina arenicola has been refined to an R value of 0.16 using the data from 5.0 to 2.5 A resolution (R = 0.21 from 10.0 to 2.5 A resolution). Hb-C crystallizes in space group P2(l) with cell constants a = 45.74, b = 45.23 and c = 40.92 A and beta = 104.4 degrees with two monomers packed in the unit cell (V(m) = 2.34 A(3) Da(-1)). The phases were determined by the multiple isomorphous replacement method with Hg(2+) the major derivative. The structure consists of 157 amino acids with N- and C-terminal regions and eight alpha-helices forming a heme pocket. The unique feature of this structure is the hemichrome form with the proximal and distal histidines coordinated to the heme Fe atom, which is nearly in the plane of the porphyrin ring. A total of 111 solvent molecules were added to the structure using difference density peaks of at least 3sigma over background. Interestingly, all the heme groups present in the crystal are nearly coplanar.

Crystallization of biosynthetic arginine decarboxylase from Escherichia coli

Putrescine is the immediate precursor for the synthesis of polyamines and is normally generated by the action of ornithine decarboxylase. However, putrescine can also be produced by the conversion of arginine to agmatine by arginine decarboxylase (bADC) followed by the release of urea by agmatine ureohydrolase. Amino-acid sequence homology with the eukaryotic ornithine decarboxylases suggests that bADC may be a model for this group of decarboxylases. We report here the crystallization of arginine decarboxylase from E. coli. Crystals up to 1 mm in size are grown by vapor equilibration using Li(2)SO(4) and polyethylene glycols as precipitants. The crystals exhibit diffraction maxima beyond 3 A resolution and belong to space group P4(1(3))2(1)2 with a = 192.4 and c = 121.0 A. These unit-cell dimensions together with the estimated density of the crystals suggest the presence of one tetramer of bADC (71 kDa subunit(-1)) per asymmetric unit (V(m) = 2.0 A(3) Da(-1)).