The structure of human recombinant interleukin lp 8) has been refined by a restrained least-squares method to a crystallographic R factor of 17.2% to 2.0 A resolution. One-hundred sixty-eight solvent molecules have been located, and isotropic temperature factors for each atom have been refined. The overall structure is composed of 12 ,8-strands that can best be described as forming the four triangular faces of a tetrahedron with hydrogen bonding resembling normal antiparallel fl-sheets only at the vertices. Interleukin 1 (IL-1) is a member of a family of cellular mediators known as cytokines. Two distinct species of IL-1 have been characterized, IL-la and IL-1,3. They are about 23% identical in amino acid sequence (1, 2). Both molecules are expressed as a 31-kDa precursor upon activation of a producer cell. The precursor is processed to give a 17.4-kDa carboxyl fragment, which is the mature IL-1f3 molecule (17.8-kDa carboxyl fragment for IL-la). The IL-la precursor is also active, whereas the precursor of IL-1,8 is inactive (3).Processing of the precursor is not well understood. There is no leader sequence (4), and cleavage is believed to be carried out by serine proteases (5, 6). It has been shown that IL-la and IL-1,i bind to the same IL-i-specific receptor (7 Crystals were grown from buffered ammonium sulfate as described (27). The space group is P43, with a = b = 54.9 A and c = 76.8 A with one molecule per asymmetric unit. The structure was solved to 3.0 A resolution (28) by the multiple isomorphous replacement method with two heavy atom derivatives, p-hydroxymercuribenzoate and UO2(NO3)2 combined with phase improvement using solvent flattening (29).High-resolution diffraction data (to 2.0 A) were collected on the X-11 beamline at the Deutsche Electronen Synchrotron at the European Molecular Biology Laboratory Outstation, Hamburg, Federal Republic of Germany, set to a wavelength of 1.464 A. Five randomly oriented crystals were used. Exposure time was 67-157 sec per film pack, depending upon beam strength. An oscillation angle of 1.50 with no overlap between successive films was used. Diffraction data were collected on Kodak DEFO film with two films per film pack. The films were digitized on an Optronics P-1000 film microdensitometer with a 50-gkm raster and a 3.0 optical density scale. The orientation of the crystals was determined by averaging the results of the autoindexing procedure of Kabsch (30) for the first, the last, and a middle film from any one crystal. Typically, 10 films (15°) of data could be collected from one crystal before radiation damage was deemed too severe as judged by visual inspection of the highresolution data on the films. The MoSCO film processing package as integrated into the CCP4 protein crystallographic program package (Darsbury) was used. The results of film processing are shown in Table 1. Despite the random orientation of the crystals, the oscillation data set was about 90% complete, with most of the missing data being at low resolution due to saturation of the spots...

The crystal structure of human recombinant interleukin‐1 beta has been determined at 3.0 A resolution by the isomorphous replacement method in conjunction with solvent flattening techniques. The model prior to refinement has a crystallographic R‐factor of 42.3%. The structure is composed of 12 beta‐strands forming a complex network of hydrogen bonds. The core of the structure can best be described as a tetrahedron whose edges are each formed by two antiparallel beta‐strands. The interior of this structure is filled with hydrophobic side chains. There is a 3‐fold repeat in the folding of the polypeptide chain. Although this folding pattern suggests gene triplication, no strong internal sequence homology between topologically corresponding residues exists. The folding topology of interleukin‐1 beta is very similar to that described by McLachlan (1979) J. Mol. Biol., 133, 557‐563, for soybean trypsin inhibitor.

Structures have been determined of Bacillus stearothermophilus "apo" and holo lactate dehydrogenase. The holo-enzyme had been co-crystallized with the activator fructose 1,6-bisphosphate. The "apo" lactate dehydrogenase structure was solved by use of the known apo-M4 dogfish lactate dehydrogenase molecule as a starting model. Phases were refined and extended from 4 A to 3 A resolution by means of the noncrystallographic molecular 222 symmetry. The R-factor was reduced to 28.7%, using 2.8 A resolution data, in a restrained least-squares refinement in which the molecular symmetry was imposed as a constraint. A low occupancy of coenzyme was found in each of the four subunits of the "apo"-enzyme. Further refinement proceeded with the isomorphous holo-enzyme from Bacillus stearothermophilus. After removing the noncrystallographic constraints, the R-factor dropped from 30.3% to a final value of 26.0% with a 0.019 A and 1.7 degrees r.m.s. deviation from idealized bond lengths and angles, respectively. Two sulfate ions per subunit were included in the final model of the "apo"-form--one at the substrate binding site and one close to the molecular P-axis near the location of the fructose 1,6-bisphosphate activator. The final model of the holo-enzyme incorporated two sulfate ions per subunit, one at the substrate binding site and another close to the R-axis. One nicotinamide adenine dinucleotide coenzyme molecule per subunit and two fructose 1,6-bisphosphate molecules per tetramer were also included. The phosphate positions of fructose 1,6-bisphosphate are close to the sulfate ion near the P-axis in the "apo" model. This structure represents the first reported refined model of an allosteric activated lactate dehydrogenase. The structure of the activated holo-enzyme showed far greater similarity to the ternary complex of dogfish M4 lactate dehydrogenase with nicotinamide adenine dinucleotide and oxamate than to apo-M4 dogfish lactate dehydrogenase. The conformations of nicotinamide adenine dinucleotide and fructose 1,6-bisphosphate were also analyzed.

Dihydrodipicolinate synthase (EC 4.2.1.52), the first enzyme unique to lysine biosynthesis in bacteria and higher plants, has been purified to homogeneity from etiolated pea (Pisum sativum) seedlings using a combination of conventional and affinity chromatographic steps. This is the first report on a homogeneous preparation of native dihydrodipicolinate synthase from a plant source. The pea dihydrodipicolinate synthase has an apparent molecular weight of 127,000 and is composed of three identical subunits of 43,000 as determined by gel filtration and crosslinking experiments. The tnmenc quaternary structure resembles the trimenc structure of other aldolases, such as 2-keto-3-deoxy-6-phosphogluconic acid aldolase, which catalyze similar aldol condensations. The amino acid compositions of dihydrodipicolinate synthase from pea and Escherichia coli are similar, the most significant difference concems the methionine content: dihydrodipicolinate synthase from pea contains 22 moles of methionine residue per mole of native protein, contrary to the E. coli enzyme, which does not contain this amino acid at all. Dihydrodipicolinate synthase from pea is highly specific for the substrates pyruvate and L-aspartate-fl-semialdehyde; it follows Michaelis-Menten kinetics for both substrates. The pyruvate and L-aspartate-ft-semialdehyde have Michaelis constant values of 1.70 and 0.40 millimolar, respectively. L-Lysine, S-(2-aminoethyl)-L-cysteine, and La-(2-aminoethoxyvinyl)glycine are strong allostenc inhibitors of the enzyme with 50% inhibitory values of 20, 160, and 155 millimolar, respectively. The inhibition by L-lysine and L-a-(2-aminoethoxyvinyl)glycine is noncompetitive towards L-aspartateBl-semialdehyde, whereas S-(2-aminoethyl)-L-cysteine inhibits dihydrodipicolinate synthase competitively with respect to L-aspartate-jt-semialdehyde. Furthermore, the addition of (2R,3S,6S)-2,6-diamino-3-hydroxy-heptandioic acid (1.2 millimolar) and (2S,6R/S)-2,6-diamino-6-phosphono-hexanic acid (1.2 millimolar) activates dihydrodipicolinate synthase from pea by a factor of 1.4 and 1.2, respectively. This is the first reported activation process found for dihydrodipicolinate synthase.

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