The refinement and the structure of the dimer of alpha-chymotrypsin at 1.67-A resolution

The two molecules of the asymmetric unit of the pH 3.5 conformer of alpha-chymotrypsin have been refined at 1.67-A resolution using restrained least squares methods with Hendrickson's program (PROLSQ). The final R factor is 0.179 (including 247 water molecules). The folding of the main chain of the independent molecules is the same within experimental error but the same does not generally apply to the side chain stereochemistry. From this we conclude that the folding of a protein structure is basically independent of most of the detailed stereochemistry of its side chains. The side chains of the interface region between the independent molecules display pronounced asymmetry. This asymmetry suggests that dynamic and asymmetrical structural changes take place at the time of oligomerization leading to more energetically favorable interactions for the dimer. Comparison of the structures of the independent molecules of alpha-chymotrypsin with the structure of monomeric gamma-chymotrypsin revealed that although the folding of the three molecules is essentially the same, numerous and significant differences pervade the side chain stereochemistry attributable to general flexibility. The specificity site of alpha-chymotrypsin is occupied by ordered water molecules in a similar way to gamma-chymotrypsin and other proteins. Some of these water molecules are displaced when substrate binds to the enzyme, while the others appear to help identify and position the aromatic side chain in catalysis.     

Horse heart metmyoglobin. A 2.8-A resolution three-dimensional structure determination

The structure of horse heart metmyoglobin has been determined with a molecular replacement approach and subsequently refined using rigid body and restrained-parameter least squares methods to a conventional crystallographic R-factor of 0.16 for all observed reflections in the 6.0-2.8-A resolution range. The polypeptide chain of this protein is found to be organized into eight helical regions (labeled A-H) which collectively form a hydrophobic pocket in which the heme prosthetic group is bound. Our results show that the overall thermal motions of individual residues of horse heart metmyoglobin are correlated with their mean distances from the heme group. In comparisons with the structure of sperm whale metmyoglobin it has been found that horse heart metmyoglobin has unique polypeptide chain conformations in four regions. These include residues in the immediate vicinity of the amino and carboxyl termini, residues about Lys-16, and residues 117-124 which are in the interhelical region between helices G and H. Many of these conformational changes appear to occur as a consequence of a different pattern of salt-bridging interactions between charged residues on the surface of horse heart metmyoglobin. The overall average positional deviation observed between corresponding alpha-carbons in the polypeptide chains of horse heart and sperm whale metmyoglobin is 0.50 A. This value for atoms of the porphyrin core of the central heme group is 0.39 A. A total of 12 well defined water molecules and 1 sulfate ion are included in the current structural model of horse heart metmyoglobin. One of these water molecules is found to be coordinated to the heme iron atom and hydrogen bonded to the side chain of His-64. The sulfate ion is hydrogen bonded to amide groups at the amino-terminal end of the E-helix and, as well, forms similar interactions with the amino-terminal end of the D-helix of an adjacent protein molecule in the crystalline lattice.     

The structure of a complex of bovine alpha-thrombin and recombinant hirudin at 2.8-A resolution.

Crystals of the complex of bovine alpha-thrombin with recombinant hirudin variant 1 have space group C222(1) with cell constants a = 59.11, b = 102.62, and c = 143.26 A. The orientation and position of the thrombin component was determined by molecular replacement and the hirudin molecule was fit in 2 magnitude of Fo - magnitude of Fc electron density maps. The structure was refined by restrained least squares and simulated annealing to R = 0.161 at 2.8-A resolution. The binding of hirudin to thrombin is generally similar to that observed in the crystals of human thrombin-hirudin. Several differences in the interactions of the COOH-terminal polypeptide of hirudin, specifically of residues Asp-55h, Phe-56h, Glu-57h, and Glu-58h, and a few differences in the interactions of the hirudin core, specifically of residues Asp-5h, Ser-19h, and Asn-20h, with thrombin from human thrombin-hirudin suggest that there is some flexibility in the binding of these 2 molecules. Most of the residues in the 9 subsites that bind fibrinopeptide A7-16 to thrombin also interact with the NH2-terminal domain of hirudin. The S1 subsite is a notable exception in that only 1 of its 6 residues, namely Ser-214, interacts with hirudin. The only difference between human and bovine thrombins that appears to influence the binding of hirudin is the replacement of Lys-149E by an acidic glutamate in the bovine enzyme.     

Crystal structure of an open conformation of citrate synthase from chicken heart at 2.8-A resolution

The X-ray structure of a new crystal form of chicken heart muscle citrate synthase, grown from solutions containing citrate and coenzyme A or L-malate and acetyl coenzyme A, has been determined by molecular replacement at 2.8-A resolution. The space group is P4(3) with a = 58.9 A and c = 259.2 A and contains an entire dimer of molecular weight 100,000 in the asymmetric unit. Both "closed" conformation chicken heart and "open" conformation pig heart citrate synthase models (Brookhaven Protein Data Bank designations 3CTS and 1CTS) were used in the molecular replacement solution, with crystallographic refinement being initiated with the latter. The conventional crystallographic R factor of the final refined model is 19.6% for the data between 6- and 2.8-A resolution. The model has an rms deviation from ideal values of 0.034 A for bond lengths and of 3.6 degrees for bond angles. The conformation of the enzyme is essentially identical with that of a previously determined "open" form of pig heart muscle citrate synthase which crystallizes in a different space group, with one monomer in the asymmetric unit, from either phosphate or citrate solution. The crystalline environment of each subunit of the chicken enzyme is different, yet the conformation is the same in each. The open conformation is therefore not an artifact of crystal packing or crystallization conditions and is not species dependent. Both "open" and "closed" crystal forms of the chicken heart enzyme grow from the same drop, showing that both conformations of the enzyme are present at equilibrium in solution containing reaction products or substrate analogues.     

Structure of a tetrahedral transition state complex of alpha-chymotrypsin dimer at 1.8-A resolution

A 1.8-A resolution x-ray crystallographic restrained least squares refinement has been carried out on the phenylethane boronic acid (PEBA) complex of alpha-chymotrypsin dimer (alpha-CHT), and it has been compared to the 1.67-A resolution structure of the native enzyme. PEBA has a high binding affinity for alpha-CHT, and the boronate forms a tetrahedral complex with Ser-195 OG of one molecule of the dimer; the boronate in the other molecule is severely disordered and does not form a tetrahedral complex. The former could be a model of the transition state of catalysis. The complex of PEBA X alpha-CHT displays significant nonequivalence in conformation of side chains between the independent molecules comparable to the native enzyme, but, like the latter, shows a high degree of fidelity in the folding of the main chain. The orientation of the phenyl ring, CA and CB of PEBA, in the specificity sites of the two molecules is similar, suggesting that recognition is fairly insensitive to small departures from local symmetry; the same does not apply to the boronate functionalities suggesting that greater precision is required for catalysis. The folding of the molecule remains the same upon PEBA binding, but some of the side chains respond nonequivalently. The latter is a consequence of the inherent nonequivalence of the native dimer and the asymmetrical nature of the PEBA binding.     

Crystal structure of Enterococcus hirae enolase at 2.8 A resolution

We report the crystal structure of an enolase from Enterococcus hirae, which is the first report of a structure determination among gram-positive bacteria. We isolated the enolase gene and determined the base sequence. The amino acid sequence deduced from the DNA sequence suggests that this enolase is composed of 431 amino acids. The amino acid sequence is very similar to those of enolases from eukaryotic and prokaryotic organisms, being 65% and 50% identical to enolases from Escherichia coli and yeast, respectively. The enolase prepared from E. hirae lysate yielded crystals containing one dimer per asymmetric unit. X-ray diffraction patterns were obtained at 2.8 A resolution on a SPring-8 synchrotron radiation source. Crystals belong to space group I4 with unit cell dimensions of a = b = 153.5 A, c = 90.7 A. The E. hirae, yeast, E. coli and lobster enolase structures are very similar. The E. hirae enolase takes an "Open" conformation. The regions in the structure that differ most from other enolases are loops L4 (132-140) and L3 (244-265). Considering the positions of these loops relative to the active site, they seem to have no direct involvement in function. Our findings show that the three dimensional structure of an important enzyme in the glycolytic pathway is evolutionarily conserved among eukaryotes and prokaryotes, including gram-positive bacteria.     

Molecular structure of serum transferrin at 3.3-A resolution

Serum transferrin is a metal-binding glycoprotein, molecular weight ca. 80,000, whose primary function is the transport of iron in the plasma of vertebrates. The X-ray crystallographic structure of diferric rabbit serum transferrin has been determined to a resolution of 3.3 A. The molecule has a beta alpha structure of similar topology to human lactoferrin and is composed of two homologous lobes that each bind a single ferric ion. Each lobe is further divided into two dissimilar domains, and the iron-binding site is located within the interdomain cleft. The iron is bound by two tyrosines, a histidine, and an aspartic acid residue. The location of the 19 disulfide bridges is described, and their possible structural roles are discussed in relation to the transferrin family of proteins. Mapping of the intron/exon splice junctions onto the molecule provides some topological evidence in support of the putative secondary role for transferrin in stimulating cell proliferation.     

The structure of ferrocytochrome b5 at 2.8 A resolution.

Crystals of cytochrome b5 reduced by sodium dithionite are isomorphous with the oxidized form. An electron density difference map between the two forms was calculated at 2.8 A resolution. There are no changes in main chain conformation or internal side chain orientation upon reduction. However, an ion becomes attached at the entrance of the heme crevice causing displacement of a surface lysine side chain on an adjacent molecule. The ion, identified as a cation by the nature of its coordinating ligands, appears to neutralize one of the heme propionate groups which is partially buried. It is proposed that the negatively charged propionate serves to neutralize the net formal positive charge on the heme iron in the oxidized cytochrome and that the neutralization of the heme iron upon reduction then leads to binding of a cation to the propionate.     

Draft crystal structure of the vault shell at 9-A resolution

Vaults are the largest known cytoplasmic ribonucleoprotein structures and may function in innate immunity. The vault shell self-assembles from 96 copies of major vault protein and encapsulates two other proteins and a small RNA. We crystallized rat liver vaults and several recombinant vaults, all among the largest non-icosahedral particles to have been crystallized. The best crystals thus far were formed from empty vaults built from a cysteine-tag construct of major vault protein (termed cpMVP vaults), diffracting to about 9-A resolution. The asymmetric unit contains a half vault of molecular mass 4.65 MDa. X-ray phasing was initiated by molecular replacement, using density from cryo-electron microscopy (cryo-EM). Phases were improved by density modification, including concentric 24- and 48-fold rotational symmetry averaging. From this, the continuous cryo-EM electron density separated into domain-like blocks. A draft atomic model of cpMVP was fit to this improved density from 15 domain models. Three domains were adapted from a nuclear magnetic resonance substructure. Nine domain models originated in ab initio tertiary structure prediction. Three C-terminal domains were built by fitting poly-alanine to the electron density. Locations of loops in this model provide sites to test vault functions and to exploit vaults as nanocapsules.     

The structure of guanosine-thymidine mismatches in B-DNA at 2.5-A resolution

The structure of the deoxyoligomer d(C-G-C-G-A-A-T-T-T-G-C-G) was determined at 2.5-A resolution by single crystal x-ray diffraction techniques. The final R factor is 18% with the location of 71 water molecules. The oligomer crystallizes in a B-DNA-type conformation, with two strands interacting to form a dodecamer duplex. The double helix consists of four A X T and six G X C Watson-Crick base pairs and two G X T mismatches. The G X T pairs adopt a "wobble" structure with the thymine projecting into the major groove and the guanine into the minor groove. The mispairs are accommodated in the normal double helix by small adjustments in the conformation of the sugar phosphate backbone. A comparison with the isomorphous parent compound containing only Watson-Crick base pairs shows that any changes in the structure induced by the presence of G X T mispairs are highly localized. The global conformation of the duplex is conserved. The G X T mismatch has already been studied by x-ray techniques in A and Z helices where similar results were found. The geometry of the mispair is essentially identical in all structures so far examined, irrespective of the DNA conformation. The hydration is also similar with solvent molecules bridging the functional groups of the bases via hydrogen bonds. Hydration may be an important factor in stabilizing G X T mismatches. A characteristic of Watson-Crick paired A X T and G X C bases is the pseudo 2-fold symmetry axis in the plane of the base pairs. The G X T wobble base pair is pronouncedly asymmetric. This asymmetry, coupled with the disposition of functional groups in the major and minor grooves, provides a number of features which may contribute to the recognition of the mismatch by repair enzymes.     

The crystal structure of ribonuclease B at 2.5-A resolution

The glycosylated form of bovine pancreatic ribonuclease, RNase B, was crystallized from polyethylene glycol 4000 at low ionic strength in space group C2 with unit cell dimensions of a = 101.81 A, b = 33.36 A, c = 73.60 A, and beta = 90.4 degrees. The crystals, which contained two independent molecules of RNase B as the asymmetric unit, were solved by a combination of multiple isomorphous replacement and molecular replacement approaches. The structures of the two molecules were refined to 2.5-A resolution and a conventional R factor of 0.22 using a constrained-restrained least squares procedure (CORELS). Complexes were also investigated of RNase B plus ruthenium pentaamine and between RNase B and a substrate analogue iodouridine. The polypeptide backbones of the two molecules of RNase B in the asymmetric unit were found to be statistically identical and their differences from RNase A to be statistically insignificant. The carbohydrate chains of both molecules extended into solvent cavities in the crystal lattice and appear to be disordered for the most part. The oligosaccharides appear to exert no influence on the structure of the protein. Iodouridine was observed to bind identically in the pyrimidine site of both RNase B molecules and in a way apparently the same as that previously observed for RNase A. Ruthenium pentaamine bound at histidine 105 of both RNase B molecules in the asymmetric unit, but at a number of secondary sites as well. An array of bound ions was observed by Fo-Fc difference Fourier syntheses. These ions were proximal to lysine and arginine residues at the surface of the proteins while a pair of strong ion binding sites were seen to fall exactly in the active site clefts of both RNase B molecules in the asymmetric unit.     

Crystal structure of recombinant rabbit interferon-gamma at 2.7-A resolution

The crystal structure of recombinant rabbit interferon-gamma was solved by the multiple isomorphous replacement technique at 2.7-A resolution and refined to a crystallographic R-factor of 26.2%. The interferon crystallizes with one-half of the functional dimer in the asymmetric unit, with the two polypeptide chains of the dimer related by a crystallographic 2-fold symmetry axis. The structure is predominantly alpha-helical with extensive interdigitation of the alpha-helical segments of the two polypeptide chains.     

The structure of prothrombin fragment 1 at 3.5-A resolution

The structure of prothrombin fragment 1 has been determined at 3.5-A resolution by multiple isomorphous replacement methods with four heavy atom derivatives. The final average figure of merit is 0.72. There is a large cylindrical solvent region with an average diameter of 35-40 A along the entire length of the c axis (85 A) centered at about x = y = 1/2. The connected density forming the wall of this channel is not of sufficient extent to account for the 156 residues of fragment 1 and the two accompanying carbohydrate chains totaling 5000 in molecular weight. Deglycosylated fragment 1 crystallizes isomorphously with fragment 1, and a difference map between the two revealed that the sugar chains are severely disordered and reside in the solvent channel. Although the disordered carbohydrate and the complexity of five disulfides in a 126-residue sequence have hampered the complete tracing of the peptide chain, two-thirds of the molecule has been accounted for in the form of an unusually oblate ellipsoid of about 15 X 30 X 35 A. The folding of the molecule has little secondary structure (one alpha-helix (7%), 20% beta-structure) in agreement with dichroism measurements and one of the points of carbohydrate attachment is suggested from the deglycosylated difference map.     

The crystal structure of erabutoxin a at 2.0-A resolution

The three-dimensional structure of erabutoxin a, a single-chain, 62-residue protein neurotoxin from snake venom, has been determined to 2.0-A resolution by x-ray crystal structure analysis. Molecular replacement methods were used, and the structure refined to a residual R = 0.17. The sites of 62 water molecules and 1 sulfate ion have been located and refined. The structure of erabutoxin a is very similar to that established earlier for erabutoxin b. These toxins from venom of the same snake differ in sequence only at residue 26, which is Asn in erabutoxin a and His in erabutoxin b. The substitution leads to only minor variations in intramolecular hydrogen bonding. Furthermore, the distribution of thermal parameters and the implied regional mobilities are similar in the two structures. In particular, the highly mobile character of the peripheral segment Pro44-Gly49 in both structures supports the specific role proposed for this segment in neurotoxin binding to the acetylcholine receptor. Forty-eight of the solvent sites determined are first surface positions; approximately one-half of these are equivalent to solvent sites in erabutoxin b.     

X-ray crystal structure of D-xylose isomerase at 4-A resolution

The structure of D-xylose isomerase from Streptomyces rubiginosus has been determined at 4-A resolution using multiple isomorphous phasing techniques. The folding of the polypeptide chain has been established and consists of two structural domains. The larger domain consists of eight beta-strand alpha-helix (beta alpha) units arranged in a configuration similar to that found for triose phosphate isomerase, 2-keto-3-deoxy-6-phosphogluconate aldolase, and pyruvate kinase. The smaller domain forms a loop away from the larger domain but overlapping the larger domain of another subunit so that a tightly bound dimer is formed. The tetramer then consists of two such dimers. The location of the active site in the enzyme has been tentatively identified from studies using a crystal grown from a solution containing the inhibitor xylitol.     

Molecular structure of an apolipoprotein determined at 2.5-A resolution

The three-dimensional structure of an apolipoprotein isolated from the African migratory locust Locusta migratoria has been determined by X-ray analysis to a resolution of 2.5 A. The overall molecular architecture of this protein consists of five long alpha-helices connected by short loops. As predicted from amino acid sequence analyses, these helices are distinctly amphiphilic with the hydrophobic residues pointing in toward the interior of the protein and the hydrophilic side chains facing outward. The molecule falls into the general category of up-and-down alpha-helical bundles as previously observed, for example, in cytochrome c'. Although the structure shows the presence of five long amphiphilic alpha-helices, the alpha-helical moment and hydrophobicity of the entire molecule fall into the range found for normal globular proteins. Thus, in order for the amphiphilic helices to play a role in the binding of the protein to a lipid surface, there must be a structural reorganization of the protein which exposes the hydrophobic interior to the lipid surface. The three-dimensional motif of this apolipoprotein is compatible with a model in which the molecule binds to the lipid surface via a relatively nonpolar end and then spreads on the surface in such a way as to cause the hydrophobic side chains of the helices to come in contact with the lipid surface, the charged and polar residues to remain in contact with water, and the overall helical motif of the protein to be maintained.     

The crystal structure of pea lectin at 3.0-A resolution

The structure of pea lectin has been determined to 3.0-A resolution based on multiple isomorphous replacement phasing to 6.0-A resolution and a combination of single isomorphous replacement, anomalous scattering, and density modification to 3.0-A resolution. The pea lectin model has been optimized by restrained least squares refinement against the data between 7.0- and 3.0-A resolution. The final model at 3.0 A gives an R factor of 0.24 and a root mean square deviation from ideal bond distances of 0.02 A. The two monomers in the asymmetric unit are related by noncrystallographic 2-fold symmetry to form a dimer. Monomers were treated independently in modeling and refinement, but are found to be virtually identical at this resolution. The molecular structure of the pea lectin monomer is very similar to that of concanavalin A, the lectin from the jack bean. Similarities extend from secondary and tertiary structures to the occurrence of a cis-peptide bond and the pattern of coordination of the Ca2+ and Mn2+ ions. Differences between the two lectin structures are confined primarily to the loop regions and to the chain termini, which are different and give rise to the unusual permuted relationship between the pea lectin and concanavalin A protein sequences.     

The structure of murine interleukin-1 beta at 2.8 A resolution.

The three-dimensional structure of recombinant murine interleukin-1 beta has been solved by X-ray crystallographic techniques to 2.8 A resolution and refined to a crystallographic R factor of 0.192. Although murine interleukin-1 beta crystallizes in the same space group as human interleukin-1 beta with almost identical unit cell dimensions, the packing of the molecules is quite different. The murine interleukin-1 beta structure was solved by molecular replacement using the refined structure of human interleukin-1 beta as trial structure, and found to be related to the human structure by a nearly perfect twofold rotation about the crystallographic y-axis and a 14 degrees rotation about the z-axis, with no translation. The folding of murine interleukin-1 beta is similar to that found for the human variant, consisting of 12 beta strands wrapped around a core of hydrophobic side chains in a tetrahedron-like fashion. Significant differences with respect to the human structure are seen at the N terminus and in 4 of the 11 loops connecting the 12 beta strands.     

Refined structure of alkaline phosphatase from Escherichia coli at 2.8 A resolution

The structure of alkaline phosphatase from Escherichia coli has been determined to 2.8 A resolution. The multiple isomorphous replacement electron density map of the dimer at 3.4 A was substantially improved by molecular symmetry averaging and solvent flattening. From these maps, polypeptide chains of the dimer were built using the published amino acid sequence. Stereochemically restrained least-squares refinement of this model against native data, starting with 3.4 A data and extending in steps to 2.8 A resolution, proceeded to a final overall crystallographic R factor of 0.256. Alkaline phosphatase-phosphomonoester hydrolase (EC 3.1.3.1) is a metalloenzyme that forms an isologous dimer with two reactive centers 32 A apart. The topology of the polypeptide fold of the subunit is of the alpha/beta class of proteins. Despite the similarities in the overall alpha/beta fold with other proteins, alkaline phosphatase does not have a characteristic binding cleft formed at the carboxyl end of the parallel sheet, but rather an active pocket that contains a cluster of three functional metal sites located off the plane of the central ten-stranded sheet. This active pocket is located near the carboxyl ends of four strands and the amino end of the antiparallel strand, between the plane of the sheet and two helices on the same side. Alkaline phosphatase is a non-specific phosphomonoesterase that hydrolyzes small phosphomonoesters as well as the phosphate termini of DNA. The accessibility calculations based on the refined co-ordinates of the enzyme show that the active pocket barely accommodates inorganic phosphate. Thus, the alcoholic or phenolic portion of the substrate would have to be exposed on the surface of the enzyme. Two metal sites, M1 and M2, 3.9 A apart, are occupied by zinc. The third site, M3, 5 A from site M2 and 7 A from site M1, is occupied by magnesium or, in the absence of magnesium, by zinc. As with other zinc-containing enzymes, histidine residues are ligands to zinc site M1 (three) and to zinc site M2 (one). Ligand assignment and metal preference indicate that the crystallographically found metal sites M1, M2 and M3 correspond to the spectroscopically deduced metal sites A, B and C, respectively. Arsenate, a product analog and enzyme inhibitor, binds between Ser102 and zinc sites M1 and M2. The position of the guanidinium group of Arg 166 is within hydrogen-bonding distance from the arsenate site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Three-dimensional structure of aspartate aminotransferase from Escherichia coli at 2.8 A resolution

The crystal structure of aspartate aminotransferase of Escherichia coli was determined by X-ray structure analysis at 2.8 A resolution. The structure was solved by the molecular replacement method and refined to an R-factor of 0.27, and it was found that the overall structure of AspAT of E. coli is similar to that of those of higher animals.