The structure of an iron-containing alcohol dehydrogenase from a hyperthermophilic archaeon in two chemical states.

An iron-containing alcohol dehydrogenase (FeADH) from the hyperthermophilic archaeon Thermococcus thioreducens was crystallized in unit cells belonging to space groups P21, P212121 and P43212, and the crystal structures were solved at 2.4, 2.1 and 1.9 Šresolution, respectively, by molecular replacement using the FeADH from Thermotoga maritima (Schwarzenbacher et al., 2004) as a model. In the monoclinic and orthorhombic crystals the dehydrogenase (molecular mass 41.5 kDa) existed as a dimer containing a twofold noncrystallographic symmetry axis, which was crystallographic in the tetragonal crystals. In the monoclinic and orthorhombic asymmetric units one molecule contained iron and an NADP molecule, while the other did not. The tetragonal crystals lacked both iron and NADP. The structure is very similar to that of the FeADH from T. maritima (average r.m.s. difference for Cα atoms of 1.8 Šfor 341 aligned atoms). The iron, which is internally sequestered, is bound entirely by amino acids from one domain: three histidines and one aspartic acid. The coenzyme is in an extended conformation, a feature that is common to the large superfamily of NADH-dependent dehydrogenases that share a classical nucleotide-binding domain. A long broad tunnel passes entirely through the enzyme between the two domains, completely encapsulating the coenzyme.

The X-ray crystal structure of human endothelin 1, a polypeptide hormone regulator of blood pressure.

Human endothelin is a 21-amino-acid polypeptide, constrained by two intra-chain disulfide bridges, that is made by endothelial cells. It is the most potent vasoconstrictor in the body and is crucially important in the regulation of blood pressure. It plays a major role in a host of medical conditions, including hypertension, diabetes, stroke and cancer. Endothelin was crystallized 28 years ago in the putative space group P6122, but the structure was never successfully solved by X-ray diffraction. Using X-ray diffraction data from 1992, the structure has now been solved. Assuming a unit cell belonging to space group P61 and a twin fraction of 0.28, a solution emerged with two, almost identical, closely associated molecules in the asymmetric unit. Although the data extended to beyond 1.8 Šresolution, a model containing 25 waters was refined to 1.85 Šresolution with an R of 0.216 and an Rfree of 0.284. The disulfide-constrained `core' of the molecule, amino-acid residues 1-15, has a main-chain conformation that is essentially the same as endothelin when bound to its receptor, but many side-chain rotamers are different. The carboxy-terminal `tail' comprising amino-acid residues 16-21 is extended as when receptor-bound, but it exhibits a different conformation with respect to the `core'. The dimer that comprises the asymmetric unit is maintained almost exclusively by hydrophobic interactions and may be stable in an aqueous medium.

The crystal structure of the ß subunit of luteinizing hormone and a model for the intact hormone.

The ß subunit of bovine luteinizing hormone (LH) was crystallized and its structure solved to 3.15 â€‹Å resolution by molecular replacement using human chorionic gonadotropin (hCG) ß subunit as search model. The asymmetric unit contains two copies of the ß subunit that are related by a non-crystallographic symmetry (NCS) two-fold axis, both copies of which contain proteolytic cleavages after amino acid 100. It is noteworthy that the oligosaccharide moieties covalently attached at asparagine 13 were particularly pronounced in the electron density, allowing seven sugar residues to be defined. The α subunit of LH, which is common to all glycosylated gonadotropin hormones, was placed by superposition of hCG on the LH beta subunits, thereby yielding a model for the intact hormone.

The structure of human apolipoprotein C-1 in four different crystal forms.

Human apolipoprotein C1 (APOC1) is a 57 amino acid long polypeptide that, through its potent inhibition of cholesteryl ester transferase protein, helps regulate the transfer of lipids between lipid particles. We have now determined the structure of APOC1 in four crystal forms by X-ray diffraction. A molecule of APOC1 is a single, slightly bent, α-helix having 13-14 turns and a length of about 80 Å. APOC1 exists as a dimer, but the dimers are not the same in the four crystals. In two monoclinic crystals, two helices closely engage one another in an antiparallel fashion. The interactions between monomers are almost entirely hydrophobic with sparse electrostatic complements. In the third monoclinic crystal, the two monomers spread at one end of the dimer, like a scissor opening, and, by translation along the crystallographic a axis, form a continuous, contiguous sheet through the crystal. In the orthorhombic crystals, two molecules of APOC1 are related by a noncrystallographic 2-fold axis to create an arc of about 120 Å length. This symmetrical dimer utilizes interactions not present in dimers of the monoclinic crystals. Versatility of APOC1 monomer association shown by these crystals is suggestive of physiological function.

Investigation into the binding of dyes within protein crystals.

It was found that the crystals of at least a dozen different proteins could be thoroughly stained to an intense color with a panel of dyes. Many, if not most, of the stained protein crystals retained the dyes almost indefinitely when placed in large volumes of dye-free mother liquor. Dialysis experiments showed that most of the dyes that were retained in crystals also bound to the protein when free in solution; less frequently, some dyes bound only in the crystal. The experiments indicated a strong association of the dyes with the proteins. Four protein crystals were investigated by X-ray diffraction to ascertain the mode of binding. These were crystals of lysozyme, thaumatin, trypsin inhibited with benzamidine and satellite tobacco mosaic virus. In 30 X-ray analyses of protein crystal-dye complexes, in only three difference Fourier maps was any difference electron density present that was consistent with the binding of dye molecules, and even in these three cases (thaumatin plus thioflavin T, xylene cyanol and m-cresol purple) the amount of dye observed was inadequate to explain the intense color of the crystals. It was concluded that the dye molecules, which are clearly inside the crystals, are disordered but are paradoxically tightly bound to the protein. It is speculated that the dyes, which exhibit large hydrophobic cores and peripheral charged groups, may interact with the crystalline proteins in the manner of conventional detergents.

The structure of the Pfp1 protease from the hyperthermophilic archaeon Thermococcus thioreducens in two crystal forms.

The Pfp1 protease, a cysteine protease of unknown specificity from the hyperthermophilic archaeon Thermococcus thioreducens, was crystallized in two distinctive crystal forms: from concentrated citrate in one case and PEG in the other. X-ray data were collected from both crystal forms at room temperature to about 1.9 Šresolution using a laboratory source and detector, and the structures were solved by molecular replacement using the Pfp1 protease from Pyrococcus horikoshii as the search model. In the T. thioreducens protease structures, Cys18 residues on adjacent molecules in the asymmetric units form intermolecular disulfide bonds, thereby yielding hexamers composed of three cross-linked, quasi-dyad-related dimers with crystallographically exact threefold axes and exhibiting almost exact 32 symmetry. The corresponding residue in P. horikoshii Pfp1 is Tyr18. An individual active site containing Cys100 and His101 also includes a Glu74 residue contributed by a quasi-twofold-related, non-cross-linked subunit. Two catalytic triads are therefore closely juxtaposed about the quasi-twofold axis at the interface of these subunits, and are relatively sequestered within the hexamer cavity. The cysteine in the active site is observed to be oxidized in both of the crystal forms that were studied.

Satellite tobacco mosaic virus refined to 1.4†Šresolution.

Satellite tobacco mosaic virus (STMV) is among the smallest viruses, having 60 identical subunits arranged with T = 1 icosahedral symmetry. Its crystal structure was solved at 290 K and was refined using, in part, crystals grown in microgravity. Electron-density maps revealed nearly 57% of the genomic ssRNA. Using six flash-cooled crystals, diffraction data were recorded to 1.4 Šresolution and independent refinements of the STMV model were carried out versus the previous 1.8 Šresolution data representing merged data from 21 crystals (271,689 unique reflections), data consisting of corresponding reflections to 1.8 Šresolution from the cooled crystals and 1.4 Šresolution data from the cooled crystals comprised of 570,721 unique reflections. Models were independently refined with full NCS constraints using the program CNS and in restrained mode using the programs CNS, REFMAC5 and SHELX-97, with the latter two procedures including anisotropic temperature factors. Significant additional structural detail emerged from the analyses, including a unique cation and anion arrangement on fivefold axes and a precise assessment of icosahedral symmetry exactness in the crystal lattice. STMV represents the highest resolution native virus structure currently known by a substantial margin, and it permits the evaluation of a precise atomic model of a spherical virus at near-atomic resolution for the first time.

The crystallographic structure of Panicum Mosaic Virus (PMV).

The structure of Panicum Mosaic Virus (PMV) was determined by X-ray diffraction analysis to 2.9Å resolution. The crystals were of pseudo symmetry F23; the true crystallographic unit cell was of space group P2(1) with a=411.7Å, b=403.9Å and c=412.5Å, with ß=89.7°. The asymmetric unit was two entire T=3 virus particles, or 360 protein subunits. The structure was solved by conventional molecular replacement from two distant homologues, Cocksfoot Mottle Virus (CfMV) and Tobacco Necrosis Virus (TNV), of ∼20% sequence identity followed by phase extension. The model was initially refined with exact icosahedral constraints and then with icosahedral restraints. The virus has Ca(++) ions octahedrally coordinated by six aspartic acid residues on quasi threefold axes, which is completely different than for either CfMV or TNV. Amino terminal residues 1-53, 1-49 and 1-21 of the A, B and C subunits, respectively, and the four C-terminal residues (239-242) are not visible in electron density maps. The additional ordered residues of the C chain form a prominent "arm" that intertwines with symmetry equivalent "arms" at icosahedral threefold axes, as was seen in both CfMV and TNV. A 17 nucleotide hairpin segment of genomic RNA is icosahedrally ordered and bound at 60 equivalent sites at quasi twofold A-B subunit interfaces at the interior surface of the capsid. This segment of RNA may serve as a conformational switch for coat protein subunits, as has been proposed for similar RNA segments in other viruses.

A model for the structure of satellite tobacco mosaic virus.

Satellite tobacco mosaic virus (STMV) is an icosahedral T=1 single-stranded RNA virus with a genome containing 1058 nucleotides. X-ray crystallography revealed a structure containing 30 double-helical RNA segments, with each helix having nine base pairs and an unpaired nucleotide at the 3' end of each strand. Based on this structure, Larson and McPherson proposed a model of 30 hairpin-loop elements occupying the edges of the icosahedron and connected by single-stranded regions. More recently, Schroeder et al. have combined the results of chemical probing with a novel helix searching algorithm to propose a specific secondary structure for the STMV genome, compatible with the Larson-McPherson model. Here we report an all-atom model of STMV, using the complete protein and RNA sequences and the Schroeder RNA secondary structure. As far as we know, this is the first all-atom model for the complete structure of any virus (100% of the atoms) using the natural genomic sequence.

Structure of a crystal form of human methemoglobin indicative of fiber formation.

Human methemoglobin was crystallized in a unique unit cell and its structure was solved by molecular replacement. The hexagonal unit cell has unit-cell parameters a = b = 54.6, c = 677.4 Å, with symmetry consistent with space group P6122. The unit cell has the second highest aspect ratio of all unit cells contained in the PDB. The 12 molecules in the unit cell describe a right-handed helical filament having no polarity, which is different from the filament composed of HbS fibers, which is the only other well characterized fiber of human hemoglobin. The filaments reported here can be related to canonical sickle-cell hemoglobin filaments and to an alternative sickle-cell filament deduced from fiber diffraction by slight modifications of intermolecular contacts.

X-ray crystallographic analyses of pig pancreatic alpha-amylase with limit dextrin, oligosaccharide, and alpha-cyclodextrin.

Further refinement of the model using maximum likelihood procedures and reevaluation of the native electron density map has shown that crystals of pig pancreatic alpha-amylase, whose structure we reported more than 15 years ago, in fact contain a substantial amount of carbohydrate. The carbohydrate fragments are the products of glycogen digestion carried out as an essential step of the protein's purification procedure. In particular, the substrate-binding cleft contains a limit dextrin of six glucose residues, one of which contains both alpha-(1,4) and alpha-(1,6) linkages to contiguous residues. The disaccharide in the original model, shared between two amylase molecules in the crystal lattice, but also occupying a portion of the substrate-binding cleft, is now seen to be a tetrasaccharide. There are, in addition, several other probable monosaccharide binding sites. Furthermore, we have further reviewed our X-ray diffraction analysis of alpha-amylase complexed with alpha-cyclodextrin. alpha-Amylase binds three cyclodextrin molecules. Glucose residues of two of the rings superimpose upon the limit dextrin and the tetrasaccharide. The limit dextrin superimposes in large part upon linear oligosaccharide inhibitors visualized by other investigators. By comprehensive integration of these complexes we have constructed a model for the binding of polysaccharides having the helical character known to be present in natural substrates such as starch and glycogen.

Structure of bovine pancreatic ribonuclease complexed with uridine 5'-monophosphate at 1.60 A resolution.

Bovine pancreatic ribonuclease A (RNase A) was crystallized from a mixture of small molecules containing basic fuchsin, tobramycin and uridine 5'-monophosphate (U5P). Solution of the crystal structure revealed that the enzyme was selectively bound to U5P, with the pyrimidine ring of U5P residing in the pyrimidine-binding site at Thr45. The structure was refined to an R factor of 0.197 and an R(free) of 0.253.

Structure of pig heart citrate synthase at 1.78 A resolution.

Pig heart citrate synthase was crystallized from a small-molecule cocktail containing cystamine dihydrochloride, aspartame and benzamidine hydrochloride. The structure was refined to an R factor of 0.179 (R(free) = 0.222) using synchrotron data to a resolution of 1.78 A. The model includes the full-length protein, a chloride ion, a sulfate ion, 305 water molecules and an unexpected moiety attached through a disulfide linkage to Cys184, which was modeled as a half-cystamine molecule generated by disulfide exchange with the cystamine in the small-molecule cocktail.

High-resolution structure of proteinase K cocrystallized with digalacturonic acid.

Proteinase K, a subtilisin-like fungal protease, was crystallized from a cocktail of small molecules containing digalacturonic acid (DGA). The crystal structure was determined to 1.32 A resolution and refined to an R factor of 0.158. The final model contained, beside the protein, two calcium ions, 379 water molecules, a molecule of DGA and a partially occupied HEPES molecule. The DGA molecule has one sugar moiety disposed exactly on a crystallographic twofold axis; the second ring was not observed. The DGA molecule is bound to two protein molecules across the twofold axis through hydrogen-bonding networks involving Ser150 and water molecules. One of the calcium-ion sites has not been reported previously. This study further illustrates the involvement of small molecules in the crystallization of macromolecules through their ability to form intermolecular lattice interactions.

Development of an alternative approach to protein crystallization.

We are developing an alternate strategy for the crystallization of macromolecules that does not, like current methods, depend on the optimization of traditional variables such as pH and precipitant concentration, but is based on the hypothesis that many conventional small molecules might establish stabilizing, intermolecular, non covalent crosslinks in crystals, and thereby promote lattice formation. To test the hypothesis, we carried out preliminary experiments encompassing 18,240 crystallization trials using 81 different proteins, and 200 chemical compounds. Statistical analysis of the results demonstrated the validity of the idea. In addition, we conducted X-ray diffraction analyses of some of the crystals grown in the experiments. These clearly showed incorporation of conventional molecules into the protein crystal lattices, and further validated the underlying hypothesis. We are currently extending the investigations to include a broader and more diverse set of proteins, an expanded search of conventional and biologically active small molecules, and a wider range of precipitants. The strategy proposed here is essentially orthogonal to current approaches and has an objective of doubling the success rate of today.

A new crystal form of bovine pancreatic RNase A in complex with 2'-deoxyguanosine-5'-monophosphate.

The structure of bovine pancreatic RNase A has been determined in complex with 2'-deoxyguanosine-5'-monophosphate (dGMP) at 1.33 A resolution at room temperature in a previously unreported unit cell belonging to space group P3(1). There are two molecules of nucleotide per enzyme molecule, one of which lies in the active-site cleft in the productive binding mode, whilst the guanine base of the other dGMP occupies the pyrimidine-specific binding site in a nonproductive mode such that it forms hydrogen bonds to the phosphate group of the first dGMP. This is the first RNase A structure containing a guanine base in the B2 binding site. Each dGMP molecule is involved in intermolecular interactions with adjacent RNase A molecules in the lattice and the two nucleotides appear to direct the formation of the crystal lattice. Because GMP may be produced during degradation of RNA, this association could represent an inhibitor complex and thereby affect the observed enzyme kinetics.

Bence Jones KWR protein structures determined by X-ray crystallography.

A Bence Jones protein isolated in the early 1960s from a patient (initials KWR) suffering from plasma-cell dyscrasia was crystallized and its structure was analyzed in four different unit cells by X-ray diffraction. The final models of the molecule in all crystal forms were virtually the same, although the elbow angles relating the constant and variable domains of the Bence Jones dimers varied over a range of 10 degrees. The tetragonal form had an R factor of 22.6% and an R(free) of 28.3% at 2.2 A resolution. Phosphate or sulfate ions (depending on the crystallization conditions) were found in the antigen-combining sites in all crystals, as well as an unidentified ligand tightly bound in the hydrophobic 'deep pocket' beneath the antigen-binding site. The ligand was treated as a phenol molecule. Two trigonal crystal forms were among those solved. One was grown at pH 4.0 and the other was only obtained after sitting for more than eight months at room temperature. The latter crystal was composed of molecules that were degraded in their constant domains. Both low pH and proteolytic degradation of constant domains are known to promote the polymerization of some Bence Jones proteins into amyloid fibrils. Indeed, in both trigonal crystal forms the molecules are organized with pseudo-hexagonal symmetry about the unique crystallographic axes in a manner suggestive of such fibrils. The arrangement of Bence Jones dimers is also consistent with other observations regarding Bence Jones amyloid-fibril structure and current models.

A novel strategy for the crystallization of proteins: X-ray diffraction validation.

Recently, the hypothesis was advanced that protein crystallization could be driven by the inclusion of small molecules rich in hydrogen-bonding, hydrophobic and electrostatic bonding possibilities. Conventional organic and biologically active molecules would promote lattice formation by their mediation of intermolecular interactions in crystals. The results of an extensive series of crystallization experiments strongly supported the idea. Here, difference Fourier X-ray diffraction analyses of nine crystals grown in the experiments are presented, which convincingly demonstrate the validity of the hypothesis and illustrate some of the ways in which small molecules can participate in lattice interactions.

Molecular dynamics simulations of the complete satellite tobacco mosaic virus.

This work presents an all-atom molecular dynamics simulation of a complete virus, the satellite tobacco mosaic virus. Simulations with up to 1 million atoms for over 50 ns demonstrate the stability of the entire virion and of the RNA core alone, while the capsid without RNA exhibits a pronounced instability. Physical properties of the simulated virus particle including electrostatic potential, radial distribution of viral components, and patterns of correlated motion are analyzed, and the implications for the assembly and infection mechanism of the virus are discussed.

The structure of an antitumor C(H)2-domain-deleted humanized antibody.

C(H)2-domain-deleted CC49 (HuCC49DeltaCH2), a recombinant humanized antibody that recognizes the TAG-72 antigen expressed on a variety of human carcinomas, is secreted from cultured cells as a mixture of two homodimeric isoforms. Isoform A contains two covalent interchain disulfide bonds at heavy chain positions 239 and 242, while isoform B fails to develop any interchain disulfide bonds but has 239-242 intrachain disulfide bonds instead. Form A is currently in preclinical development as a therapeutic agent for treating colorectal carcinoma, though form B shows equal efficacy. HuCC49DeltaCH2 form B can be crystallized from sodium formate only in the presence of detergents. X-ray diffraction data were collected on a single cryo-cooled crystal grown with Triton X-100 and the structure was solved by molecular replacement. The model has refined to R=0.246 (R(free)=0.297) for 2.8A data. The antibodies pack in the crystal around crystallographic 2-fold axes as tetramers with approximate 222 symmetry. Atomic force microscopy studies show that this tetrameric structure is the crystal building block and also exists free in the mother liquor. The tetramer is composed of two rings, back-to-back, with a thickness of approximately 83A. Each ring is composed of two antibodies with the complementarity-determining regions (CDR) of the two Fabs of one antibody interacting with the CDR regions of the second antibody in a head-to-head fashion. These rings are approximately 167A long and 112A wide. The C(H)3 domain is inverted with respect to the Fabs when compared to the usual orientation found in conventional antibodies. The polypeptides joining the C(H)3 domains to the Fab portions of the antibody are not seen and are almost certainly disordered. The antigen combining site of HuCC49DeltaCH2 is very similar, but not identical, in topology and charge distribution to that of antibody B72.3, which binds a similar epitope on TAG-72. The combining site consists of a deep cleft, heavily lined with aromatic amino acid side-chains but bounded by numerous charged groups.