Crystal structure of beta-D-cellotetraose hemihydrate with implications for the structure of cellulose II.

The crystal structure of beta-D-cellotetraose shows the same molecule packing as cellulose II, with two antiparallel molecules in the unit cell: For cellulose II, the orientation of the C6-O6 bonds has been described as gauche-trans and trans-gauche, respectively, for the two antiparallel molecules, which otherwise have identical conformations. In contrast, in beta-D-cellotetraose all C6-O6 bonds are gauche-trans, but the conformations of the two antiparallel molecules are different. Energy minimization and molecular dynamics studies suggest that the structure of cellulose II should be reinvestigated in light of these findings.

An amylose antiparallel double helix at atomic resolution.

In the crystal structure of the polyiodide complex (p-nitrophenyl-alpha-maltohexaose(2)) . Ba(I(3))(2) . 22H(2)O, the maltohexaose units form an antiparallel, left-handed double helix with O-2 ... O-3 and O-6 ... O-6 hydrogen bonding and a central cavity that encloses two triiodide units. This structure contrasts with the parallel, left-handed double helix with no central cavity proposed for the A-and B-starch helix and the left-handed single helix in V-amylose and may be relevant for the stabilization of glycogen Structure.

Structure of the Tet repressor-tetracycline complex and regulation of antibiotic resistance.

The most frequently occurring resistance of Gram-negative bacteria against tetracyclines is triggered by drug recognition of the Tet repressor. This causes dissociation of the repressor-operator DNA complex and enables expression of the resistance protein TetA, which is responsible for active efflux of tetracycline. The 2.5 angstrom resolution crystal structure of the homodimeric Tet repressor complexed with tetracycline-magnesium reveals detailed drug recognition. The orientation of the operator-binding helix-turn-helix motifs of the repressor is inverted in comparison with other DNA binding proteins. The repressor-drug complex is unable to interact with DNA because the separation of the DNA binding motifs is 5 angstroms wider than usually observed.

Flavones inhibit the hexameric replicative helicase RepA.

Helicases couple the hydrolysis of nucleoside triphosphates (NTPs) to the unwinding of double-stranded nucleic acids and are essential in DNA metabolism. Thus far, no inhibitors are known for helicases except heliquinomycin isolated from Streptomyces sp. As the three-dimensional structure of the hexameric replicative DNA helicase RepA encoded by the broad host-range plasmid RSF1010 is known, this protein served as a model helicase to search for inhibitory compounds. The commercially available flavone derivatives luteolin, morin, myricetin and dimyricetin (an oxidation product of myricetin) inhibited the ATPase and double-stranded DNA unwinding activities of RepA. Dimyricetin was the most effective inhibitor for both activities. Single-stranded DNA-dependent RepA ATPase activity is inhibited non-competitively by all four compounds. This finding contrasts the inhibition of phosphoinositide 3-kinase by flavones that fit into the ATP binding pocket of this enzyme. Myricetin also inhibited the growth of a Gram-positive and a Gram-negative bacterial species. As we found other hexameric and non-hexameric prokaryotic helicases to be differentially sensitive to myricetin, flavones may provide substructures for the design of molecules helpful for unraveling the mechanism of helicase action and of novel pharmacologically useful molecules.

Theoretical investigations on the conformation of 1,5,N(4),N(4)-tetramethylcytosine.

Theoretical investigations (Perturbative Configuration Interaction over Localized Orbitals (PCILO) and Intermediate Neglect of Differential Overlap (INDO) methods) of the conformation of tetramethylcystosine, an overcrowded molecule with planar structure, have been carried out. The physical features of rotational bending potentials of the dimethylamino group are discussed. Particularly, the controversial problem concerning the planarity of the molecule is investigated. The obtained results show that the planarity of tetramethylcytosine is an intrinsic property of the molecule. Nevertheless, because of the repulsion between the methyl groups, the planar structure of tetramethylcytosine is slightly destabilized. Further, the functional dependence of the dipole moment of tetramethylcytosine on rotation and bending of the dimethylamino group has been analyzed.

Autolysis and inhibition of proteinase K, a subtilisin-related serine proteinase isolated from the fungus Tritirachium album Limber.

The activity of proteinase K (EC 3.4.21.14), a subtilisin-related serine proteinase, was assayed with azoalbumin that showed non-expected behavior in substrate saturation curve because of interaction between albumin molecules. Succinyl-(Ala)n-p-nitroanilide with n = 2 and 3, yielded specific activities of 3.5, 13 units/mg protein, respectively, reflecting a chain length dependence. The influence of peptide chain length on binding to proteinase K was also observed using mono- and dipeptide chloromethyl ketone inhibitors. They showed a maximum inhibition. They showed a maximum inhibition of proteinase K in solution of only about 50% even at a more than 20-fold molar excess. With the above substrates, the Vmax is not affected in presence of 10, 20 and 30% methanol, but the Km differs remarkably, suggesting competitive inhibition. The activity of proteinase K shows a maximum at 37 degrees C, and a temperature profile with more than 80% maximum activity in the range 20-60 degrees C. Autolysis of the enzyme is observed during sample preparation for SDS-gel electrophoresis and at low concentration (0.01 mg/ml) in aqueous solution. It does not occur at higher proteinase K concentrations at or above 1.0 mg/ml, consistent with crystallographic studies.

Synthesis and kinetic study of transition state analogs for ribonuclease T1.

Based on the proposal that ribonucleases cleave the RNA phosphodiester bond with a mechanism involving pentacovalent phosphorous as transition state, complexes of guanosine and inosine with vanadate-(IV, V), molybdate-(VI), tungstate-(VI), chromate-(VI) and hexacyanochromate-(III) were synthesized and probed as inhibitors of recombinant ribonuclease T1, obtained from an E. coli. overproducing strain. The apparent dissociation constants of these inhibitors and RNase T1, as determined by Michaelis-Menten kinetics, vary between 0.5-0.9 microM and indicate very strong binding, 100- to 1000-fold stronger than the binding of guanosine (Kd = 545 microM) and inosine (Kd = 780 microM), and 50-100-fold stronger than the binding of the product 3' GMP (Kd = 55 microM). Therefore the synthesized inhibitors may be considered as genuine transition state analogs for the enzyme.

Crystal structure of a zwitterionic detergent: n-octyl-2-hydroxyethylsulfoxide.

The crystal structure of n-octyl-2-hydroxyethylsulfoxide (1) (space group P2(1)/c, a = 16.516(7), b = 9.053(4), c = 8.222(4) A, beta = 97.58 degrees) was determined by X-ray diffraction methods to 0.94 A resolution and refined to R = 0.050 (Rw = 0.052). In the crystal lattice the molecules are not arranged tail-to-tail, as usually observed with amphiphilic compounds, but head-to-tail and aligned in the a-axis direction. They form layers in the b, c plane which are interdigitated such that adjacent molecules in one layer are in antiparallel orientation. The packing is stabilized by intermolecular hydrogen bonds O-H--O-S. No solvent molecules were detectable.

Thermodynamic analysis of the equilibrium, association and dissociation of 2'GMP and 3'GMP with ribonuclease T1 at pH 5.3.

Fluorescence titrations and temperature-jump relaxation experiments were performed as a function of temperature on ribonuclease T1 with the inhibitors 2'GMP and 3'GMP to obtain information on the energetics and molecular events controlling the binding of those inhibitors. Results from the titration and temperature-jump experiments were in agreement concerning the equilibrium constant. The larger equilibrium constant for 2'GMP is enthalpic in origin and is due to both a higher on rate and a lower off rate as compared to 3'GMP. On rates for both inhibitors appear to be below the diffusion controlled limit, apparently due to conformational changes in the portion of the active site responsible for recognition of the guanine base. Comparison of the measured enthalpic and entropic terms associated with the equilibrium constant determined from the fluorescence titrations are in disagreement with those calculated from the on and off rates indicating the presence of an induced conformational change in the 2'GMP-enzyme complex. This second conformational change appears to be due to additional interactions between 2'GMP and the catalytic portion of the active site, which may also be responsible for the differences in the binding constant, the on rate and the off rate between 2'GMP and 3'GMP.

Colloidal properties of human transferrin receptor in detergent free solution.

The colloidal properties of transferrin receptor, isolated from human placenta, in detergent free solution has been investigated by light scattering techniques and analytical ultracentrifugation. In detergent free solution at 293.2 K, hTfR forms stable aggregates with an apparent hydrodynamic radius of 17 nm. The molecular mass was determined by ultracentrifugation to lie between (1722+/-87) kDa (sedimentation equilibrium) and (1675+/-46) kDa (sedimentation velocity). This implies that the aggregates are build up from nine hTfR dimers. Based on model calculations, which are in good agreement with the experimental data, we propose a torus-like structure for the aggregates. Upon pH shift from pH 7.5 to 5.0 or removal of the N-linked carbohydrate chains, formation of larger aggregates is induced. These aggregates can be described in terms of porous fractal structures. We propose a simple model, which accounts for that behaviour assuming that the aggregation is mainly due to the reduction of negative surface charge.

An integrated approach to structural genomics.

Structural genomics aims at determining a set of protein structures that will represent all domain folds present in the biosphere. These structures can be used as the basis for the homology modelling of the majority of all remaining protein domains or, indeed, proteins. Structural genomics therefore promises to provide a comprehensive structural description of the protein universe. To achieve this, a broad scientific effort is required. The Berlin-based "Protein Structure Factory" (PSF) plans to contribute to this effort by setting up a local infrastructure for the low-cost, high-throughput analysis of soluble human proteins. In close collaboration with the German Human Genome Project (DHGP) protein-coding genes will be expressed in Escherichia coli or yeast. Affinity-tagged proteins will be purified semi-automatically for biophysical characterization and structure analysis by X-ray diffraction methods and NMR spectroscopy. In all steps of the structure analysis process, possibilities for automation, parallelization and standardization will be explored. Major new facilities that are created for the PSF include a robotic station for large-scale protein crystallization, an NMR center and an experimental station for protein crystallography at the synchrotron storage ring BESSY II in Berlin.

Evidence for rapid association-dissociation of ribonuclease T1 from a recombinant strain of Escherichia coli.

On the basis of photon correlation experiments and computer simulations, we provide evidence for a rapid dimerization of the enzyme ribonuclease T1 isolated from an Escherichia coli overproducing strain. An attractive potential in addition to the usual repulsive hardcore and electrostatic potentials was found to be necessary for interpreting the concentration dependence of the diffusion coefficient of the enzyme. Computer searches of surface complementarity suggest that dimer formation of ribonuclease T1 takes place due to an extensive surface contact of approximately 700 A2. Energy minimization of the ribonuclease T1 dimer shows that large conformational changes are not induced upon self-association of the enzyme. The two molecules in the dimer are orientated back-to-back, and this is expected to lead to an active enzyme form.

Three-dimensional structure of ribonuclease T1 complexed with guanylyl-2',5'-guanosine at 1.8 A resolution.

The enzyme ribonuclease T1 (RNase T1) isolated from Aspergillus oryzae was cocrystallized with the specific inhibitor guanylyl-2',5'-guanosine (2',5'-GpG) and the structure refined by the stereochemically restrained least-squares refinement method to a crystallographic R-factor of 14.9% for X-ray data above 3 sigma in the resolution range 6 to 1.8 A. The refined model consists of 781 protein atoms, 43 inhibitor atoms in a major site and 29 inhibitor atoms in a minor site, 107 water oxygen atoms, and a metal site assigned as Ca. At the end of the refinement, the orientation of His, Asn and Gln side-chains was reinterpreted on the basis of two-dimensional nuclear magnetic resonance data. The crystal packing and enzyme conformation of the RNase T1/2',5'-GpG complex and of the near-isomorphous RNase T1/2'-GMP complex are comparable. The root-mean-square deviation is 0.73 A between equivalent protein atoms. Differences in the unit cell dimensions are mainly due to the bound inhibitor. The 5'-terminal guanine of 2',5'-GpG binds to RNase T1 in much the same way as in the 2'-GMP complex. In contrast, the hydrogen bonds between the catalytic center and the phosphate group are different and the 3'-terminal guanine forms no hydrogen bonds with the enzyme. This poor binding is reflected in a 2-fold disorder of 2',5'-GpG (except the 5'-terminal guanine), which originates from differences in the pucker of the 5'-terminal ribose. The pucker is C2'-exo for the major site (2/3 occupancy) and C1'-endo for the minor site (1/3 occupancy). The orientation of the major site is stabilized through stacking interactions between the 3'-terminal guanine and His92, an amino acid necessary for catalysis. This might explain the high inhibition rate observed for 2',5'-GpG, which exceeds that of all other inhibitors of type 2',5'-GpN. On the basis of distance criteria, one solvent peak in the electron density was identified as metal ion, probably Ca2+. The ion is co-ordinated by the two Asp15 carboxylate oxygen atoms and by six water molecules. The co-ordination polyhedron displays approximate 4m2 symmetry.

Comparative studies of ribulose-1,5-biphosphate carboxylase/oxygenase from Alcaligenes eutrophus H16 cells, in the active and CABP-inhibited forms.

RuBisCO (D-ribulose-1,5-biphosphate carboxylase/oxygenase; EC 4.1.1.39) has been isolated from the autotrophic hydrogen-oxidizing bacterium Alcaligenes eutrophus H16. Combining photon correlation and sedimentation analysis transport parameters of the enzyme were investigated in the active, (E.CO2.Mg2+) as a ternary complex, and inactive state, (E.CO2.Mg2+.CABP) as a quaternary complex, where RuBisCO is complexed with the transition state analogue CABP (2-C-carboxy-D-arabinitol-1,5-biphosphate). Within experimental error, no difference has been detected between the diffusion and sedimentation coefficients (D020,w = 2.72(+/- 0.07) x 10(-7) cm2 s-1, s020,w = 17.8(+/- 0.5)S) of active and CABP-complexed enzyme thus leading to the conclusion that the molecule, at least in solution, does not assume a different conformation when complexed with CABP.

Decamer-like conformation of a nona-peptide bound to HLA-B*3501 due to non-standard positioning of the C terminus.

The N and C termini of peptides presented by major histocompatibility complex (MHC) class I molecules are held within the peptide binding groove by a network of hydrogen bonds to conserved MHC residues. However, the published structure of the human allele HLA-B*3501 complexed with the nef octa-peptide VPLRPMTY, revealed non-standard positioning for both peptide termini. To investigate whether these deviations are indeed related to the length of the nef-peptide, we have determined the structure of HLA-B*3501 presenting a nona-peptide to 2.5 A resolution. A comparison of HLA-B*3501/peptide complexes with structures of other HLA molecules exhibits allele-specific properties of HLA-B*3501, as well as peptide-induced structural changes. Independent of the length of the bound peptide, HLA-B*3501 positions the peptide C terminus significantly closer to the alpha1-helix and nearer to the A pocket than observed for other HLA class I/peptide complexes. This reorientation is accompanied by a shift within the N-terminal part of the alpha2-helix towards the middle of the binding groove. Due to the short distance between the N and C termini, the nona-peptide is compressed and forced to zig-zag vertically within the binding groove. Its conformation rather resembles that of a deca-peptide than of other nona-peptides bound to class I molecules. Superposition of both HLA-B*3501/peptide complexes additionally reveals a significant, peptide-dependent deviation between the N-terminal parts of the alpha1-helices which might be due to different positioning of the peptide N termini. Taken together, these data illustrate the strong interdependence between the HLA class I molecule and the bound peptide.

Conformational differences between alpha-cyclodextrin in aqueous solution and in crystalline form. A molecular dynamics study.

The computer simulation technique of molecular dynamics is a powerful tool to delineate the conformational differences between a molecule in different environments. As an illustration, the difference between an alpha-cyclodextrin molecule in aqueous solution and in crystalline form is determined. Two molecular dynamics simulations are compared. In one simulation, one alpha-cyclodextrin form in a "truncated octahedron box" containing 611 water molecules is simulated over 90 picoseconds to mimic the solution structure. In the other simulation, the crystalline form is modelled by a molecular dynamics simulation of four unit cells in space group P2(1)2(1)2(1) containing 16 alpha-cyclodextrin molecules and 96 water molecules over a period of 15 picoseconds. The solution structure of alpha-cyclodextrin deviates by about 0.1 nm from that in the crystal and shows twice as much mobility of the atoms. The experimentally observed twist of glucose unit 5 out of alignment with the other five glucose units in the alpha-cyclodextrin torus that is present in the crystal simulation, disappears in the simulation in solution, but the glucosidic torsion angles around the ring remain asymmetric. The hydrogen-bonding patterns in crystal and in solution are rather different. This means that in a crystal structure, the molecule and its (hydration) hydrogen-bonding scheme represent only one static minimum energy picture, whereas the molecular dynamics simulations yield a description of all the many hydrogen-bonding configurations that can occur in solution.

Crystal structure of the hexameric replicative helicase RepA of plasmid RSF1010.

Unwinding of double-stranded DNA into single-stranded intermediates required for various fundamental life processes is catalyzed by helicases, a family of mono-, di- or hexameric motor proteins fueled by nucleoside triphosphate hydrolysis. The three-dimensional crystal structure of the hexameric helicase RepA encoded by plasmid RSF1010 has been determined by X-ray diffraction at 2.4 A resolution. The hexamer shows an annular structure with 6-fold rotational symmetry and a approximately 17 A wide central hole, suggesting that single-stranded DNA may be threaded during unwinding. Homologs of all five conserved sequence motifs of the DnaB-like helicase family are found in RepA, and the topography of the monomer resembles RecA and the helicase domain of the bacteriophage T7 gp4 protein. In a modeled complex, ATP molecules are located at the subunit interfaces and clearly define adenine-binding and ATPase catalytic sites formed by amino acid residues located on adjacent monomers; most remarkable is the "arginine finger" Arg207 contributing to the active site in the adjacent monomer. This arrangement of active-site residues suggests cooperativity between monomers in ATP hydrolysis and helicase activity of RepA. The mechanism of DNA unwinding remains elusive, as RepA is 6-fold symmetric, contrasting the recently published asymmetric structure of the bacteriophage T7 gp4 helicase domain.

Ribonuclease T1 with free recognition and catalytic site: crystal structure analysis at 1.5 A resolution.

The free form of ribonuclease T1 (RNase T1) has been crystallized at neutral pH, and the three-dimensional structure of the enzyme has been determined at 1.5 A nominal resolution. Restrained least-squares refinement yielded an R value of 14.3% for 12,623 structure amplitudes. The high resolution of the structure analysis permits a detailed description of the solvent structure around RNase T1, the reliable rotational setting of several side-chain amide and imidazole groups and the identification of seven disordered residues. Among these, the disordered and completely internal Val78 residue is noteworthy. In the RNase T1 crystal structures determined thus far it is always disordered in the absence of bound guanosine, but not in its presence. A systematic analysis of hydrogen bonding reveals the presence in RNase T1 of 40 three-center and an additional seven four-center hydrogen bonds. Three-center hydrogen bonds occur predominantly in the alpha-helix, where their minor components close 3(10)-type turns, and in beta-sheets, where their minor components connect the peptide nitrogen and carbonyl functions of the same residue. The structure of the free form is compared with complexes of RNase T1 with filled base recognition site and/or catalytic site. Several structural rearrangements occurring upon inhibitor or substrate binding are clearly apparent. In conjunction with the available biochemical knowledge, they are used to describe probable steps occurring early during RNase T1-catalyzed phosphate transesterification.

Differential binding of S-adenosylmethionine S-adenosylhomocysteine and Sinefungin to the adenine-specific DNA methyltransferase M.TaqI.

The crystal structures of the binary complexes of the DNA methyltransferase M.TaqI with the inhibitor Sinefungin and the reaction product S-adenosyl-L-homocysteine were determined, both at 2.6 A resolution. Structural comparison of these binary complexes with the complex formed by M.TaqI and the cofactor S-adenosyl-L-methionine suggests that the key element for molecular recognition of these ligands is the binding of their adenosine part in a pocket, and discrimination between cofactor, reaction product and inhibitor is mediated by different conformations of these molecules; the methionine part of S-adenosyl-L-methionine is located in the binding cleft, whereas the amino acid moieties of Sinefungin and S-adenosyl-L-homocysteine are in a different orientation and interact with the active site amino acid residues 105NPPY108. Dissociation constants for the complexes of M.TaqI with the three ligands were determined spectrofluorometrically. Sinefungin binds more strongly than S-adenosyl-L-homocysteine or S-adenosyl-L-methionine, with KD=0.34 microM, 2.4 microM and 2.0 microM, respectively.

Crystal structure of calf spleen purine nucleoside phosphorylase in a complex with hypoxanthine at 2.15 A resolution.

Trimeric calf spleen purine nucleoside phosphorylase has been complexed with hypoxanthine via phosphorolysis of inosine in the presence of phosphate. The resulting, "Michaelis" complex (three hypoxanthine molecules per trimer), presumed to be formed under these conditions, crystallized in the cubic space group P2(1)3, with unit cell dimension a = 94.11 A and one monomer in the asymmetric crystal unit; the biologically active trimer is located on the crystallographic 3-fold axis. High-resolution X-ray diffraction data were collected using synchrotron radiation (EMBL outstation, Hamburg, c/o DESY). The crystal structure has been determined by molecular replacement and refined at 2.15 A resolution to an R-value of 0.18. In the hypoxanthine binding site, a cis-peptide bond between Asn243 and Lys244 is observed. Side-chains of GIu201 and Asn243, as well as one integral water molecule located in the base binding site, form hydrogen bonds with the hypoxanthine N-1 H, N-7 H and O-6. A second water molecule links the base positions N-3 and N-9 with an adjacent pocket, which presumably is the phosphate-binding site. This pocket is filled completely by a cluster of six water molecules. Hence all possible donor/acceptor-positions of hypoxanthine are saturated by hydrogen-bonding to protein side-chains or integral water molecules. Purine nucleoside phosphorylase isolated form human tissues is a primary target for chemotherapeutic intervention, and the more stable calf enzyme has similar physico-chemical and kinetic properties, as well as response to inhibitors. Hence the high-resolution structure presented here may serve for design of inhibitors with potential pharmacological applications.