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Biomedical subjects

W Saenger

Publications and source records attributed to W Saenger.

At least 91 records · Page 5Linked to original sources
PubMed1996

Thermally induced hydrogen exchange processes in small proteins as seen by FTIR spectroscopy.

Fourier-transform infrared (FTIR) spectroscopy has been used to study the thermally induced exchange characteristics of those backbone amide protons which persist H-D exchange at ambient conditions in ribonuclease A, in wild type ribonuclease T1 and some of its variants, and in the histone-like protein HBsu. The H-D exchange processes were induced by increasing the thermal energy of the protein solutions in two ways: (i) by linearly increasing the temperature, and (ii) by a temperature jump. To trace the H-D exchange in the proteins, various infrared absorption bands known to be sensitive to H-D exchange were used as specific monitors. Characteristic H-D exchange curves were obtained from which the endpoints (TH/D) of H-D exchange could be determined. The H-D exchange curves, the TH/D-values and the phase transition temperatures Tm were used to estimate the structural flexibility and stability of the given proteins. It is suggested that time-resolved FTIR spectroscopy can be used to determine global stability parameters of proteins.

Amides↗
PubMed1996

Structure model of a complex between the factor for inversion stimulation (FIS) and DNA: modeling protein-DNA complexes with dyad symmetry and known protein structures.

A method is presented to predict overall conformations of protein-DNA complexes on the basis of the known three-dimensional structures of the proteins. The method is restricted to proteins with a common twofold symmetry axis, which show only minor conformational changes upon binding to DNA. The method uses a numerical finite difference solution of the linearized Poisson-Boltzmann equation and subsequent energy minimization cycles. Structural parameters-the rotation angle of the DNA relative to the protein around the common symmetry axis, the protein-DNA distance, and intermolecular hydrogen-bonding contacts-are presented for two test cases, DNA bound to CAP (catabolite gene activator protein) and to the Cro-repressor of bacteriophage 434. The DNA curvature in the starting model of the docking procedure was chosen as a smoothed approximation of the conformation found in the X-ray structures of these complexes. The method is further used to predict the unknown structure of the complex between the factor for inversion stimulation (FIS) and DNA, which is bent upon binding to FIS. In contrast to the test cases, the unknown curvature of the starting model is derived from a calibration of electrostatic precalculations for different proteins according to crystallographically observed DNA bending. The results of the modeling are in good accordance with the experimentally observed overall structure of protein-DNA complexes for the two test cases; for FIS, they correspond to several of the experimentally proposed protein-DNA contacts.

DNA↗
PubMed1996

Photosystem I at 4 A resolution represents the first structural model of a joint photosynthetic reaction centre and core antenna system.

The 4 A X-ray structure model of trimeric photosystem I of the cyanobacterium Synechococcus elongatus reveals 31 transmembrane, nine surface and three stromal alpha-helices per monomer, assigned to the 11 protein subunits: PsaA and PsaB are related by a pseudo two-fold axis normal to the membrane plane, along which the electron transfer pigments are arranged. 65 antenna chlorophyll a (Chl a) molecules separated by < or = 16 A form an oval, clustered net continuous with the electron transfer chain through the second and third Chl a pairs of the electron transfer system. This suggests a dual role for these Chl a both in excitation energy and electron transfer. The architecture of the protein core indicates quinone and iron-sulphur type reaction centres to have a common ancestor.

Cell Membrane↗
PubMed1995

Calf spleen purine nucleoside phosphorylase: purification, sequence and crystal structure of its complex with an N(7)-acycloguanosine inhibitor.

Calf spleen purine nucleoside phosphorylase was purified to homogeneity and its amino acid sequence was determined. The complex of the enzyme with an N(7)-acycloguanosine inhibitor crystallized in the cubic space group P2(1)3, with unit cell dimension a = 94.02 A and one monomer in the asymmetric crystal unit. The biologically active trimer is formed by the crystallographic three-fold axis. The structure was solved by molecular replacement methods, using the model of the human erythrocyte enzyme, and refined at a resolution of 2.9 A to an R-factor of 0.21. The orientation of the inhibitor at the active site is examined in relation to the catalytic activity of the enzyme in the phosphorolysis of N(7)-beta-D-purine nucleosides.

Amino Acid Sequence↗
PubMed1995

A model for DNA binding and enzyme action derived from crystallographic studies of the TaqI N6-adenine-methyltransferase.

The crystal structures of the DNA-N6-adenine-methyltransferase M.TaqI, in complexes with the cofactor S-adenosyl-L-methionine (AdoMet) and the competitive inhibitor sinefungin (Sf) show identical folding of the polypeptide chains into two domains. The N-terminal domain carries the cofactor-binding site, the C-terminal domain is thought to be implicated in sequence-specific DNA binding. Model building of the M.TaqI-DNA complex suggests that the adenine to be methylated swings out of the double helix as found previously in the cytosine-C5-MTase HhaI DNA co-crystal structure. A torsion of the methionine moiety of the cofactor is required to bring the methyl group within reach of the swung-out base and allow methyl group transfer.

Amino Acid Sequence↗
PubMed1995

The complex formed between Tet repressor and tetracycline-Mg2+ reveals mechanism of antibiotic resistance.

In recent years Gram-negative bacteria have developed several resistance mechanisms against the broad-spectrum antibiotic tetracycline (Tc). The most abundant mechanism involves a membrane-associated protein (TetA) that exports the antibiotic out of the bacterial cell before it can attach to the ribosomes and inhibit polypeptide elongation. The expression of the TetA protein is regulated by the Tet repressor (TetR). It occurs as a homodimer and binds with two alpha-helix-turn-alpha-helix motifs (HTH) to two tandemly orientated DNA operators, thereby blocking the expression of the associated genes, one encoding for TetA and the other for TetR. If Tc in complex with a divalent cation binds to TetR, a conformational change occurs and the induced TetR is then unable to bind to DNA. TetR of class D, TEtRD, was cocrystallized with tetracycline (7HTc) and Mg2+ in space group I4(1)22 and studied by X-ray diffraction. One TetRD monomer occupies the crystal asymmetric unit, and the dimer is formed by a crystallographic 2-fold rotation. The crystal structure was determined by multiple isomorphous replacement at 2.5 A resolution, and on this basis the structure of the nearly isomorphous complex with 7-chlorotetracycline, TetRD/(Mg 7CITc)+, has been refined to an R-factor of 18.3% using all reflections to 2.1 A resolution. TetRD folds into ten alpha-helices with connecting turns and loops. The N-terminal three alpha-helices of the repressor form the DNA-binding domain, including the HTH with an inverse orientation compared with HTH in other DNA-binding proteins. The distance of 39 A between the two recognition helices explains the inability of the induced TetR to bind to B-form DNA. The core of the protein is formed by helices alpha 5 to alpha 10. It is responsible for dimerization and contains, for each monomer, a binding pocket that accommodates Tc in the presence of a divalent cation. The structure of the TetRD/(Mg 7CITc)+ complex reveals the octahedral coordination of Mg2+ by Tc (chelating O-11, and O-12), His100 N epsilon and by three water molecules; in addition there is an extended network of hydrogen bonding and van der Waals interactions formed between 7CITc and TetR. The detailed view of the Tc-binding pocket and the interactions between the antibiotic and the repressor offers the first solid basis for rational tetracycline design, with the aim of circumventing resistance.

Amino Acid Sequence↗
PubMed1995

Universal catalytic domain structure of AdoMet-dependent methyltransferases.

The DNA methyltransferases, M.HhaI and M.TaqI, and catechol O-methyl-transferase (COMT) catalyze the transfer of a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to carbon-5 of cytosine, to nitrogen-6 of adenine, and to a hydroxyl group of catechol, respectively. The catalytic domains of the bilobal proteins, M.HhaI and M.TaqI, and the entire single domain of COMT have similar folding with an alpha/beta structure containing a mixed central beta-sheet. The functional residues are located in equivalent regions at the carboxyl ends of the parallel beta-strands. The cofactor binding sites are almost identical and the essential catalytic amino acids coincide. The comparable protein folding and the existence of equivalent amino acids in similar secondary and tertiary positions indicate that many (if not all) AdoMet-dependent methyltransferases have a common catalytic domain structure. This permits tertiary structure prediction of other DNA, RNA, protein, and small-molecule AdoMet-dependent methyltransferases from their amino acid sequences.

Amino Acid Sequence↗
PubMed1995

Characterization of non-inducible Tet repressor mutants suggests conformational changes necessary for induction.

Non-inducible tetracycline repressor (TetR) mutants were grouped in three structurally distinct classes. We quantitated in vivo operator binding, inducibility, and in vitro tetracycline binding of mutants from each class. Mutation of residues close to tetracycline (class 1) leads to reduced affinity for the drug. Mutation of residues located at the connection of the DNA-reading head with the protein core (class 2) and at the dimerization interface (class 3) bind inducer with the same affinity as wild-type TetR. These mutations interfere with the induced, but not the operator-binding conformation of TetR. The affinity of some class 1 mutants for tetracycline is less affected than their inducibility, suggesting that the mutated residues are important for triggering those conformational changes necessary for induction.

Amino Acid Sequence↗
PubMed1994

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.

Carbohydrate Conformation↗
PubMed1994

Three-dimensional structure of the adenine-specific DNA methyltransferase M.Taq I in complex with the cofactor S-adenosylmethionine.

The Thermus aquaticus DNA methyltransferase M.Taq I (EC 2.1.1.72) methylates N6 of adenine in the specific double-helical DNA sequence TCGA by transfer of --CH3 from the cofactor S-adenosyl-L-methionine. The x-ray crystal structure at 2.4-A resolution of this enzyme in complex with S-adenosylmethionine shows alpha/beta folding of the polypeptide into two domains of about equal size. They are arranged in the form of a C with a wide cleft suitable to accommodate the DNA substrate. The N-terminal domain is dominated by a nine-stranded beta-sheet; it contains the two conserved segments typical for N-methyltransferases which form a pocket for cofactor binding. The C-terminal domain is formed by four small beta-sheets and alpha-helices. The three-dimensional folding of M.Taq I is similar to that of the cytosine-specific Hha I methyltransferase, where the large beta-sheet in the N-terminal domain contains all conserved segments and the enzymatically functional parts, and the smaller C-terminal domain is less structured.

Bacterial Proteins↗
PubMed1994

Crystal structure of calcium-free proteinase K at 1.5-A resolution.

Proteinase K from the fungus Tritirachium album Limber binds two Ca2+ ions, one strongly (Ca 1) and the other weakly (Ca 2). Removal of these cations reduces the stability of proteinase K as shown by thermal denaturation, but the proteolytic activity is unchanged. The x-ray structures of native and Ca(2+)-free proteinase K at 1.5-A resolution show that there are no cuts in the polypeptide backbone (i.e. no autolysis), Ca 1 has been replaced by Na+, while Ca 2 has been substituted by a water associated with a larger but locally confined structural change at that site. A small but concerted geometrical shift is transmitted from the Ca 1 site via eight secondary structure elements to the substrate recognition site (Gly100-Tyr104, and Ser132-Gly136) but not to the catalytic triad (Asp39,His69,Ser224). This is accompanied by positional changes of localized waters.

Binding Sites↗
PubMed1994

Impact of point mutations on the structure and thermal stability of ribonuclease T1 in aqueous solution probed by Fourier transform infrared spectroscopy.

We undertook a detailed comparative analysis of the infrared spectra of wild-type ribonuclease T1 and three mutants: two single mutants, Tyr-45-->Trp (Y45W) and Trp-59-->Tyr (W59Y), and a double mutant, Tyr-45-->Trp/Trp-59-->Tyr (Y45W/W59Y). These mutants were selected because they are known to affect the activity of the enzyme. The structural differences were evaluated by using peptide backbone and side-chain "marker" bands as conformation-sensitive monitors. All mutations lead to a decrease of the thermal transition temperature, though the mutation Tyr-45-->Trp affects the Tm to a lesser degree than the replacement of Trp-59 by Tyr, both in the single (W59Y) and in the double (Y45W/W59Y) mutant. Small changes in the protein backbone conformation and in the microenvironment of certain amino acids, induced by the point mutations, could be detected. In particular, we found subtle differences in the hydrogen bonding pattern of the beta-strands in the mutants W59Y and Y45W/W59Y, compared to that in wild-type RNase T1 and in the mutant Y45W. Practically identical spectra in the amide I region were obtained for the double mutant Y45W/W59Y and the single mutant W59Y, demonstrating that it is the change from Trp to Tyr in position 59 (located at the interface between the alpha-helix and a beta-strand) which affects the overall protein conformation. The mutation Tyr to Trp in position 45, on the other hand, has practically no impact on the polypeptide backbone conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspergillus oryzae↗
PubMed1994

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.

Antiporters↗
PubMed1994

X-ray crystallographic and calorimetric studies of the effects of the mutation Trp59-->Tyr in ribonuclease T1.

Two mutants of ribonuclease T1 (RNaseT1), [59-tyrosine]ribonuclease T1 (W59Y) and [45-tryptophan,59-tyrosine]ribonuclease T1 (Y45W/W59Y) possess between 150% and 190% wild-type activity. They have been crystallised as complexes of the inhibitor 2'-guanylic acid and analysed by X-ray diffraction at resolutions of 0.23 nm and 0.24 nm, respectively. The space group for both is monoclinic, P2(1), with two molecules/asymmetric unit, W59Y: a = 4.934 nm, b = 4.820 nm, c = 4.025 nm, beta = 90.29 degrees. Y45W/W59Y: a = 4.915 nm, b = 4.815 nm, c = 4.015 nm, beta = 90.35 degrees. Compared to wild-type RNaseT1 in complex with 2'-guanylic acid (2'GMP) both mutant inhibitor complexes indicate that the replacement of Trp59 by Tyr leads to a 0.04-nm inward shift of the single alpha-helix and to significant differences in the active-site geometry, inhibitor conformation and inhibitor binding. Calorimetric studies of a range of mutants [24-tryptophan]ribonuclease T1 (Y24W), [42-tryptophan]ribonuclease T1 (Y42W), [45-tryptophan]ribonuclease T1 (Y45W), [92-alanine]ribonuclease T1 (H92A) and [92-threonine]ribonuclease T1 (H92T) with and without the further mutation Trp59-->Tyr showed that mutant proteins for which Trp59 is replaced by Tyr exhibit slightly decreased thermal stability.

Amino Acid Sequence↗
PubMed1993

The complex between ribonuclease T1 and 3'GMP suggests geometry of enzymic reaction path. An X-ray study.

The crystal structure of the complex between ribonuclease T1 and 3'GMP suggests that (a) a substrate GpN is bound to the active site of ribonuclease T1 in a conformation that actively supports the catalytic process, (b) the reaction occurs in an in-line process, (c) His40 N epsilon H+ activates O2'-H, (d) Glu58 carboxylate acts as base and His92 N epsilon H+ as acid in a general acid-base catalysis. The crystals have the monoclinic space group P2(1), a = 4.968 nm, b = 4.833 nm, c = 4.048 nm, beta = 90.62 degrees with two molecules in the asymmetric unit. The structure was determined by molecular replacement and refined to R = 15.3% with 11,338 data > or = 1 sigma (Fo) in the resolution range 1.0-0.2 nm; this includes 180 water molecules and two Ca2+. The structure of ribonuclease T1 is as previously observed. 3'GMP is bound in syn conformation; guanine is located in the specific recognition site, the ribose adopts C4'-exo puckering, the ribose phosphate is extended with torsion angle epsilon in trans. The O2'-H group is activated by accepting and donating hydrogen bonds from His40 N epsilon H+ and to Glu58 O epsilon 1; the phosphate is hydrogen bonded to Glu58 O epsilon 2H, Arg77 N epsilon H+ and N eta 2H+, Tyr38 O eta H, His92 N eta H+. The conformation of ribose phosphate is such that O2' is at a distance of 0.31 nm from phosphorus, and opposite the P-OP3 bond which accepts a hydrogen bond from His92 N epsilon H+; we infer from a model building study that this bond is equivalent to the scissile P-O5' in a substrate GpN.

Binding Sites↗
PubMed1993

Studies on the inhibitory action of mercury upon proteinase K.

In proteinase K, Cys73 is located "below" the imidazole of the active site His69. In a 2.4-A resolution x-ray crystal structure of the complex formed between the enzyme and HgAc2, two Hg(II) positions are found: a fully occupied site, covalently bound to Cys73 (S gamma), which disrupts the catalytic triad (Asp39-His69-Ser224), and a 2-fold disordered (25 and 35% occupancy), noncovalent complexation to His72, Cys73, and Thr76 of lower affinity. The enzyme is inhibited noncompetitively at low concentrations and competitively above stoichiometric concentrations of Hg(II), but it retains 7% residual activity. This can be rationalized if the molecule is flexible enough to permit transient formation of the catalytic triad. Except for the active site, only minor structural changes are observed upon binding of Hg(II), but the thermal stability is reduced by 4 degrees C.

Binding Sites↗
PubMed1993

Crystal structures of cyclomaltoheptaose (beta-cyclodextrin) complexed with ethylene glycol.8.0H2O and glycerol.7.2H2O.

Single-crystal X-ray diffraction studies were carried out for the title compounds at room temperature. The crystal packings are of the cage-type and isomorphous to that of beta-cyclodextrin (beta CD) hydrate. In both crystal structures, disorder and extensive thermal vibrations of the complexed guest molecules are observed. In beta CD-ethylene glycol.8H2O, one ethylene glycol molecule (disordered over two discrete sites) and three water molecules (four discrete sites) are included in the beta CD cavity. Within the beta CD cavity, all oxygen sites (ordered and disordered) are in positions occupied by water molecules in beta CD.12H2O; this is only possible because the ethylene glycol molecule adopts the low-energy conformation with the O-C-C-O torsion angle approximately 60 degrees and an O...O separation of 2.9 A, in which its hydroxyl groups can directly substitute for two hydrogen-bonding water molecules. In beta CD-glycerol.7.2H2O, one glycerol molecule (disordered over two discrete sites) and two water molecules (two fully occupied sites) are included in the beta CD cavity. The general situation in both compounds parallels that found earlier in beta CD-ethanol.8H2O. It is assumed that the disorder is dynamic, i.e., associated with jumps between the partially occupied molecular sites.

Carbohydrate Sequence↗