Biomedical subjects
Publications and source records attributed to C Kisker.
Studies on several recently discovered error-prone DNA polymerases reveal novel structures that may explain the low fidelity of this general class of enzymes, a number of which are involved in the replicative bypass (translesion synthesis) of base damage in DNA.
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Polymerase gamma, which replicates and repairs mitochondrial DNA, requires the Pol gamma B subunit for processivity. We determined the crystal structure of mouse Pol gamma B, a core component of the mitochondrial replication machinery. Pol gamma B shows high similarity to glycyl-tRNA synthetase and dimerizes through an unusual intermolecular four-helix bundle. A human Pol gamma B mutant lacking the four-helix bundle failed to dimerize in solution or to stimulate the catalytic subunit Pol gamma A, but retained the ability to bind with Pol gamma A to a primer-template construct, indicating that the functional holoenzyme contains two Pol gamma B molecules. Other mutants retained stimulatory activity but lost the ability to bind folded ssDNA. These results suggest that the Pol gamma B dimer contains distinct sites for Pol gamma A binding, dimerization, and DNA binding.
Nucleotide excision repair (NER) is a universal DNA repair mechanism found in all three kingdoms of life. Its ability to repair a broad range of DNA lesions sets NER apart from other repair mechanisms. NER systems recognize the damaged DNA strand and cleave it 3', then 5' to the lesion. After the oligonucleotide containing the lesion is removed, repair synthesis fills the resulting gap. UvrB is the central component of bacterial NER. It is directly involved in distinguishing damaged from undamaged DNA and guides the DNA from recognition to repair synthesis. Recently solved structures of UvrB from different organisms represent the first high-resolution view into bacterial NER. The structures provide detailed insight into the domain architecture of UvrB and, through comparison, suggest possible domain movements. The structure of UvrB consists of five domains. Domains 1a and 3 bind ATP at the inter-domain interface and share high structural similarity to helicases of superfamilies I and II. Not related to helicase structures, domains 2 and 4 are involved in interactions with either UvrA or UvrC, whereas domain 1b was implicated for DNA binding. The structures indicate that ATP binding and hydrolysis is associated with domain motions. UvrB's ATPase activity, however, is not coupled to the separation of long DNA duplexes as in helicases, but rather leads to the formation of the preincision complex with the damaged DNA substrate. The location of conserved residues and structural comparisons with helicase-DNA structures suggest how UvrB might bind to DNA. A model of the UvrB-DNA interaction in which a beta-hairpin of UvrB inserts between the DNA double strand has been proposed recently. This padlock model is developed further to suggest two distinct consequences of domain motion: in the UvrA(2)B-DNA complex, domain motions lead to translocation along the DNA, whereas in the tight UvrB-DNA pre-incision complex, they lead to distortion of the 3' incision site.
The prototype of the gamma-class of carbonic anhydrase has been characterized from the methanogenic archaeon Methanosarcina thermophila. Previously reported kinetic studies of the gamma-class carbonic anhydrase are consistent with this enzyme having a reaction mechanism similar to that of the mammalian alpha-class carbonic anhydrase. However, the overall folds of these two enzymes are dissimilar, and apart from the zinc-coordinating histidines, the active site residues bear little resemblance to one another. The crystal structures of zinc-containing and cobalt-substituted gamma-class carbonic anhydrases from M. thermophila are reported here between 1.46 and 1.95 A resolution in the unbound form and cocrystallized with either SO(4)(2)(-) or HCO(3)(-). Relative to the tetrahedral coordination geometry seen at the active site in the alpha-class of carbonic anhydrases, the active site of the gamma-class enzyme contains additional metal-bound water ligands, so the overall coordination geometry is trigonal bipyramidal for the zinc-containing enzyme and octahedral for the cobalt-substituted enzyme. Ligands bound to the active site all make contacts with the side chain of Glu 62 in manners that suggest the side chain is likely protonated. In the uncomplexed zinc-containing enzyme, the side chains of Glu 62 and Glu 84 appear to share a proton; additionally, Glu 84 exhibits multiple conformations. This suggests that Glu 84 may act as a proton shuttle, which is an important aspect of the reaction mechanism of alpha-class carbonic anhydrases. A hydrophobic pocket on the surface of the enzyme may participate in the trapping of CO(2) at the active site. On the basis of the coordination geometry at the active site, ligand binding modes, the behavior of the side chains of Glu 62 and Glu 84, and analogies to the well-characterized alpha-class of carbonic anhydrases, a more-defined reaction mechanism is proposed for the gamma-class of carbonic anhydrases.
The excessive use of antibiotics has enabled bacteria to develop resistance through a variety of mechanisms. The most common bacteriostatic action of the broad-spectrum antibiotic tetracycline (Tc) is by the inactivation of the bacterial ribosome so that the protein biosynthesis is interrupted and the bacteria die. The most common mechanism of resistance in gram-negative bacteria against Tc is associated with the membrane-intrinsic protein TetA, which exports invaded Tc out of the bacterial cell before it can attack its target, the ribosome. The expression of TetA is tightly regulated by the homodimeric Tet repressor (TetR)(2), which binds specifically with two helix-turn-helix motifs of operator DNA (tetO; K(ass) approximately 10(11) M(-1)) located upstream from the tetA gene on a plasmid or transposon. When Tc diffuses into the cell it chelates Mg(2+) and the complex [MgTc](+) binds to (TetR)(2) to form the induced complex (TetR small middle dot[MgTc](+))(2). This process is associated with conformational changes, which sharply reduce the affinity of (TetR)(2) to tetO, so that expression of TetA can take place, thus conferring resistance to the bacteria cells against Tc. Crystallographic studies show sequence-specific protein-nucleic acid interactions in the (TetR)(2) small middle dottetO complex and how the binding of two [MgTc](+) to (TetR)(2) enforces conformational changes that are stabilized by cooperative binding of two chains of eight water molecules each so that the formed (TetR small middle dot[MgTc](+))(2) is no longer able to recognize and bind to tetO. Since the switching mechanisms of the TetR/[MgTc](+) system is so tight, it has proven very useful in the regulation of eukaryotic gene expression and may also be applicable in gene therapy.
Nucleotide excision repair (NER) is a highly conserved DNA repair mechanism. NER systems recognize the damaged DNA strand, cleave it on both sides of the lesion, remove and newly synthesize the fragment. UvrB is a central component of the bacterial NER system participating in damage recognition, strand excision and repair synthesis. We have solved the crystal structure of UvrB in the apo and the ATP-bound forms. UvrB contains two domains related in structure to helicases, and two additional domains unique to repair proteins. The structure contains all elements of an intact helicase, and is evidence that UvrB utilizes ATP hydrolysis to move along the DNA to probe for damage. The location of conserved residues and structural comparisons allow us to predict the path of the DNA and suggest that the tight pre-incision complex of UvrB and the damaged DNA is formed by insertion of a flexible beta-hairpin between the two DNA strands.
The enoyl-acyl carrier protein reductase (ENR) is involved in bacterial fatty acid biosynthesis and is the target of the antibacterial diazaborine compounds and the front-line antituberculosis drug isoniazid. Recent studies suggest that ENR is also the target for the broad-spectrum biocide triclosan. The 1.75 A crystal structure of EnvM, the ENR from Escherichia coli, in complex with triclosan and NADH reveals that triclosan binds specifically to EnvM. These data provide a molecular mechanism for the antibacterial activity of triclosan and substantiate the hypothesis that its activity results from inhibition of a specific cellular target rather than non-specific disruption of the bacterial cell membrane. This has important implications for the emergence of drug-resistant bacteria, since triclosan is an additive in many personal care products such as toothpastes, mouthwashes and soaps. Based on this structure, rational design of triclosan derivatives is possible which might be effective against recently identified triclosan-resistant bacterial strains.
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This work gives an overview of the recent achievements which have contributed to the understanding of the structure and function of molybdenum and tungsten enzymes. Known structures of molybdo-pterin cofactor-containing enzymes will be described briefly and the structural differences between representatives of the same and different families will be analyzed. This comparison will show that the molybdo-pterin cofactor-containing enzymes represent a very heterogeneous group with differences in overall enzyme structure, cofactor composition and stoichiometry, as well as differences in the immediate molybdenum environment. Two recently discovered molybdo-pterin cofactor-containing enzymes will be described with regard to molecular and EPR spectroscopic properties, pyrogallol-phloroglucinol transhydroxylase from Pelobacter acidigallici and acetylene hydratase from Pelobacter acetylenicus. On the basis of its amino acid sequence, transhydroxylase can be classified as a member of the dimethylsulfoxide reductase family, whereas classification of the tungsten/molybdenum-containing acetylene hydratase has to await the determination of its amino acid sequence.
The molybdenum-containing enzyme sulfite oxidase catalyzes the conversion of sulfite to sulfate, the terminal step in the oxidative degradation of cysteine and methionine. Deficiency of this enzyme in humans usually leads to major neurological abnormalities and early death. The crystal structure of chicken liver sulfite oxidase at 1.9 A resolution reveals that each monomer of the dimeric enzyme consists of three domains. At the active site, the Mo is penta-coordinated by three sulfur ligands, one oxo group, and one water/hydroxo. A sulfate molecule adjacent to the Mo identifies the substrate binding pocket. Four variants associated with sulfite oxidase deficiency have been identified: two mutations are near the sulfate binding site, while the other mutations occur within the domain mediating dimerization.
The coupling of ATP hydrolysis to electron transfer by the enzyme nitrogenase during biological nitrogen fixation is an important example of a nucleotide-dependent transduction mechanism. The crystal structure has been determined for the complex between the Fe-protein and MoFe-protein components of nitrogenase stabilized by ADP x AIF4-, previously used as a nucleoside triphosphate analogue in nucleotide-switch proteins. The structure reveals that the dimeric Fe-protein has undergone substantial conformational changes. The beta-phosphate and AIF4- groups are stabilized through intersubunit contacts that are critical for catalysis and the redox centre is repositioned to facilitate electron transfer. Interactions in the nitrogenase complex have broad implications for signal and energy transduction mechanisms in multiprotein complexes.
Molybdenum-containing enzymes catalyze basic metabolic reactions in the nitrogen, sulfur, and carbon cycles. With the exception of the nitrogenase cofactor, molybdenum is incorporated into proteins as the molybdenum cofactor that contains a mononuclear molybdenum atom coordinated to the sulfur atoms of a pterin derivative named molybdopterin. Certain microorganisms can also utilize tungsten in a similar fashion. Molybdenum-cofactor-containing enzymes catalyze the transfer of an oxygen atom, ultimately derived from or incorporated into water, to or from a substrate in a two-electron redox reaction. On the basis of sequence alignments and spectroscopic properties, four families of molybdenum-cofactor-containing enzymes have been identified. The available crystallographic structures for members of these families are discussed within the framework of the active site structure and catalytic mechanisms of molybdenum-cofactor-containing enzymes. Although the function of the molybdopterin ligand has not yet been conclusively established, interactions of this ligand with the coordinated metal are sensitive to the oxidation state, indicating that the molybdopterin may be directly involved in the enzymatic mechanism.
The periplasmic dimethyl sulfoxide reductase (DMSOR) from the photosynthetic purple bacterium Rhodobacter capsulatus functions as the terminal electron acceptor in its respiratory chain. The enzyme catalyzes the reduction of highly oxidized substrates like dimethyl sulfoxide to dimethyl sulfide. At a molybdenum redox center, two single electrons are transferred from cytochrome C556 to the substrate dimethyl sulfoxide, generating dimethyl sulfide and (with two protons) water. The enzyme was purified and crystallized in space group P4(1)2(1)2 with unit cell dimensions of a = b = 80.7 A and c = 229.2 A. The crystals diffract beyond 1.8 A with synchrotron radiation. The three-dimensional structure was solved by a combination of multiple isomorphous replacement and molecular replacement techniques. The atomic model was refined to an R-factor of 0.169 for 57,394 independent reflections. The spherical protein consists of four domains with a funnel-like cavity that leads to the freely accessible metal-ion redox center. The bis(molybdopterin guanine dinucleotide) molybdenum cofactor (1541 Da) of the single chain protein (85,033 Da) has the molybdenum ion bound to the cis-dithiolene group of only one molybdopterin guanine dinucleotide molecule. Three additional ligands, two oxo groups and the oxygen of a serine side-chain, are bound to the molybdenum ion. The second molybdopterin system is not part of the ligand sphere of the metal center with its sulfur atoms at distances of 3.5 A and 3.8 A away. It might be involved in electron shuttling from the protein surface to the molybdenum center.
The molybdoenzyme dimethylsulfoxide (DMSO) reductase contributes to the release of dimethylsulfide, a compound that has been implicated in cloud nucleation and global climate regulation. The crystal structure of DMSO reductase from Rhodobacter sphaeroides reveals a monooxo molybdenum cofactor containing two molybdopterin guanine dinucleotides that asymmetrically coordinate the molybdenum through their dithiolene groups. One of the pterins exhibits different coordination modes to the molybdenum between the oxidized and reduced states, whereas the side chain oxygen of Ser147 coordinates the metal in both states. The change in pterin coordination between the Mo(VI) and Mo(IV) forms suggests a mechanism for substrate binding and reduction by this enzyme. Sequence comparisons of DMSO reductase with a family of bacterial oxotransferases containing molybdopterin guanine dinucleotide indicate a similar polypeptide fold and active site with two molybdopterins within this family.
A carbonic anhydrase from the thermophilic archaeon Methanosarcina thermophila that exhibits no significant sequence similarity to known carbonic anhydrases has recently been characterized. Here we present the structure of this enzyme, which adopts a left-handed parallel beta-helix fold. This fold is of particular interest since it contains only left-handed crossover connections between the parallel beta-strands, which so far have been observed very infrequently. The active form of the enzyme is a trimer with three zinc-containing active sites, each located at the interface between two monomers. While the arrangement of active site groups differs between this enzyme and the carbonic anhydrases from higher vertebrates, there are structural similarities in the zinc coordination environment, suggestive of convergent evolution dictated by the chemical requirements for catalysis of the same reaction. Based on sequence similarities, the structure of this enzyme is the prototype of a new class of carbonic anhydrases with representatives in all three phylogenetic domains of life.
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.
We demonstrate in a quantitative in vitro induction assay that tetracycline-Fe2+ is a more than 1000-fold stronger inducer of Tet repressor compared to tetracycline-Mg2+. Oxidative cleavage of the Tet repressor-tetracycline-Fe2+ complex with H2O2 and ascorbate results in an Fe(2+)-dependent specific fragmentation of the protein. The maximal yield of about 15% and a reaction time of less than 30 s are only observed in the presence of the drug, whereas about 1% cleavage is obtained after 30 min in the presence of Fe2+ without tetracycline. Cleavage is not inhibited by several radical scavengers, suggesting a highly localized reactivity of the redox-active oxo intermediates in the proximity of the Fe(2+)-tc chelater where they are generated. The products can be separated by HPLC only after denaturation, indicating that the complex is not disrupted by cleavage. Residues at which the cleavage takes place are identified using the masses of the fragments determined by electrospray mass spectrometry and their N-terminal sequences. The major cleavage site maps to residues 104 and 105 of Tet repressor. Less efficient cleavages occur at residues 56 and 136, and the least efficiently cleaved sites are around residues 144 and 147. The cleavage efficiencies correlate to the distances and orientations of the respective peptide bonds to Mg2+ in the crystal structure of the Tet repressor-tetracycline-Mg2+ complex. We discuss potential reaction mechanisms leading to protein cleavage.