Identification and localization of differences between Escherichia coli and Salmonella typhimurium genomes by suppressive subtractive hybridization

The availability of bacterial genome sequences raises an important new problem - how can one move from completely sequenced microorganisms as a reference to the hundreds and thousands of other strains or isolates of the same or related species that will not be sequenced in the near future? An efficient way to approach this task is the comparison of genomes by subtractive hybridization. Recently we developed a sensitive and reproducible subtraction procedure for comparison of bacterial genomes, based on the method of suppression subtractive hybridization (SSH). In this work we demonstrate the applicability of subtractive hybridization to the comparison of the related but markedly divergent bacterial species Escherichia coli and Salmonella typhimurium. Clone libraries representing sequence differences were obtained and, in the case of completely sequenced E. coli genome, the differences were directly placed in the genome map. About 60% of the differential clones identified by SSH were present in one of the genomes under comparison and absent from the other. Additional differences in most cases represent sequences that have diverged considerably in the course of evolution. Such an approach to comparative bacterial genomics can be applied both to studies of interspecies evolution - to elucidate the "strategies" that enable different genomes to fit their ecological niches - and to development of diagnostic probes for the rapid identification of pathogenic bacterial species.     

Balanced regulation of microtubule dynamics during the cell cycle: a contemporary view

Assembly of mitotic and meiotic spindles into an elliptical bipolar shape is an example of morphogenetic processes that involve local chromosomal regulation of microtubule dynamics for proper spatial microtubule assembly. Global microtubule dynamics during the cell cycle and local microtubule dynamics during spindle assembly are regulated by a balance between microtubule stabilizing and destabilizing factors. How a chromosome-induced phosphorylation gradient may be generated and modulate spindle microtubule assembly through balanced regulation of the activity of microtubule-associated proteins and Stathmin/Op 18 is analyzed.     

The recombinant third domain of human alpha-fetoprotein retains the antiestrotrophic activity found in the full-length molecule

Previous studies have shown that alpha-fetoprotein (AFP) interferes with estrogen (E2)-stimulated growth, including E2-stimulated breast cancer growth. In an effort to localize the antiestrotrophic portion of the molecule, the C-terminal one-third (200 amino acids) of human AFP, known as Domain III, was produced in a baculovirus expression system as a fusion protein containing an amino terminal histidine tag. The histidine tag was included to facilitate purification by metal ion affinity chromatography. The purified recombinant Domain III fusion protein was functionally similar to full-length natural AFP isolated from human cord sera or from cultured human hepatoma cells (HepG2) in that they all produced significant and quantitatively similar inhibition of E2-stimulated growth of immature mouse uterus. Furthermore, the dose-response profiles of the recombinant Domain III AFP and natural full-length AFP were similar. Preincubation of either protein in a molar excess of E2 lowered the minimally effective antiestrotrophic dose and produced a difference spectrum consistent with a change in conformation. These findings indicate that the antiestrotrophic activity of AFP is contained within the third domain of the molecule, and they have obvious implications for the production of biologically active peptides derived from this portion of the AFP molecule.     

Crystal structure of ADP-ribosylated ribosomal translocase from Saccharomyces cerevisiae

The crystal structure of ADP-ribosylated yeast elongation factor 2 in the presence of sordarin and GDP has been determined at 2.6 A resolution. The diphthamide at the tip of domain IV, which is the target for diphtheria toxin and Pseudomonas aeruginosa exotoxin A, contains a covalently attached ADP-ribose that functions as a very potent inhibitor of the factor. We have obtained an electron density map of ADP-ribosylated translation factor 2 revealing both the ADP-ribosylation and the diphthamide. This is the first structure showing the conformation of an ADP-ribosylated residue and confirms the inversion of configuration at the glycosidic linkage. Binding experiments show that the ADP-ribosylation has limited effect on nucleotide binding affinity, on ribosome binding, and on association with exotoxin A. These results provide insight to the inhibitory mechanism and suggest that inhibition may be caused by erroneous interaction of the translation factor with the codon-anticodon area in the P-site of the ribosome.     

The transducer domain is important for clamp operation in human DNA topoisomerase IIalpha

DNA topoisomerase II is a multidomain homodimeric enzyme that changes DNA topology by coupling ATP hydrolysis to the transport of one DNA helix through a transient double-stranded break in another. The process requires dramatic conformational changes including closure of an ATP-operated clamp, which is comprised of two N-terminal domains from each protomer. The most N-terminal domain contains the ATP-binding site and is directly involved in clamp closure, undergoing dimerization upon ATP binding. The second domain, the transducer domain, forms the walls of the N-terminal clamp and connects the clamp to the enzyme core. Although structurally conserved, it is unclear whether the transducer domain is involved in clamp mechanism. We have purified and characterized a human topoisomerase II alpha enzyme with a two-amino acid insertion at position 408 in the transducer domain. The enzyme retains both ATPase and DNA cleavage activities. However, the insertion, which is situated far from the N-terminal dimerization area, severely disrupts the function of the N-terminal clamp. The clamp-deficient enzyme is catalytically inactive and lacks most aspects of interdomain communication. Surprisingly, it seems to have retained the intersubunit communication, allowing it to bind ATP cooperatively in the presence of DNA. The results show that even distal parts of the transducer domain are important for the dynamics of the N-terminal clamp and furthermore indicate that stable clamp closure is not required for cooperative binding of ATP.     

Structural determinants of the anti-HIV activity of a CCR5 antagonist derived from Toxoplasma gondii

The protozoan parasite Toxoplasma gondii possesses a protein, cyclophilin-18 (C-18), which binds to the chemokine receptor CCR5, induces interleukin-12 production from murine dendritic cells, and inhibits fusion and infectivity of human immunodeficiency virus 1 (HIV-1) R5 viruses by co-receptor antagonism. Site-directed mutagenesis was employed to identify the domains in C-18 responsible for its CCR5 binding and antiviral functions. To do so we focused on amino acid differences with Plasmodium falciparum cyclophilin, which, although 53% identical with C-18, has minimal binding activity for CCR5, and we generated 22 mutants with substitutions in the regions of non-homology located on the putative surface of the molecule. Two mutations situated on the face of C-18, predicted to be involved in its interaction with the ligand cyclosporin A, were shown to be critical for CCR5-binding and the inhibition of HIV-1 fusion and infectivity. In contrast, four mutations in C-18 specifically designed to abolish the peptidyl-prolyl cis-trans-isomerase activity of the protein failed to inactivate its CCR5 binding and HIV inhibitory activities. Interleukin-12 induction by C-18, on the other hand, was abrogated by mutations effecting either the CCR5 binding or enzymatic function of the molecule. These findings shed light on the structural basis of the molecular mimicry of the chemokine function by a pathogen-derived protein and provide a basis for further modification of C-18 into an antiviral agent.     

Localization of a K+ -binding site involved in dephosphorylation of the sarcoplasmic reticulum Ca2+ -ATPase

K+ plays an important role for the function of the sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA), but its binding site within the molecule has remained unidentified. We have located the binding site for a K+ ion in the P-domain by means of x-ray crystallography using crystals prepared in the presence of the K+ congener Rb+. Backbone carbonyls from the loop containing residues 711-715 together with the side chain of Glu732 define the K+/Rb+ site in the Ca2+ -ATPase conformation with bound Ca2+, ADP, and AlF4-. Functional analysis of Ca2+ -ATPase mutants with alterations to Glu732 shows that this site is indeed important for the stimulatory effect of K+ on the dephosphorylation rate. Comparison with the Ca2+ -ATPase in a dephosphorylated E2 conformation suggests that the K+ site is involved in the correct movement and positioning of the A-domain during translocation and dephosphorylation.     

CymA of Klebsiella oxytoca outer membrane: binding of cyclodextrins and study of the current noise of the open channel

CymA, the outer membrane component of the cyclodextrin (CD) uptake and metabolism system of Klebsiella oxytoca, was reconstituted into lipid bilayer membranes. The channel properties of this unusual porin were studied in detail. The binding of CDs to the channel resulted in its complete block for ion transport. This result allowed the detailed investigation of carbohydrate binding, and the stability constants for the binding of cyclic and linear carbohydrates to the binding site inside the channel were calculated from titration experiments of the membrane conductance with the carbohydrates. Highest stability constant was observed for alpha-cyclodextrin (alpha-CD; K = 32,000 1/M) followed by beta-cyclodextrin (beta-CD; K = 1970 1/M) and gamma-cyclodextrin (gamma-CD; K = 310 1/M). Linear maltooligosaccharides bound also to CymA but with much smaller stability constants as compared to cyclic ones. The noise of the current through CymA in multi- and single-channel experiments was investigated using fast Fourier transformation. The current through the open channels had a rather high spectral density, which was a Lorentzian function of the frequency up to 2000 Hz. Upon addition of cyclic dextrins to the aqueous phase the spectral density decreased in a dose-dependent manner, which made it impossible to evaluate the binding kinetics. Experiments with single CymA-channels demonstrated the channel is highly asymmetric concerning channel flickers and current noise.     

Cholesterol-induced protein sorting: an analysis of energetic feasibility

The mechanism(s) underlying the sorting of integral membrane proteins between the Golgi complex and the plasma membrane remain uncertain because no specific Golgi retention signal has been found. Moreover one can alter a protein's eventual localization simply by altering the length of its transmembrane domain (TMD). M. S. Bretscher and S. Munro (SCIENCE: 261:1280-1281, 1993) therefore proposed a physical sorting mechanism based on the hydrophobic match between the proteins' TMD and the bilayer thickness, in which cholesterol would regulate protein sorting by increasing the lipid bilayer thickness. In this model, Golgi proteins with short TMDs would be excluded from cholesterol-enriched domains (lipid rafts) that are incorporated into transport vesicles destined for the plasma membrane. Although attractive, this model remains unproven. We therefore evaluated the energetic feasibility of a cholesterol-dependent sorting process using the theory of elastic liquid crystal deformations. We show that the distribution of proteins between cholesterol-enriched and cholesterol-poor bilayer domains can be regulated by cholesterol-induced changes in the bilayer physical properties. Changes in bilayer thickness per se, however, have only a modest effect on sorting; the major effect arises because cholesterol changes also the bilayer material properties, which augments the energetic penalty for incorporating short TMDs into cholesterol-enriched domains. We conclude that cholesterol-induced changes in the bilayer physical properties allow for effective and accurate sorting which will be important generally for protein partitioning between different membrane domains.     

A human topoisomerase II alpha heterodimer with only one ATP binding site can go through successive catalytic cycles

Eukaryotic DNA topoisomerase II is a dimeric nuclear enzyme essential for DNA metabolism and chromosome dynamics. It changes the topology of DNA by coupling binding and hydrolysis of two ATP molecules to the transport of one DNA duplex through a temporary break introduced in another. During this process the structurally and functionally complex enzyme passes through a cascade of conformational changes, which requires intra- and intersubunit communication. To study the importance of ATP binding and hydrolysis in relation to DNA strand transfer, we have purified and characterized a human topoisomerase II alpha heterodimer with only one ATP binding site. The heterodimer was able to relax supercoiled DNA, although less efficiently than the wild type enzyme. It furthermore possessed a functional N-terminal clamp and was sensitive to ICRF-187. This demonstrates that human topoisomerase II alpha can pass through all the conformations required for DNA strand passage and enzyme resetting with binding and hydrolysis of only one ATP. However, the heterodimer lacked the normal stimulatory effect of DNA on ATP binding and hydrolysis as well as the stimulatory effect of ATP on DNA cleavage. The results can be explained in a model, where efficient catalysis requires an extensive communication between the second ATP and the DNA segment to be cleaved.