The kinetic basis for dual recognition in colicin endonuclease-immunity protein complexes

The antibacterial activity of E colicin endonucleases (DNases) is counteracted by the binding of immunity proteins; the affinities of cognate and non-cognate complexes differing by up to ten orders of magnitude. Here, we address the mechanism of complex formation using a combination of protein engineering, pre-steady-state kinetics and isothermal titration calorimetry, in order to understand the underlying basis for specificity. Contrary to previous work, we show that a pre-equilibrium mechanism does not explain the binding kinetics. Instead, the data are best explained by a modified induced-fit mechanism where cognate and non-cognate complexes alike form a non-specific, conformationally dynamic encounter complex, most likely centred on conserved interactions at the interface. The dynamics appear to be an intrinsic property of the encounter complex where the proteins move relative to one another, thereby sampling different conformations rather than being "induced" by binding. This allows optimal alignment of interface specificity sites, without producing energetically costly conformational changes, essential for high-affinity binding. Importantly, specificity is achieved without slowing the rate of association, an important requirement for rapid inhibition of the colicin in the producing bacterial cell. A rigid-body rotation model is also consistent with the observation that specificity contacts in colicin-immunity protein complexes can involve different regions of the interface. Such a kinetic discrimination mechanism explains the ability of DNase-specific immunity proteins to display dual recognition specificity, wherein they are broadly cross-reactive yet are highly specific, achieving femtomolar binding affinities in complexes with their cognate DNases.     

Highly discriminating protein-protein interaction specificities in the context of a conserved binding energy hotspot

We explore the thermodynamic basis for high affinity binding and specificity in conserved protein complexes using colicin endonuclease-immunity protein complexes as our model system. We investigated the ability of each colicin-specific immunity protein (Im2, Im7, Im8 and Im9) to bind the endonuclease (DNase) domains of colicins E2, E7 and E8 in vitro and compared these to the previously studied colicin E9. We find that high affinity binding (Kd < or = 10(-14) M) is a common feature of cognate colicin DNase-Im protein complexes as are non-cognate protein-protein associations, which are generally 10(6)-10(8)-fold weaker. Comparative alanine scanning of Im2 and Im9 residues involved in binding the E2 DNase revealed similar behaviour to that of the two proteins binding the E9 DNase; helix III forms a conserved binding energy hotspot with specificity residues from helix II only contributing favourably in a cognate interaction, a combination we have termed as "dual recognition". Significant differences are seen, however, in the number and side-chain chemistries of specificity sites that contribute to cognate binding. In Im2, Asp33 from helix II dominates colicin E2 specificity, whereas in Im9 several hydrophobic residues, including position 33 (leucine), help define its colicin specificity. A similar distribution of specificity sites was seen using phage display where, with Im2 as the template, a library of randomised sequences was generated in helix II and the library panned against either the E2 or E9 DNase. Position 33 was the dominant specificity site recovered in all E2 DNase-selected clones, whereas a number of Im9 specificity sites were recovered in E9 DNase-selected clones, including position 33. In order to probe the relationship between biological specificity and in vitro binding affinity we compared the degree of protection afforded to bacteria against colicin E9 toxicity by a set of immunity proteins whose affinities for the E9 DNase differed by up to ten orders of magnitude. This analysis indicated that the Kd required for complete biological protection is <10(-10)M and that the "affinity window" over which the selection of novel immunity protein specificities likely evolves is 10(-6)-10(-10)M. This comprehensive survey of colicin DNase-immunity protein complexes illustrates how high affinity protein-protein interactions can be very discriminating even though binding is dominated by a conserved hotspot, with single or multiple specificity sites modulating the overall binding free energy. We discuss these results in the context of other conserved protein complexes and suggest that they point to a generic specificity mechanism in divergently evolved protein-protein interactions.     

The cytotoxic domain of colicin E9 is a channel-forming endonuclease

Bacterial toxins commonly translocate cytotoxic enzymes into cells using channel-forming subunits or domains as conduits. Here we demonstrate that the small cytotoxic endonuclease domain from the bacterial toxin colicin E9 (E9 DNase) shows nonvoltage-gated, channel-forming activity in planar lipid bilayers that is linked to toxin translocation into cells. A disulfide bond engineered into the DNase abolished channel activity and colicin toxicity but left endonuclease activity unaffected; NMR experiments suggest decreased conformational flexibility as the likely reason for these alterations. Concomitant with the reduction of the disulfide bond is the restoration of conformational flexibility, DNase channel activity and colicin toxicity. Our data suggest that endonuclease domains of colicins may mediate their own translocation across the bacterial inner membrane through an intrinsic channel activity that is dependent on structural plasticity in the protein.     

DNA-DNA hybridization evidence of the rapid rate of muroid rodent DNA evolution

Single-copy nuclear DNAs (scnDNAs) of eight species of arvicoline and six species of murine rodents were compared using DNA-DNA hybridization. The branching pattern derived from the DNA comparisons is congruent with the fossil evidence and supported by comparative biochemical, chromosomal, and morphological studies. The recently improved fossil record for these lineages provides seven approximate divergence dates, which were used to calibrate the DNA-hybridization data. The average rate of scnDNA divergence was estimated as 2.5%/Myr. This is approximately 10 times the rate in the hominoid primates. These results agree with previous reports of accelerated DNA evolution in muroid rodents and extend the DNA-DNA hybridization data set of Brownell.      

Core alpha1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts

Carbohydrates have been suggested to account for some IgE cross- reactions between various plant, insect, and mollusk extracts, while some IgG antibodies have been successfully raised against plant glycoproteins. A rat monoclonal antibody raised against elderberry abscission tissue (YZ1/2.23) and rabbit polyclonal antiserum against horseradish peroxidase were screened for reactivity in enzyme-linked immunosorbent assay against a range of plant glycoproteins and extracts as well as neoglycoproteins, bee venom phospholipase, and several animal glycoproteins. Of the oligosaccharides tested, Man3XylFucGlcNAc2(MMXF3) derived from horseradish peroxidase was the most potent inhibitor of the reactivity of both YZ1/2.23 and anti- horseradish peroxidase to native horseradish peroxidase glycoprotein. The reactivity of YZ1/2. 23 and anti-horseradish peroxidase against Sophora japonica lectin was most inhibited by a neoglycoconjugate of bromelain glycopeptide cross-linked to bovine serum albumin, while the defucosylated form of this conjugate was inactive as an inhibitor. A wide range of plant extracts was found to react against YZ1/2.23 and anti-horseradish peroxidase, with particularly high reactivities recorded for grass pollen and nut extracts. All these reactivities were inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate. Bee venom phospholipase and whole bee venom reacted weakly with YZ1/2.23 but more strongly with anti-horseradish peroxidase in a manner inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate, while hemocyanin from Helix pomatia reacted poorly with YZ1/2.23 but did react with anti-horseradish peroxidase. It is concluded that the alpha1, 3-fucose residue linked to the chitobiose core of plant glycoproteins is the most important residue in the epitope recognized by the two antibodies studied, but that the polyclonal anti-horseradish peroxidase antiserum also contains antibody populations that recognize the xylose linked to the core mannose of many plant and gastropod N-linked oligosaccharides.      

Structural comparison of fibroblast growth factor-specific heparan sulfates derived from a growing or differentiating neuroepithelial cell line

Heparan sulfate (HS) glycosaminoglycans are essential modulators of fibroblast growth factor (FGF) activity both in vivo and in vitro, and appear to act by cross-linking particular forms of FGF to appropriate FGF receptors. We have recently isolated and characterized two separate HS pools derived from immortalized embryonic day 10 mouse neuroepithelial 2.3D cells: one from cells in log growth phase, which greatly potentiates the activity of FGF-2, and the other from cells undergoing contact-inhibition and differentiation, which preferentially activates FGF-1. These two pools of HS have very similar functional activities to those species isolated from primary neuroepithelial cells at corresponding stages of active proliferation or differentiation. We present here a structural comparison between these cell line HS species to establish the nature of the changes that occur in the biosynthesis of HS. A combination of chemical and enzymatic cleavage, low pressure chromatography and strong anion-exchange HPLC were used to generate full chain models of each species. Overall, the HS pools synthesized in the dividing cell line pools possessed less complex sulfation than those derived from more differentiated, growth arrested cells.      

An RNA folding method capable of identifying pseudoknots and base triples

MOTIVATION: Recently, we described a Maximum Weighted Matching (MWM) method for RNA structure prediction. The MWM method is capable of detecting pseudoknots and other tertiary base-pairing interactions in a computationally efficient manner (Cary and Stormo, Proceedings of the Third International Conference on Intelligent Systems for Molecular Biology, pp. 75-80, 1995). Here we report on the results of our efforts to improve the MWM method's predictive accuracy, and show how the method can be extended to detect base interactions formerly inaccessible to automated RNA modeling techniques. RESULTS: Improved performance in MWM structure prediction was achieved in two ways. First, new ways of calculating base pair likelihoods have been developed. These allow experimental data and combined statistical and thermodynamic information to be used by the program. Second, accuracy was improved by developing techniques for filtering out spurious base pairs predicted by the MWM program. We also demonstrate here a means by which the MWM folding method may be used to detect the presence of base triples in RNAs. AVAILABILITY: http://www.cshl.org/mzhanglab/tabaska/j axpage. html CONTACT: tabaska@cshl.org      

The evolutionary history of Drosophila buzzatii. XXX. Mitochondrial DNA polymorphism in original and colonizing populations

Both original and colonizer populations of Drosophila buzzatii have been analyzed for mtDNA restriction polymorphisms. Most of the mtDNA nucleotide variation in original populations of NW Argentina can be explained by intrapopulation diversity and only a small fraction can be accounted for by between-population diversity. Similar results are obtained using either the estimated number of nucleotide substitutions per site or considering each restriction site as a locus. Colonizer populations of the Iberian Peninsula are monomorphic and show only the most common haplotype from the original populations. Under the infinite island model and assuming that populations are in equilibrium, fixation indices indicate enough gene flow to explain why the populations are not structured. Yet, the possibility exists that populations have not reached an equilibrium after a founder event at the end of the last Pleistocene glaciation. Tajima's test suggests that directional selection and/or a recent bottleneck could explain the present mtDNA differentiation. Considering the significant population structure found for the chromosomal and some allozyme polymorphisms, the among- population uniformity for mtDNA variability argues in favor of the chromosomal and some allozyme polymorphisms being adaptive.