Competing modes of self-association in the regulatory domains of Bruton's tyrosine kinase: Intramolecular contact versus asymmetric homodimerization

A nuclear magnetic resonance (NMR) investigation of a fragment of the nonreceptor Tec family tyrosine kinase Btk has revealed an intricate set of coupled monomer-dimer equilibria. The Btk fragment studied contains two consecutive proline-rich motifs followed by a single Src homology 3 (SH3) domain. We provide evidence for an asymmetric homodimer in which the amino-terminal proline sequence of one monomer contacts the opposite SH3 binding pocket, whereas the carboxy-terminal proline sequence of the other monomer is engaged by the second SH3 domain across the dimer interface. We show that the asymmetric homodimer structure is mimicked by a heterodimer formed in an equimolar mixture of complimentary mutants: one carrying mutations in the amino-terminal proline stretch; the other, in the carboxy-terminal proline motif. Moreover, a monomeric species characterized by an intramolecular complex between the amino-terminal proline motif and the SH3 domain predominates at low concentration. Association constants were determined for each of the competing equilibria by NMR titration. The similarity of the determined K(a) values reveals a delicate balance between the alternative conformational states available to Btk. Thus, changes in the local concentration of Btk itself, or co-localization with exogenous signaling molecules that have high affinity for either proline sequence or the SH3 domain, can significantly alter species composition and regulate Btk kinase activity.     

Antiviral determinants of rat Mx GTPases map to the carboxy-terminal half.

Rat Mx2 and rat Mx3 are two alpha/beta interferon-inducible cytoplasmic GTPases that differ in three residues in the amino-terminal third, which also contains the tripartite GTP-binding domain, and that differ in five residues in the carboxy-terminal quarter, which also contains a dimerization domain. While Mx2 is active against vesicular stomatitis virus (VSV), Mx3 lacks antiviral activity. We mapped the functional difference between Mx2 and Mx3 protein to two critical residues in the carboxy-terminal parts of the molecules. An exchange of either residue 588 or 630 of Mx2 with the corresponding residues of Mx3 abolished anti-VSV activity, and the introduction of the two Mx2 residues on an Mx3 background partially restored anti-VSV activity. These results are consistent with the facts that Mx2 and Mx3 have similar intrinsic GTPase activities and that the GTPase domain of Mx3 can fully substitute for the GTPase domain of Mx2. Nevertheless, the amino-terminal third containing the GTP-binding domain is necessary for antiviral activity, since an amino-terminally truncated Mx2 protein is devoid of anti-VSV activity. Furthermore, Fab fragments of a monoclonal antibody known to neutralize antiviral activity block GTPase activity by binding an epitope in the carboxy-terminal half of Mx2 or Mx3 protein. The results are consistent with a two-domain model in which both the conserved amino-terminal half and the less-well-conserved carboxy-terminal half of Mx proteins carry functionally important domains.     

Mapping the functional domains of the eukaryotic elongation factor 1 beta gamma

The functional domains of the eukaryotic elongation factor (EF) 1 beta gamma have been delineated with the use of limited proteolysis, protein microsequencing, gel electrophoresis under non-denaturing conditions and antibodies against EF-1 beta and EF-1 gamma. By means of limited proteolysis, it was possible to obtain large fragments of EF-1 beta. In contrast to amino-terminal fragments, those derived from the carboxy-terminal part of EF-1 beta were still active in enhancing the guanine nucleotide exchange of GDP bound to EF-1 alpha. With the same technique of limited proteolysis, it was possible to isolate a trypsin-resistant core from EF-1 beta gamma containing polypeptide chain fragments derived from both subunits. A polyvalent antiserum against EF-1 beta and two monoclonal antibodies against EF-1 gamma were used to identify the protein fragments in this core. The monoclonal antibodies were shown to recognize different epitopes, one localized on the amino-terminal and another on the carboxy-terminal half of EF-1 gamma. The antiserum against EF-1 beta and one of the monoclonal antibodies (mAb 36E5), which recognized the amino-terminal half of EF-1 gamma, reacted with this trypsin-resistant core. We conclude that the amino-terminal halves of both EF-1 beta and EF-1 gamma are firmly attached to each other, and that the carboxy-terminal part of EF-1 beta interacts with EF-1 alpha.     

Nuclear pore complex glycoprotein p62 of Xenopus laevis and mouse: cDNA cloning and identification of its glycosylated region

cDNA clones for nuclear pore complex glycoprotein p62 of two distantly related species, mouse and Xenopus laevis, were isolated. Antibodies raised against recombinant murine p62 react on protein blots with p62 of both species and decorate pore complexes. Analysis of the predicted protein sequence indicates that vertebrate p62 is organized into two structurally different regions. The entire carboxy-terminal half (86.7% identical amino acids) and the amino-terminal 56 amino acids (62.5% identity) have been highly conserved during evolution. The amino-terminal half contains several penta amino acid repeats and is able to form beta-sheets, whereas the carboxy-terminal half is predominantly organized in alpha-helical structures in part with heptad repeats typical for intermediate filament proteins. p62 of mouse and Xenopus is glycosylated by N-acetylglucosamine additions in the amino-terminal half. The region containing these potential glycosylation sites has been identified.     

A new monoclonal antibody recognizing the amino-terminal consensus sequence of vertebrate intermediate filament proteins

The mouse monoclonal antibody ME 101 raised against human peripherin, an intermediate filament protein (IFP) specific to well defined neuronal populations, recognizes all the major classes of vertebrate IFP in immunoblotting assays. Desmin, GFAP, vimentin, peripherin and the lightest neurofilament protein (NF-L) were cleaved into carboxy- and amino-terminal halves by N-chlorosuccinimide at their unique trytophan residue. Whereas the antibody directed against the epitope common to every IFP (intermediate filament antigen or IFA) and located on the carboxy-terminal end of the rod domain recognizes the carboxy-terminal half, the ME 101 antibody, as the present study illustrates, recognizes specifically the amino-terminal half. From the amino acid sequence data of IFP, it is deduced that the cognate epitope is localized on the amino-terminal part of coil la.     

The mosquito larvicidal activity of 130 kDa delta-endotoxin of Bacillus thuringiensis var. israelensis resides in the 72 kDa amino-terminal fragment

Bacillus thuringiensis var. israelensis produces 130 kDa delta-endotoxin which is highly toxic to mosquito-larvae. The mosquito-larvicidal activity was delineated by sequential deletions from ends of the 1136 amino acids delta-endotoxin. A maximum of 459 amino acids could be removed from the carboxy-terminal of the toxin without a significant loss of the larvicidal activity. However, no more than 38 amino acids could be deleted from the amino-terminal without losing the toxicity. The truncated peptide of 72 kDa exhibited similar toxicity to the 130 kDa toxin and was between 39th and 677th amino acids.     

Characterization of structural and immunological properties of specific domains of Friend ecotropic and dual-tropic murine leukemia virus gp70s.

A detailed comparison of the gp70 proteins of cloned ecotropic Friend murine leukemia virus (FLV) and dual-tropic Friend mink focus-forming virus (FrMCF) was performed by analyzing the structural and immunological properties of amino- and carboxy-terminal domains of these molecules generated upon controlled trypsinization. The two gp70s gave characteristic fragmentation patterns; the amino-terminal fragments of FrMCF gp70 were smaller than the corresponding fragments of FLV and contained a trypsin site which resulted in a 19,000-dalton amino-terminal fragment not observed for FLV, whereas both molecules yielded an identically sized carboxy-terminal fragment. All amino-terminal fragments of both gp70 molecules contained an endo H-sensitive oligosaccharide chain; for FrMCF, a second endo H-sensitive carbohydrate was present as well at a carboxy-terminal site for approximately 50% of the molecules. Several aspects of the disulfide interactions of the two gp70s were conserved; in both cases the carboxy-terminal fragments were disulfide bonded to p15(E), there were no disulfide bonds between amino- and carboxy-terminal fragments, and the amino-terminal fragments exhibited a significant increase in mobility upon analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Analysis of the immunoreactivity of the different domains of the proteins by immunoprecipitation of the fragments with antisera prepared against xenotropic murine leukemia virus and feline leukemia virus gp70s indicated major differences in antigenicity for the amino-terminal domains of FLV and FrMCF gp70, whereas the carboxy-terminal domains were immunologically conserved. Similar analyses with antibodies specific for p15(E) and Pr15(E) demonstrate that these components are conserved as well. These data provide direct evidence that p15(E) and the C-terminal gp70 domain of FrMCF gp70 are related to the corresponding regions of the ecotropic FLV parent and indicate that the acquisition of MCF-specific properties is due to the replacement of the ecotropic amino-terminal gp70 domain with sequences related to those of xenotropic gp70s.     

Human nucleoporin p62 and the essential yeast nuclear pore protein NSP1 show sequence homology and a similar domain organization

NSP1 is an essential nuclear pore protein in yeast. We observed that anti-NSP1 antibodies label mammalian nuclear pore complexes and recognize nucleoporin p62. Also peptide antibodies raised against the NSP1 carboxy-terminal end cross-react with p62, a conserved component of the nuclear pore complex in higher eukaryotes. To further analyze the structural and functional similarity between NSP1 and mammalian nucleoporins, we cloned and sequenced nucleoporin p62 from a HeLa cDNA library. Human p62 consists of a carboxy-terminal domain homologous to the essential yeast NSP1 carboxy-terminal domain and an amino-terminal half resembling the repetitive middle domain of NSP1. The full-length p62 and a fusion protein consisting of cytosolic mouse dihydrofolate reductase (DHFR) and the p62 carboxy-terminal domain were expressed in transfected HeLa cells. Only overexpressed full-length p62, but not the DHFR-C-p62 fusion protein, binds wheat germ agglutinin (WGA). This suggests that modification by N-acetylglucosamine is mainly restricted to the repetitive amino-terminal half of p62 and implies a role of this type of repetitive sequences in nuclear transport. In the transfected HeLa cells, the DHFR-C-p62 fusion protein forms patchy aggregates that accumulate at the nuclear periphery but are also scattered through the cytoplasm. It is suggested that nucleoporin p62 may be targeted and anchored to the pore complex via its carboxy-terminal domain which reveals a hydrophobic heptad repeat organization similar to that found in lamins and other intermediate filament proteins.     

Bacillus thuringiensis proteases: Production and role in growth,sporulation and synergism

Bacillus thuringiensis (Bt) subspecies produces metalloproteases and serine alkaline proteases (endogenous) which affect sporulation and entomotoxicity against different insect orders. The production of Bt proteases is investigated in conventional medium and alternative substrates with future repercussions on Bt formulations and larval mortality. Relationship between protease activity and total cell count during Bt fermentation has been discussed while protease activity as a potential indicator of entomotoxicity has also been explored. In general, the proteases influence entomotoxicity in two divergent ways—processing of inactive protoxins to active toxin fractions (by endogenous Bt as well as exogenous larval midgut proteases) and degradation of protoxins to fragments which sometimes lack insecticidal activity (usually by Bt proteases). In fact, the function of endogenous (intra and extracellular) proteases is ambiguous and has been raising serious questions on their role in larval mortality. The review explores various schools of thoughts (traditional as well as advanced) to solve the enigma of protease interactions with crystal toxins at different levels (sporulation and insecticidal action).     

Structural requirements of the unique disulphide bond and the proline-rich motif within the alpha4-alpha5 loop for larvicidal activity of the Bacillus thuringiensis Cry4Aa delta-endotoxin

Both the disulphide bond (Cys192-Cys199) and the proline-rich motif (Pro193ProAsnPro196) in the long loop connecting the alpha4-alpha5 transmembrane hairpin of the Cry4Aa mosquito-larvicidal protein have been found to be unique among the Bacillus thuringiensis Cry delta-endotoxins. In this study, their structural requirements for larvicidal activity of the Cry4Aa toxin were investigated. C192A and C199A mutant toxins were initially generated and over-expressed in Escherichia coli cells as 130-kDa protoxins at levels comparable to that of the wild-type toxin. When their activities against Aedes aegypti larvae were determined, Escherichia coli cells expressing each mutant toxin retained the high-level toxicity. Further mutagenic analysis of the PPNP motif revealed that an almost complete loss in larvicidal activity was observed for the C199A/P193A double mutant, whereas a small reduction in toxicity was shown for the C199A/P194A and C199A/P196A mutants. Increasing the flexibility of the alpha4-alpha5 loop through C199A/P193G, C199A/P194G/P196A, C199A/P194A/P196G, and C199A/P194G/P196G mutations significantly decreased the larvicidal activity. Similar to the wild-type protoxin, all mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. These findings are the first biological evidence for a structural function in larvicidal activity of the unique disulphide bridge as well as the proline-rich motif within the alpha4-alpha5 loop of the Cry4Aa toxin.     

Ribosomal S6 kinase 1 (RSK1) activation requires signals dependent on and independent of the MAP kinase ERK.

BACKGROUND: The rsk1 gene encodes the 90 kDa ribosomal S6 kinase 1 (RSK1) protein, which contains two kinase domains. RSK1, which is involved in regulating cell survival and proliferation, lies at the end of the signaling cascade mediated by the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein (MAP) kinases. ERK activation and subsequent phosphorylation of the RSK1 carboxy-terminal catalytic loop stimulates phosphotransferase activity in the RSK1 amino-terminal kinase domain. When activated, RSK1 phosphorylates both nuclear and cytoplasmic substrates through this amino-terminal catalytic domain. It is thought that stimulation of the ERK/MAP kinase pathway is sufficient for RSK1 activation, but how ERK phosphorylation activates the RSK1 amino-terminal kinase domain is not known. RESULTS: The individual isolated RSK1 kinase domains were found to be under regulatory control. In vitro kinase assays established that ERK phosphorylates RSK1 within the carboxy-terminal kinase domain, and the phosphoinositide-dependent kinase 1 (PDK1) phosphorylates RSK1 within the amino-terminal kinase domain. In transiently transfected HEK 293E cells, PDK1 alone stimulated phosphotransferase activity of an isolated RSK1 amino-terminal kinase domain. Nevertheless, activation of full-length RSK1 in the absence of serum required activation by both PDK1 and ERK. CONCLUSIONS: RSK1 is phosphorylated by PDK1 in the amino-terminal kinase-activation loop, and by ERK in the carboxy-terminal kinase-activation loop. Activation of phosphotransferase activity of full-length RSK1 in vivo requires both PDK1 and ERK. RSK1 activation is therefore regulated by both the mitogen-stimulated ERK/MAP kinase pathway and a PDK1-dependent pathway.     

Structurally conserved aromaticity of Tyr249 and Phe264 in helix 7 is important for toxicity of the Bacillus thuringiensis Cry4Ba toxin

Functional elements of the conserved helix 7 in the poreforming domain of the Bacillus thuringiensis Cry delta- endotoxins have not yet been clearly identified. Here, we initially performed alanine substitutions of four highly conserved aromatic residues, Trp(243), Phe(246), Tyr(249) and Phe(264), in helix 7 of the Cry4Ba mosquito-larvicidal protein. All mutant toxins were overexpressed in Escherichia coli as 130-kDa protoxins at levels comparable to the wild-type. Bioassays against Stegomyia aegypti mosquito larvae revealed that only W243A, Y249A or F264A mutant toxins displayed a dramatic decrease in toxicity. Further mutagenic analysis showed that replacements with an aromatic residue particularly at Tyr(249) and Phe(264) still retained the high-level toxin activity. In addition, a nearly complete loss in larvicidal activity was found for Y249L/F264L or F264A/ Y249A double mutants, confirming the involvement in toxicity of both aromatic residues which face towards the same direction. Furthermore, the Y249L/F264L mutant was found to be structurally stable upon toxin solubilisation and trypsin digestion, albeit a small change in the circular dichroism spectrum. Altogether, the present study provides for the first time an insight into the highly conserved aromaticity of Tyr(249) and Phe(264) within helix 7 playing an important role in larvicidal activity of the Cry4Ba toxin.     

Molecular markers to determine the ecological fate of Bacillus thuringiensis ssp. kurstaki

A set of DNA markers was developed that successfully identifies Bacillus thuringiensis ssp. kurstaki (Btk) when screened against other Bacillus species and subspecies. These subspecies-specific primer sets allowed detection and characterization of Btk within an environmental background that contained many Bacillus species. Because Btk is used as an active ingredient in many commercial formulations, yet is not naturally widely distributed in North America or Europe, these markers will prove useful in investigations on the environmental persistence and ecological fate of Btk.     

Identification and characterization of proteins from Bacillus thuringiensis with high toxic activity against the sheep blowfly, Lucilia cuprina

Current control of the sheep blowfly (Lucilia cuprina) relies on chemical insecticides, however, with the development of resistance and increasing concerns about human health and environmental residues, alternative strategies to control this economically important pest are required. In this study, we have identified several isolates of Bacillus thuringiensis (Bt), collected from various Australian soil samples, that produce crystals containing 130 and 28 kDa proteins. These isolates were highly toxic to feeding larvae in both in vitro bioassays and in vivo on sheep. By N-terminal amino acid sequencing, we identified the smaller crystal band (28 kDa) as a cytological (Cyt) protein. Upon solubilization and proteolytic processing by trypsin, the 130 kDa crystal protein yielded among others, a truncated 55-60 kDa toxin moiety which exhibited larvicidal activity against sheep blowfly. The amino-terminal sequence of the trypsin-resistant protein band revealed that this Bt endotoxin was encoded by a new cry gene. The novel cry protein was present in all the strains that were highly toxic in the larval assay. We have also identified from one of the isolates, a novel secretory toxin with larvicidal activity.     

Separation of the two domains of human mucus proteinase inhibitor: inhibitory activity is only located in the carboxy-terminal domain

Human mucus proteinase inhibitor is a two-domain protein which inactivates bovine trypsin and chymotrypsin, leukocyte elastase and cathepsin G. In order to localize the site(s) responsible for these inhibitory activities, the two domains were isolated after specific cleavage of the Asp49-Pro50 bond following mild acid treatment of the bronchial inhibitor. The carboxy-terminal domain was active against leukocyte elastase, trypsin and chymotrypsin whereas the amino-terminal domain, which contained a putative antitryptic active site, was devoid of activity. This implicates that, in the whole molecule, the inhibitory activity region is localized only in the carboxy-terminal domain.     

Persistence and impact on microorganisms of Bacillus thuringiensis proteins in some Zimbabwean soils

The persistence of the Bacillus thuringiensis subsp. kurstaki (Btk) toxin (Cry1Ab protein) from Bt maize (MON810, Yieldgard®) residues incorporated in a vertisol (739 g clay kg^?1) was investigated. The maize residues were incubated in the soil for 4 weeks, and activity of the toxin in the residues was bioassayed using larvae of the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae). Corrected mortality of P. xylostella in the bioassays decreased from 76% to 30% in less than a week of incubation in the soil. In addition to the above observations, the effects of Btk, Bt subsp. israelensis (Bti), and Bt subsp. tenebrionis (Btt) proteins on the soil microbiota were examined using a vertisol, an alfisol, and an oxisol. The pre-incubated soils (7 days after moisture adjustment) were treated with crystal proteins of Btk, Bti, and Btt and incubated for further a 7-day period. Microbial biomass carbon (MBC) and counts of culturable bacteria and fungi were determined. The proteins did not show effects on MBC or bacterial and fungal counts, possibly as a result of adsorption of the proteins on soil particles, which could have rendered the proteins inaccessible for microbial utilization. Microbial biomass carbon and counts arranged in decreasing order were vertisol>oxisol>alfisol, similar to the amounts of organic C and clay in the soils. However, bacteria and fungi counts were higher in the vertisol than in the alfisol and the oxisol soils. Our observations suggest that larvicidal proteins produced by different subspecies of Bt and Bt maize could persist in tropical soils as a result of adsorption on soil clays but that there were no observable effect on the soil microbiota.     

Targeted mutagenesis of loop residues in the receptor-binding domain of the Bacillus thuringiensis Cry4Ba toxin affects larvicidal activity

Loop residues in domain II of Bacillus thuringiensis Cry delta-endotoxins have been demonstrated to be involved in insecticidal specificity. In this study, selected residues in loops beta6-beta7 (S(387)SPS(390)), beta8-beta9 (S(410), N(411), T(413), T(415), E(417) and G(418)) and beta10-beta11 (D(454)YNS(457)) in domain II of the Cry4Ba mosquito-larvicidal protein were changed individually to alanine by PCR-based directed mutagenesis. All mutant toxins were expressed in Escherichia coli JM109 cells as 130-kDa protoxins at levels comparable to the wild type. Only E. coli cells that express the P389A, S410A, E417A, Y455A or N456A mutants exhibited a loss in toxicity against Aedes aegypti mosquito larvae of approximately 30% when compared to the wild type. In addition, E. coli cells expressing double mutants, S410A/E417A or Y455A/N456A, at wild-type levels revealed a significantly higher loss in larvicidal activity of approximately 70%. Similar to the wild-type protoxin, both double mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. These results indicate that S(410) and E(417) in the beta8-beta9 loop, and Y(455) and N(456) in the beta10-beta11 loop are involved in larvicidal activity of the Cry4Ba toxin.     

Toxicity to Spodoptera exigua and Trichoplusia ni of individual P1 protoxins and sporulated cultures of Bacillus thuringiensis subsp. kurstaki HD-1 and NRD-12

The toxicities to neonate Spodoptera exigua and Trichoplusia ni of lyophilized powders obtained from sporulated liquid cultures (referred to as sporulated cultures) and Escherichia coli-expressed P1 [cryIA(a) cryIA(b) cryIA(c)] protoxins from three-gene strains of NRD-12 and HD-1 of Bacillus thuringiensis subsp. kurstaki were determined by using diet incorporation bioassays. Although sporulated cultures from both strains were more toxic to T. ni than S. exigua, there were no differences in toxicity between NRD-12 and HD-1. Toxicities of the three individual P1 protoxins against S. exigua varied by at least fivefold, with the cryIA(b) protein being the most toxic. These same protoxins varied in toxicity against T. ni by at least 16-fold, with the cryIA(c) protein being the most toxic. However, when tested against either S. exigua or T. ni, there were no differences in toxicity between an NRD-12 P1 protoxin and the corresponding HD-1 P1 protoxin. Comparing the toxicities of individual protoxins with that of sporulated cultures demonstrates that no individual protoxin was as toxic to S. exigua as the sporulated cultures. However, this same comparison against T. ni shows that both the cryIA(b) and cryIA(c) proteins are at least as toxic as the sporulated cultures. Results from this study suggest that NRD-12 is not more toxic to S. exigua than HD-1, that different protein types have variable host activity, and that other B. thuringiensis components are not required for T. ni toxicity but that other components such as spores might be required for S. exigua toxicity.     

Asn183 in alpha5 is essential for oligomerisation and toxicity of the Bacillus thuringiensis Cry4Ba toxin

The proposed toxicity mechanism of the Bacillus thuringiensis Cry insecticidal proteins involves membrane penetration and lytic pore formation of the alpha4-alpha5 hairpins in the target larval midgut cell membranes. In this study, alanine substitutions of selected polar residues (Tyr(178), Gln(180), Asn(183), Asn(185), and Asn(195)) in the hydrophobic helix-alpha5 of the Cry4Ba mosquito-larvicidal protein were initially conducted via PCR-based directed mutagenesis. Upon IPTG induction, all the 130-kDa mutant protoxins were highly expressed in Escherichia coli as cytoplasmic inclusions, with yields similar to the wild-type protoxin. When E. coli cells expressing each mutant toxin were tested against Stegomyia aegypti mosquito larvae, the larvicidal activity of the N183A mutant was almost completely abolished whereas the four other mutants showed only a small reduction in toxicity. Additionally, replacements of this critical residue with various amino acids revealed that the uncharged polar residue at position 183 in alpha5 is crucial for larvicidal activity. Further characterisation of the N183K bio-inactive mutant revealed that the 65-kDa activated toxin was unable to form oligomers in lipid vesicles and its ability to induce the release of entrapped calcein from liposomes was much weaker than that of the wild-type toxin. These results suggest that the highly conserved Asn(183) located in the middle of the transmembrane alpha5 of Cry4Ba plays a crucial role in toxicity and toxin oligomerisation in the lipid membranes.