Site-directed and deletion mutational analysis of the receptor binding domain of the interleukin-6 receptor targeted fusion toxin DAB389-IL-6

We have used site-directed and in-frame deletion mutationalanalysis in order to explore the structural features of theIL–6 portion of the diphtheria toxin-related interleukin–6(IL–6) fusion toxin DAB₃₈₉-IL–6 that are essentialfor receptorbinding and subsequent inhibition of protein synthesisin target cells. Deletion of the first 14 amino acids of theIL–6 component of the fusion toxin did not alter eitherreceptor binding affinity or cytotoxk potency. In contrast,both receptor binding and cytotoxic activity were abolishedwhen the C–terminal 30 amino acids of the fusion toxinwere deleted. In addition, we explored the relative role ofthe disulfide bridges within the IL–6 portion of DAB₃₈₉-IL–6in the stabilization of structure required for receptor-binding.The analysis of mutants in which the substitution of eitherCys⁴⁴⁰, Cys⁴⁴⁶, Cys⁴⁶⁹ or Cys⁴⁷⁹ to Ser respectively, demonstratesthat only the disulfide bridge between Cys⁴⁶⁹ and Cys⁴⁷⁹ isrequired to maintain a functional receptor binding domain. Inaddition, the internal in-frame deletion of residues 435–451,which includes Cys⁴⁴⁰ and Cys⁴⁴⁶, was found to reduce, but notabolish receptor binding affinity. These results further demonstratethat the disulfide bridge between Cys⁴⁴⁰ and Cys⁴⁴⁶ is not essentialfor receptor-binding. However, the reduced cytotoxic potencyof DAB₃₈₉-IL6(435–451) suggests that the conformationand/or receptor binding sites associated with this region ofthe fusion toxin is/are important for maintaining the wild typereceptor binding affinity and cytotoxic potency.     

Diphtheria toxin receptor-binding domain substitution with interleukin 6: genetic construction and interleukin 6 receptor-specific action of a diphtheria toxin-related interleukin 6 fusion protein

We have genetically replaced that portion of the diphtheriatoxin structural gene which encodes the native receptor-bindingdomain with a synthetic gene encoding the cytokine interleukin6 (IL-6/IFN-ß2/BSF-2). The resulting gene fusion encodesthe chimeric toxin DAB₃₈₉-IL-6. Following expression and purification,we demonstrate that DAB₃₈₉-IL-6 is selectively cytotoxic foreukaryotic cells bearing the interleukin 6 receptor. In addition,the cytotoxic action of DAB₃₈₉-IL-6 is shown to require bindingto the IL-6 receptor, internalization by receptor-mediated endocytosisand passage through an acidic compartment. Following the deliveryof the catalytically active fragment A to the cytosol of targetcells, cellular protein synthesis is inhibited by the ADP-ribosylationof elongation factor 2. While eukaryotic cells which are devoidof the IL-6 receptor are uniformly resistant to the action ofthis fusion toxin, the data presented suggest that a minimalnumber of IL-6 receptors may be necessary to mediate the internalizationof sufficient levels of DAB₃₈₉-IL-6 to result in the intoxicationof target cells.     

Protein engineering of diphtheria-toxin-related interleukin-2 fusion toxins to increase cytotoxic potency for high-affinity IL-2-receptor-bearing target cells

We have used site-directed insertion and point mutagenesis inan attempt to increase the cytotoxic potency and receptor-bindingaffinity of the diphtheria-toxin-related interleukin-2 (IL-2)fusion toxins. Previous studies have demonstrated that boththe DAB₄₈₆-IL-Z and DAB₃₈₉-IL-2 forms of the fusion toxin consistof three functional domains: the N-tenninal fragment-A-assodatedADP-ribosyltransferase, the hydrophobk-membrane-associatingdomains, and the C-terminal receptor-binding domain of humanIL-2. By insertion mutagenesis we have increased the apparentflexibility of the polypeptide chain between the membraneassociatingdomains and the receptor-binding domain of this fusion toxin.In comparison to DAB₄₈₆-IL-2, the cytotoxic potency of the insertionmutants was increased by 17-fold for high-affinity IL-2-receptor-bearingcell lines in vitro. Moreover, competitive displacement experimentsusing [¹²⁵I]rIL-2 demonstrate that the increase in cytotoxicpotency correlates with an increase in receptor-binding affinityfor both the high and intermediate forms of the IL-2 receptor.     

Maintenance of the hydrophobic face of the diphtheria toxin amphipathic transmembrane helix 1 is essential for the efficient delivery of the catalytic domain to the cytosol of target cells

Abstract The transmembrane (T) domain of diphtheria toxin (DT) comprisesnine -helices and has been shown to play an essential role inthe efficent delivery of the catalytic (C) domain ofDT acrossthe eukaryotic cell membrane and into the cytosol. We have demonstratedrecently thatthe first three amphipathic helixes of the T domain,although not necessary for either channel formation or receptorbinding, are required for the efficient transmembrane deliveryof the Cdomain.In the present study,we have performed a detailedstructure-function analysis of T domainhelix 1 (TH1) of theDT-related fusion protein DAB₃₈₉lL-2. We performed exchangeandsite-directed mutagenesis of TH1 and the resulting mutantfusion toxins were analyzed by gel electrophoresis and testedfor their efficiencies in the delivery of the C domain to thecell cytosol. We demonstrate that the overall charge distributionand hydrophobicity of amino acids in the amphipathic helix TH1,rather than a specific amino acid sequence, are critical forthe function of this helix. The insertion of a charged residuein the hydrophobic face of TH1 abolishes cytotoxic activity,whereas replacement of a hydrophobic residue by a charged aminoacid in the hydrophilic face of the helix has little, if any,effect on cytotoxic activity. In addition,we have identifiedSer220 by site-directed mutagenesis as a residue that appearsto be criticalfor correct folding of the fusion toxin. Mutationsin this position result in fusion proteins that are extremelysensitive to proteolytic attack.     

Molecular modelling of UH-301 and 5-HTla receptor interactions

A three-dimensional model of the human 5-HT_1a receptor was constructedby molecular modelling, and the molecular and electronic structuresof (R)- and (S)-5-fluoro-8-hydroxy-2-(dipropylamino)tetralin(UH-301) and of (R)- and (S)-8-hydroxy-2-(dipropylamino)tetralin(8-OH-DPAT) were examined by molecular mechanics and quantummechanics calculations and molecular dynamics simulations. Thereceptor model has seven transmembrane helices (TMHs), organizedaccording to a projection map of visual rhodopsin, and includesall loops between helices and the N- and C-terminal parts. Interactionsof UH-301 and 8-OH-DPAT with the 5-HT_1a receptor were examinedby molecular dynamics simulations and energy minimization ofreceptor–ligand complexes. 8-OH-DPAT had lower electrostaticpotentials around the hydroxyl group and stronger hydrogen bondingto the receptor model than had UH-301. The simulations indicatedthat the 5-HT_1a receptor agonists, (R)- and (S)-8-OH-DPAT and(R)-UH-301, interacted with the receptor at a site closer toAsp82 in TMH2 than did (S)-UH-301, which is a 5-HT,_1a receptorantagonist. Simulations of receptor–ligand complexes indicatedthat Asp82, Asp116, Ser199, Thr200 and De385 are essential forbinding of both agonist and antagonist to the receptor.     

Direct expression in E.coli of a functionally active protein A-snake toxin fusion protein

We constructed a recombinant expression plasmid encoding a proteinA–neurotoxin fusion protein. The fused toxin is directlyexpressed in the periplasmic space of Escherichia coli and canbe purified in the milligram range by a single immuno-affinitystep. The LD₅₀ values of the fused toxin and native toxin are130 and 20 nmol/kg mouse respectively. The K_d values characterizingtheir binding to the nicotinic acetylcholine receptor (AcChoR)are respectively 4.8 ± 0.8 and 0.07 ± 0.03 nM.In contrast, the fused and native toxins are equally well recognizedby a toxin-specific monoclonal antibody which recognizes theAcChoR binding site. The lower toxicity of the fused toxin mightresult, therefore, from a steric hindrance, due to the presenceof the bulky protein A moiety (mol. wt = 31 kd) rather thanto a direct alteration of the ‘toxic’ site. Thefused toxin is more immunogenic than native toxin, since 1 nmolof hybrid toxin and 14 nmol of native toxin give rise to comparabletiters of antitoxin antibodies which, furthermore, are equallypotent at neutralizing neurotoxicity. The work described inthis paper shows that the use of fused toxins may be of paramountimportance for future development of serotherapy against envenomationby snake bites.     

Bipartite organization of the Bacillus subtilis endo-{beta}-1,4-glucanase revealed by C-terminal mutations

The C-terminal boundary of primary sequence of the Bacillussubtilis PAP115 endo-ß-1,4-glucanase (EG) requiredfor stable catalytic activity has been mapped by site-directedmutagenesis using Escherichia coli as host. The 52 kDa cel geneproduct, EG₄₇₀ and a 33 kDa mutant (EG₃₀₀), lacking 170 residuesthrough a nonsense mutation at the leucine-330 codon of thegene, exhibited similar patterns of enzymatic activity and pHoptima using cellooligopentaose as substrate.CD spectra indicatedthat the bulk of the -helical secondary structure in EG₄₇₀ wascontained within EG₃₀₀. However, relative to EG₄₇₀, the specificactivity of EG₃₀₀ was 3- to 4-fold lower with amorphous celluloseas substrate and {small tilde}4-to5-fold higher with carboxymethylcellulose(soluble cellulose).These results along with data which showthat EG₄₇₀ binding capacity to mirocrystalline cellulose is{small tilde} 11 times more than that of EG₃₀₀, demonstratethe importance of residues 330–499 for non-catalytic bindingof cellulose. A construct of the cel gene carrying a deletionof codons 330–499 and an insertion of a nonsense codonat leucine-330, was further used to make mutants EG₂₉₆ and EG₂₉₁with nonsense codon substitutions at arginine and serine-321,respectively.Western analysis using EG-specific antiserum revealedthat relative losses in enzymatic activity of EG₂₉₆ (50%) andEG₂₉₁ (95%) could be accounted for by the extent of their proteolysis,signifying a marked destabilization of these enzymes by removalof only a few amino acids.     

The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment

The facile detection and purification of a recombinant proteinwithout detailed knowledge about its individual biochemicalproperties constitutes a problem of general interest in proteinengineering. The use of a novel kind of random peptide libraryfor the stepwise engineering of a C-terminal fusion peptidewhich confers binding activity towards streptavidin is describedin this study. Because of its widespread use as part of a varietyof conjugates and other affinity reagents, streptavidin constitutesthe binding partner of choice both for detection and purificationpurposes. The streptavidin-affinity tag was engineered at theC-terminus of the V_H domain as part of the D1.3 Fv fragmentwhich was functionally expressed in Escherichia coli. Irrespectiveof whether it was displayed by the V_H or the V_L domain, theoptimized version of the affinity peptide termed ‘Strep-tag’allowed the detection of the Fv fragment both on Western blotsand in ELISAs by a streptavidin–alkaline phosphatase conjugate.In addition, the one-step purification of the intact Fv fragmentcarrying a single Strep-tag at the C-terminus of only one ofits domains was achieved by affinity chromatography with streptavidin-agaroseusing very mild elution conditions.     

Site-directed mutagenesis of Arg60 and Cys271 in ornithine transcarbamylase from rat liver

We have investigated the putative carbamylphosphate- and ornithine-bindingdomains in ornithine transcarbamylase from rat liver using site-directedmutagenesis. Arg60, present in the phosphate-binding motif X-Ser-X-Arg-Xand therefore implicated in the binding of the phosphate moietyof carbamylphosphate has been replaced with a leucine. Thisresults in a dramatic reduction of catalytic activity, althoughthe enzyme is synthesized in cells stably transfected with themutant clone and imported, correctly processed and assembledinto a homotrimer in mitochondria. The sole cysteine residue(Cys271) has been implicated in ornithine binding by the chemicalmodification studies of Marshall and Cohen in 1972 and 1980(J. Biol. Chem., 247, 1654–1668, 1669–1682; 255,7291–7295, 7296–7300). Replacement of this residuewith serine did not eliminate enzyme activity but affected theMichaelis constant for ornithine (K_b, increasing it 5-fold from0.71 to 3.7 mM and reduced the k_cat at pH 8.5 by 20-fold. Thesechanges represent a loss in apparent binding energy for theenzyme - ornithine complex of 2.9 kcal/mol, suggesting thatCys271 is normally involved in hydrogen bonding to the substrate,ornithine. The cysteine to serine substitution also caused thedissociation constant (Kä for the competitive inhibitor,L-norvaline to be increased 10-fold, from 12 to 120 µM.The small loss in binding energy and relatively high residualcatalytic activity of the mutant strongly suggests that a numberof other residues are involved in the binding of ornithine.The effect of replacement of Cys271 with serine was restrictedto the ornithine binding site of the enzyme since both the bindingconstant for carbamyl-phosphate (K_ia) and Michaelis constant(K_a) were not appreciably different for mutant and wild-typeenzymes. The pH optimum of the wild-type enzyme (8.6) is increasedto > 9.6 in the Ser271 mutant.     

Strong inhibition of fibrinogen binding to platelet receptor {alpha}IIb{beta}3 by RGD sequences installed into a presentation scaffold

In order to probe the structural constraints on binding of RGDsequences to the platelet receptor _IIbß₃ we have usedrecombinant DNA techniques to install the RGD sequence into‘presentation scaffolds’, small proteins of known3-D structure chosen to present guest sequences in constrainedorientations. Using Escherichia coli expression systems we madesequence variants in which loop residues of the immunoglobulinV_L domain REI and of human interleukin-1ß were replaced(without changing polypeptide length) by the RGD sequence atpositions predicted, based on small molecule studies, to orientthe RGD moiety into an active conformation. These variants donot compete for fibrinogen binding to _IIbß₃ up toalmost 1 mM concentration. Unfolded or proteolytically fragmentedforms of these same proteins do compete, however, showing thatthe RGD sequences in the mutants must be prohibited from bindingby constraints imposed by scaffold structure. To suppress theeffects of such structural constraints we constructed two sequencevariants in which RGD-containing sequences 42–57 or 44–55from the snake venom platelet antagonist kistrin were inserted(this increasing the length of the loop) into the third complementaritydetermining loop of REI. Both of these variants compete stronglyfor fibrinogen binding with IC₅₀s in the nM range. These results,plus data on kistrin-related peptides also presented here, suggestthat the molecular scaffold REI is capable of providing to aninstalled sequence a structural context and conformation beneficialto binding. The results also suggest that in order to bind wellto _IIbß₃, RGD sequences in protein ligands must eitherproject significantly from the surface of the scaffold and/orretain a degree of conformational flexibility within the scaffold.Molecular scaffolds like REI should prove useful in the elucidationof structure-function relationships and the discovery of newactive sequences, and may also serve as the basis for noveltherapeutic agents.