Effect of amino acid deletions in the O-glycosylated region of Aspergillus awamori glucoamylase

Aspergillus awamori glucoamylase (GA) contains globular catalyticand starch-binding domains (residues 1–471 and 509–616,respectively). A heavily O-glycosylated sequence comprises twoparts. The first (residues 441–471) in the crystal structurewraps around an /-barrel formed by residues 1–440. Thesecond (residues 472–508) is an extended, semi-rigid linkerbetween the two domains. To investigate the functional roleof this linker, we made internal deletions to remove residues466–512 (GA1), 485–512 (GA2) and 466–483 (GA3).GA2 and GA3 were expressed in Saccharomyces cerevisiae culturesupernatants at 60 and 20% the wild-type level, respectively,while GA1 was almost undetectable. Western blots comparing extracellularand intracellular fractions indicated that the region deletedin GA3 was critical for secretion, while the region deletedin GA2 contributed to the production of a stable enzyme structure.The activities of purified GA2 and GA3 on soluble and insolublestarch were similar to those of wild-type GA, indicating thatfor soluble starch their deletions did not affect the catalyticdomain and for insoluble starch the linker does not coordinatethe activities of the catalytic and starch-binding domains.The deletions had a significant negative effect on GA2 and GA3thermos tabilities.     

Molecular modelling of Staphylococcal {delta}-toxin ion channels by restrained molecular dynamics

-Toxin is a 26-residue channel-forming peptide from Staphylococcusaureus which forms an amphipathic -helix in a membrane environment.Channel formation in planar bilayers suggests that an averageof six -toxin helices self-assemble to form transbilayer pores.Molecular models for channels formed by -toxin and by a syntheticanalogue have been generated using a simulated annealing protocolapplied via restrained molecular dynamics. These models areanalysed in terms of the predicted geometric and energetic propertiesof the transbilayer pores. Pore radius calculations of the modelsdemonstrate that rings of channel-lining residues contributea series of constrictions along the pore. Electrostatic propertiesof the pores are determined both by pore-lining charged sidechains and by the aligned helix dipoles of the parallel helixbundle. Molecular dynamics simulations (100 ps) of -toxin modelscontaining intra-pore water were performed. Analysis of theresultant dynamics trajectories further supports the proposalthat alternative conformations of pore-constricting side chainsmay be responsible for the observed conductance heterogeneityof -toxin ion channels.     

Family of G protein {alpha} chains: amphipathic analysis and predicted structure of functional domains

The G proteins transduce hormonal and other signals into regulationof enzymes such as adenylyl cyclase and retinal cGMP phosphodiesterase.Each G protein contains an subunit that binds and hydrolyzesguanine nucleotides and interacts with ß subunitsand specific receptor and effector proteins. Amphipathic andsecondary structure analysis of the primary sequences of fivedifferent chains (bovine _s, _t1 and _t2, mouse _i, and rat _o)predicted the secondary structure of a composite chain (_avg).The chains contain four short regions of sequence homologousto regions in the GDP binding domain of bacterial elongationfactor Tu (EF-Tu). Similarities between the predicted secondarystructures of these regions in _avg and the known secondary structureof EF-Tu allowed us to construct a three-dimensional model ofthe GDP binding domain of _avg. Identification of the GDP bindingdomain of _avg defined three additional domains in the compositepolypeptide. The first includes the amino terminal 41 residuesof _avg, with a predicted am phipathic helical structure; thisdomain may control binding of the chains to the ßcomplex. The second domain, containing predicted ßstrands and helices, several of which are strongly amphipathic,probably contains sequences responsible for interaction of chains with effector enzymes. The predicted structure of thethird domain, containing the carhoxy terminal 100 amino acids,is predominantly ß sheet with an amphipathic helixat the carboxy terminus. We propose that this domain is reponsiblefor receptor binding. Our model should help direct further experimentsinto the structure and function of the G protein chain.     

Site-directed mutagenesis of glutathione synthetase from Escherichia coli B: mapping of the {gamma}-L-glutamyl-L-cysteine-binding site

Lysl8, Arg86, Asn283, Ser286, Thr288 and Glu292 of glutathionesynthetase from Escherichia coli B are presumed to be highlyconcerned with the substrate, -L-glutamyl-L-cysteine (-Glu-Cys),binding by X-ray crystallography and affinity labeling studies.Using site-directed mutagenesis, we investigated functionalroles of those residues for -Glu-Cys binding. The mutant enzymesof Arg86 and Asn283 altered their kinetic parameters, especiallythe Michaelis constants of -Glu-Cys. In the case of Asn283,the residue is not likely to have an essential role in -Glu-Cysbinding but its side chain would extend to make a van der Waalscontact with bound -Glu-Cys. Chemical modification of a cysteineresidue with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) showed Arg86would not only be much responsible for -Glu-Cys binding butwould also have a role in maintaining the structural integrityof the enzyme. The other mutant enzymes showed little defectin their kinetic parameters of -Glu-Cys.     

Comparative stability of dihydrofolate reductase mutants in vitro and in vivo

Dihydrofolate reductase mutants with amino acid replacementsin the active center (Thr35 Asp mutant, Arg57 His mutant andthe mutant with triple replacement Thr35 Asp, Asn37 Ser, Arg57 His) were obtained by site-directed mutagenesis. The stabilizationeffect of trimethoprim and NADP·H on the protein tertiarystructure in vitro has been investigated. In the case of mutantswith a ‘weak’ tertiary structure (Thr35 Asp35 andthe triple mutant) the separate addition of ligands does notaffect their stability. The simultaneous addition of these ligandsto Thr35 Asp35 and the triple mutant leads to the large increasein their stability. A distinct correlation was found betweenthe in vitro studied stability of the mutant proteins to theurea- or heat-induced denaturation and the level of proteolyticdegradation of these mutants previously observed in vivo.     

An EGF-pseudomonas exotoxin A recombinant protein with a deletion in toxin binding domain specifically kills EGF receptor bearing cells

We constructed two chimeric toxins; one composed of epidermalgrowth factor (EGF) and pseudomonas exotoxin A (PE), designatedEGF-PE and the other composed of EGF and PE with a deletionof the Ia domain (cell-binding domain), designated EGF-PE (Ia).Both chimeric toxins reacted with anti-EGF and anti-PE antibodies.The cell-killing experiments showed that EGF-PE, but not EGF-PE(Ia),was cytotoxic to the murine fibroblast cell line NR6, whichcarried the PE receptor, but not the EGF receptor. However,after NR6 was transfected with DNA for the expression of humanEGF receptor, the transfected cell line, designated NRHER5,overexpressed human EGF receptors and became sensitive to EGF-PE(IA).The cytotoxicity of EGF-PE(Ia), but not EGF-PE, to NRHER5 canbe completely blocked by an excess amount of EGF. To completelyreverse the cytotoxicity of EGF-PE on NRHER5, both the EGF receptorpathway and the PE receptor pathway need to be blocked. Theseresults suggest that EGF-PE exhibits both EGF and PE bindingactivities, while EGF-PE(IA) possesses only EGF binding activity.Thus, EGF-PE(Ia) may be a better chimeric toxin than EGF-PEin terms of target specificity to EGF receptor bearing cells.We, therefore, examined the cytotoxicity of EGF-PE(Ia) to varioushuman cancer cell lines. We find that human cancer cells containingmore EGF receptors are more sensitive to EGF-PE(Ia).     

Inhibition of {beta}S-chain dependent polymerization by synergistic complementation of contact site perturbations of {alpha}-chain: application of semisynthetic chimeric {alpha}-chains

Mouse _1–30-horse _31–141 chimeric -chain, a semisyntheticsuper-inhibitory -chain, inhibits ß^S-chain dependent polymerizationbetter than both parent -chains. Although contact site sequencedifferences are absent in the _1–30 region of the chimericchain, the four sequence differences of the region _17-22 couldinduce perturbations of the side chains at ₁₆, ₂₀ and ₂₃, thethree contact sites of the region. A synergistic complementationof such contact site perturbation with that of horse _31–141probably results in the super-inhibitory activity of the chimeric-chain. The inhibitory contact site sequence differences, bythemselves, could also exhibit similar synergistic complementation.Accordingly, the polymerization inhibitory activity of Hb Le-Lamentin(LM) mutation [His20()Gln], a contact site sequence difference,engineered into human–horse chimeric -chain has been investigatedto map such a synergistic complementation. Gln20() has littleeffect on the O₂ affinity of HbS, but in human–horse chimeric-chain it reduces the O₂ affinity slightly. In the chimeric-chain, Gln20() increased sensitivity of the ßßcleft for the DPG influence, reflecting a cross-talk betweenthe ₁ß₁ interface and ßß cleft in this semisyntheticchimeric HbS. In the human -chain frame, the polymerizationinhibitory activity of Gln20() is higher compared with horse_1–30, but lower than mouse _1–30. Gln20() synergisticallycomplements the inhibitory propensity of horse _31–141.However, the inhibitory activity of LM–horse chimeric-chain is still lower than that of mouse–horse chimeric-chain. Therefore, perturbation of multiple contact sites inthe _1–30 region of the mouse–horse chimeric -chainand its linkage with the inhibitory propensity of horse _31–141has been now invoked to explain the super-inhibitory activityof the chimeric -chain. The `linkage-map' of contact sites canserve as a blueprint for designing synergistic complementationof multiple contact sites into -chains as a strategy for generatingsuper-inhibitory antisickling hemoglobins for gene therapy ofsickle cell disease.     

Production and characterization of anti-human interferon {gamma} receptor antibody fragments that inhibit cytokine binding to the receptor

Three single-chain antibody fragments that recognize the extracellularhuman interferon receptor -chain (IFNR), and inhibit the bindingof human IFN, have been produced in Escherichia coli. Thesefragments are derived from murine anti-receptor monoclonal antibodies,and comprise the variable heavy (V_H) domain linked to the variablelight (V_L) chain through a 15 amino acid linker [(GGGGS)₃].Using surface plasmon resonance technology (BIAcore), the solubleproteins were shown to retain a high affinity for recombinantIFNR, and by radioimmunoassay to possess high inhibitory activitytowards IFN-binding to human Raji cells. The antibody fragmentsmost likely recognize epitopes that overlap the cytokine bindingsite on the receptor surface. Attempts to dissect further theantibodies to isolated V_H- and V_L-chains and to synthetic linearand cyclic peptides derived from the individual complementaritydetermining regions failed to afford fragments with significantIFNR binding affinity. Nevertheless, these native-like variableregion fragments and petidomimetics derived from them are ofinterest in the design of novel IFNR antagonists.     

A Pore-forming protein with a protease-activated trigger

Hemolysin (HL) is a 293 amino acid pore-forming toxin, whichis secreted as a water-soluble monomer by Staphylococcus aureus.By forming a hexameric pore, HL damages the plasma membranesof target cells. Previous studies established that HL proteinswith nicks near the midpoint of a central glycine-rich loopare held together by a domain-domain interaction and are hemolyticallyactive. In contrast, HL proteins comprising two HL truncationmutants that overlap in the central loop have no or greatlyreduced pore-forming activity, even though the two chains againform a tight complex. Based on these findings, overlap mutantshave now been designed that are activated when redundant aminoacids in the loop are removed by proteases. Further, the identityof the activating enzyme can be specified by additional mutagenesisof the protease recognition site in the overlap sequence. Mutantsof aHL that are activated by tumor-associated proteases mightbe useful components of immunotoxins     

Expression of recombinant {alpha}Ains-crystallin and not {alpha}A-crystallin inhibits bacterial growth

A-Crystallin and A^ins-crystallin are derived from the A-crystallingene via alternative splicing. They are identical except forthe presence of a polypeptide, 23 amino acids long, encodedby the ‘insert’ exon. Evolutionary logic would suggestthat the insertion of a 23 amino acid peptide in the middleof A-crystallin, a protein evolving more slowly than eitherhistone H1, cytochrome c or hemoglobin, would lead to appreciablestructural and functional changes. However, based on physico-chemicalstudies, it is presently believed that A-crystallin and A^ins-crystallinare functionally equivalent and that the presence of the ‘insert’peptide in A^Ins-crystallin is inconsequential. We report herethat the independent expression of recombinant A^Ins-crystallin,and not A-crystallin, inhibits growth of the bacterial host.These observations were confirmed in co-expression experiments,wherein both the proteins were expressed in the same cell. Interestingly,growth inhibition is reversible. Importantly, the data demonstratethat it is catalytic amounts and not the gross accumulationof A^Ins-crystalline which causes growth inhibition. Given theprior knowledge that A-crystallin and A^Ins-crystallin differby a peptide of 23 amino acids, these data suggest that the‘insert peptide’ in A^Ins-crystallin imparts propertieson this protein that are different from A-crystallin.     

Mutational analysis of structure--activity relationships in human tumor necrosis factor-alpha

To determine the region of human tumor necrosis factor-alpha(TNF-), essential for cytotoxic activity against mouse L-M cells,single amino-acid-substituted TNF- mutant proteins (muteins)were produced in Escherichia coli by protein engineering techniques.An expression plasmid for TNF- was mutagenized by passage throughan E.coli mutD5 mutator strain and by oligonucleotide-directedmutagenesis. Approximately 100 single amino-acid-substitutedTNF- muteins were produced and assayed for cytotoxic activity.The cytotoxic activities of purified TNF- muteins, e.g. TNF-31T,-32Y, -82D, -85H, -115L, -141Y, -144K and -146E, were < 1%of that of parent TNF-. These results indicate that the integrityof at least four distinct regions of the TNF- molecule is requiredfor full biological activity. These regions are designated asfollows: region I, from position 30 to 32; region II, from position82 to 89; region III, from position 115 to 117; region FV, fromposition 141 to 146. In addition, TNF-141Y could not completelycompete with parent TNF- for binding to the receptor. This demonstratesthat region IV, and at least aspartk acid at position 141, mustbe involved in the TNF receptor binding site.     

Folding and stability of the active N-terminal domain of tissue inhibitor of metalloproteinases-1 and -2

The truncated forms of tissue inhibitor of metalloproteinase-1and -2 (TIMP-1 and -2), comprising the N-terminal active domain,are ideal molecules for structural analysis by intrinsic fluorescenceas each contains a single conserved tryptophan residue. In thispaper we describe studies on their conformational stability,unfolding/refolding kinetics and the environment of the uniquetryptophan as judged by its fluorescence properties in the nativestate and exposure to an external quencher, acrylamide. Twoforms of TIMP-2 were studied: TIMP-2 T21 derived from the full-lengthcDNA clone isolated from a mixed-tumour library, and TIMP-2A21 containing the highly conserved V18IRAK22 sequence. In allthree TIMP proteins the tryptophan environments in the nativestate appeared to be similar, but substantial differences wereseen in their conformational stabilities and refolding kinetics.TIMP-1 was approximately twice as stable as TIMP-2 T21 and 1.4-foldmore stable than TIMP-2 A21. This stability difference betweenTIMP-1 and TIMP-2 was shown to be independent of N-linked glycosylation.TTMP-1 and TIMP-2 A21 both showed simple two-state refoldingkinetics, whereas TIMP-2 T21 refolding was more complex andbiphasic in character. These differences between TIMP-2 T21and A21 suggest that residue 21 is a structurally importantsite in the TIMP protein.All three truncated molecules can beconsidered as stable independent folding domains ideally suitedfor further structural analysis     

Recombinant hirudin: kinetic mechanism for the inhibition of human thrombin

Recombinant hirudin variant-2(Lys⁴⁷ ), was found to be a competitiveinhibitor of human -thrombin with respect to peptidyl p-Miitroanilidesubstrates. These results contrast with those of Degryse andcoworkers that suggest that recombinant hirudin variant-2(Lys⁴⁷)inhibited thrombin by a non-competitive mechanism [Degryse etal. (1989) Protein Engng, 2, 459–465], -Thrombin, whichcan arise from -thrombin by autolysis, was shown to have anaffinity for recombinant hirudin variant-2(Lys⁴⁷) that was fourorders of magnitude lower than that of -thrombin. It was demonstratedthat the apparent noncompetitive mechanism observed previouslywas probably caused by a contamination of the thrombin preparationby -thrombin. Comparison of the inhibition of -thrombin by recombinanthirudins variant-2(Lys⁴⁷) and variant-1, which differ from oneanother in eight out of 65 amino acids, indicated that the twovariants have essentially the same kinetic parameters.     

The amino acid sequence required for 5' -> 3' exonuclease activity of Bacillus caldotenax DNA polymerase

We studied the 5' 3' exonuclease activity of Bacillus caldotenaxDNA polymerase by site-directed mutagenesis. Among seven mutantsconstructed, two mutant DNA polymerases with an amino acid substitutionof Glyl84 Asp or Glyl92 Asp were confirmed to be deficientin this exonuclease. The two positions corresponded to thoseof the Escherichia coli DNA polymerase I mutants defective in5' 3' exonuclease, polA480ex and polA214. These results provideexperimental support for the proposed amino acid sequence essentialfor the 5' 3' exonuclease activity associated with eubacterialpolymerase I–like DNA polymerases (family A), includingE.coli and Thermits aquaticus.     

Altered specificities of genetically engineered {alpha}1 antitrypsin variants

Seven active site variants of human ₁-antitrypsin (₁AT) wereproduced in Escherichia coli following site-specific mutagenesisof the ₁AT complementary DNA. ₁AT (Ala ³⁵⁸), ₁AT (Ile³⁵⁸ and₁AT (Val³⁵⁸), were efficient inhibitors of both neutrophil andpancreatic elastases, but not of cathepsin G. ₁AT (Ala³⁵⁸, Val³⁵⁸)and ₁AT (Phe³⁵⁸ specifically inhibited pancreatic elastase andcathepsin G respectively. The most potent inhibitor of neutrophilelastase was ₁AT (Leu³⁵⁸), which also proved to be effectiveagainst cathepsin G. The ₁AT (Arg³⁵⁸) variant inactivated thrombinwith kinetics similar to antithrombin III in the presence ofheparin. Electrophoretic analysis showed that SDS-stable highmol. wt complexes were formed between the mutant inhibitorsand the cognate proteases in each case. These data indicatethat effective inhibition occurs when the ₁AT P1 residue (position358) corresponds to the primary specificity of the target protease.Moreover, alteration of the P3 residue (position 356) can furthermodify the reactivity of the inhibitor. Two of the variantshave therapeutic potential: ₁AT (Leu³⁵⁸ may be more useful thanplasma ₁AT in the treatment of destructive lung disorders and₁ (Arg³⁵⁸ could be effective in the control of thrombosis.     

Recombinant-derived interleukin-1{alpha} stabilized against specific deamidation

Recombinant-derived human interleukln-1 (IL-1), purified fromEscherichia coli, was resolved by isoelectric focusing on polyacrylamidegels into two species of isoelectric points (pI) 5.45 and 5.20,which constituted 75% and 25% of the total IL-1 protein respectively.The pI 5.45 and pI 5.20 species were separated by chromatofocusingand subjected to N-terminal sequence analysis. The pI 5.45 speciescontained the expected Asn residue at position 36 of the matureprotein sequence whereas the pI 5.20 species contained an Aspresidue at the same position. A mutant protein in which Asn-36was substituted for a Ser residue was isolated from E.coli andshown to be homogeneous on isoelectric focusing analysis witha pI = 5.45. ¹H-n.m.r. and circular dichroism analyses of wild-typeand the mutant IL-1 indicated a similar conformation which wasalso indicated by the identical receptor binding affinitiesof IL-1 with Asn, Asp or Ser in position 36. The mutant proteinwas stabilized against specific base catalysed and temperature-induceddeamidation, and may be more suitable than the wild-type positionfor physical and structural studies.     

The role of the sequence extensions in {beta}-crystallin assembly

The modular construction of the eye lens ß-crystallinsmakes them good candidates for protein engineering to ascertainthe rules of assembly of oligomers. X-ray studies have shownthat although the polypeptide chains of ßB2-crystallinand -crystallins fold to form similar N- and C-terminal domains,the conformation of the connecting peptides are such that the-crystallins are monomers and the ß-crystallin isa dimer. Unlike -crystallins, the numerous -crystallins haveextensions of variable sequence from the globular domains. Wehave tested the effect of removing the N- and C-terminal extensionsfrom rat ßB2-crystallin using a bacterial expressionsystem. Abundant proteins were produced in Escherichia coliusing the pET or pQE vectors. Full-length and truncated proteinswere purified and checked for refolding using circular dichroism.Sizing of the truncated proteins using gel filtration chroma-tographyshowed that the absence of either the N- or C-terminal extensiondoes not affect dimerization of ßB2-crystallin.     

Sequence requirements for cytochromes P450IIA1 and P450IIA2 catalytic activity: evidence for both specific and non-specific substrate binding interactions through use of chimeric cDNAs and cDNA expression

Cytochrome P450s IIA1 and IIA2, encoded by the CYP2A1 and CYP2A2genes, display 88% amino acid sequence similarities. The dissimilaritiesof sequence between these two enzymes are primarily localizedwithin four discrete regions of the polypeptides that are separatedby regions of absolute sequence identity. IIA1 specificallyhydroxylates the prototype substrate testosterone at the 7 and6 position with a predominance of 7 metabolite. IIA2, on theother hand, hydroxylates this steroid at eight positions onthe molecule, with one of the most abundant metabolites being15hydroxytestosterone. To determine those amino acids responsiblefor the difference in testosterone hydroxylation specificities,chimeras were constructed between IIA1 and IIA2 cDNAs and expressedin cell culture using vaccinia-virus-mediated cDNA expression.Chimeras, in which the first 355 amino acids correspond to asingle enzyme, maintain the specificity associated with thatenzyme. Of six chimeras which have substitutions between aminoacids 161 and 276, two are inactive and the remaining four givesimilar metabolite profiles, in which both 7 and 15 hydroxylationspecificities have been lost. Two of these four chimeras arediametric apposites, suggesting that modification of eitherthe N-terminal or central regions of the enzymes results inconformational changes that prevent the specific binding interactionsresponsible for the narrow regioselectivity associated withIIA1 and 15-hydroxytestosterone formation associated with IIA2.     

A point mutation of human interferon {gamma} abolishes receptor recognition

We identified a single amino acid mutation that abolished thebioactivity of human IFN. The mutation was identified by screeninga mutagenized IFN expression library for molecules with alteredbiological activity. The mutant protein was expressed at highlevels in Escherichia coli, and remained soluble upon purification.However, the protein was completely inactive in all IFN assaysinvestigated, exhibiting < 0.0006% of the specific activityof native IFN antiviral activity. Sequencing the plasmid DNAencoding this mutant protein showed that the histidine at position111 of native human IFN is changed to aspartic acid (IFN/H111D).Other mutations at this site showed that only hydrophobic aminoacids at position 111 maintain significant, though low, biologicalactivity. Structural characterization of the IFN/H111D proteinby NMR as well as CD spectroscopy demonstrated that the proteinhas limited conformational differences from native IFN. Modelsof the X-ray crystal structure of human IFN [Ealick, P.E., W.J.Cook,S.Vijay-Kumar, M.Carson, T.L.Nagabhushan, P.P.Trotta and C.E.Bugg(1991) Science, 252, 698–702] suggest that this histidineresidue is located at a severe 55° bend in the C-terminalF helix. We conclude that H111 lies within or affects the receptorbinding domain of human IFN.