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.     

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.     

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.     

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.     

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     

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.     

Structure-function analysis of human IL-1{alpha}: identification of residues required for binding to the human type I IL-1 receptor

Using oligonucleotide-directed mutagenesis, the binding siteon human interleukin-1 (IL-1) for the human type I IL-1 receptor(IL-1R) has been analyzed. Substitution of seven amino acids(Arg12, Ile14, Asp60, Asp61, Ile64, Lys96 and Trp109) resultedin a significant loss of binding to the receptor. Based on crystallographicinformation, the side chains of these residues are clusteredin one region of IL-1 and exposed on the surface of the protein.Five of the residues in the IL-1 binding site align with thebinding residues previously determined in human IL-1ß,demonstrating that the type I IL-1R recognizes homologous regionsin both ligands. Unexpectedly, only three of the aligned residuesare identical between IL-1 and IL-1ß. These observationssuggest that the composition of contact residues in the bindingsite is unique for each ligand–receptor complex in theIL-1 system.     

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.     

Calcium binding mode of {gamma}-carboxyglutamic acids in conantokins

Conantokin-T (con-T) and conantokin-G (con-G) are two highlyhomologous peptide toxins found in Conus venom. The former isa 21-residue peptide with four -carboxyglutamic acid (Gla) residues(at positions 3, 4, 10 and 14), while the latter is a 17-residuepeptide with five -carboxyglutamic acid residues (at positions3, 4, 7, 10 and 14). Despite the apparent similarity in numberand relative positions of the -carboxyglutamic acid residues,¹¹³Cd-NMR studies indicated a distinct metal binding behaviorfor con-G and con-T. There appears to be four binding sitesin con-G in contrast to one metal binding site in con-T. Toelucidate the mode of calcium binding by the -carboxyglutamicacid residues in these conantokins, we designed various analogouspeptides with their -carboxyglutamic acid replaced by otheramino acid residues. ¹¹³Cd-NMR experiments on conantokin analoguesreveal that the major difference in the number of metal bindingsites between con-G and con-T is due to the residue at position7. We also performed molecular simulations to calculate therelative binding free energies of several potential bindingsites. Based on our theoretical and experimental results, wepropose a `four-site' binding model for conantokin-G and a `single-site'binding model for conantokin-T.     

Protein modification from mutational analysis of an autologous peptide fragment

In the -complementation of ß-galactosidase an N-terminalpeptide fragment (-peptide) of the wild-type enzyme interactswith a defective ß-galactosidase enzyme to restorecapacity for subunit assembly and activity. We have used previouslya random mutagenesis and screening approach to identify a pentapeptideresidue tract in the -peptide that was highly tolerant of residuesubstitution, with some mutations conferring improved function.This tract is of clear importance for -peptide function butis apparently dispensible in the intact parental enzyme. Toinvestigate this further, we selected tract mutations and placedthem into intact ß-galactosidase, at the correspondingN-terminal position as in the -peptide. We then tested whethersuch specific tract sequences conferred properties to the wholeenzyme which could be predicted from the behaviour of the defectiveenzyme complemented with the corresponding mutant -peptide.This was shown for mutations which positively or negativelyaffected enzyme stability. Additionally, a subset of mutationswhich affected complementation efficiency in vivo were predictedto affect the formation of higher-order structures in the intactprotein, and this was observed experimentally. Mutations whichdecreased peptide complementation dramatically decreased thelevel of formation of multimers in the intact protein and amutation which increased peptide complementation produced markedenhancement of multimer formation in a protein with a preexistingimpairment in higher-order structure formation. Such subtleeffects are difficult to detect directly in the whole proteinby randomization/selection approaches, but in the complementingpeptide the role of the residues within the pentapeptide tractis effectively amplified. Identification of residue tracts exhibitingfunctional tolerance to amino acid substitution in an activepeptide fragment can thus be combined with transferral of potentiallyuseful mutant peptide sequences back into the intact protein.Manipulation of a complementation system in this manner affordsa sensitive approach towards targeted improvement of proteins.     

Biosynthetic incorporation of 7-azatryptophan into the phage lambda lysozyme: estimation of tryptophan accessibility, effect on enzymatic activity and protein stability

The phage lambda lysozyme (L) contains four tryptophans. Thesehave been efficiently replaced by 7-azatryptophan (7aW) throughbiosynthetic incorporation into the overexpressed protein. Comparativeanalysis of the effect of temperature or pH on the fluorescenceof the wild-type L and 7aWs-containing protein (aL) shows thatthe stability of the protein is only mildly reduced by 7aW incorporationabove pH 5 but that it is strongly decreased below pH 4 on protonationof inaccessible 7aWs. The aL fluorescence depends on pH as aconsequence of its effect on the denaturation equilibrium, onthe state of protonation of accessible 7aWs in the native stateand of all 7aWs in the denatured state. The pH dependence ofthe fluorescenceis used to estimate the number of accessibletryptophans in the protein. The result agrees with that derivedfrom tryptophan NH exchange measurements by ¹H-NMR. The acidlimb of the activity-pH profile is characterized by a sharpdrop that might arise from a cooperative acidinduced denaturation.The difference in acid stability of aL versus L is used to ruleout this acid denaturation hypothesis as tryptophan replacementdoes not affect the lytic activity on chloroform-sensitizedEscherichia coli cells or its pH profile.     

Highly controlled carbodiimide reaction for the modification of lysozyme. Modification of Leu129 or Asp119

In the cross-linking reaction of lysozyme between Leu¹²⁹ (-COO^–)and Lys¹³ (-NH₃⁺ using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimidehydrochloride (EDC), a side reaction of the peptide bond inversionfrom to ß between A and Gly¹⁰² was greatly reducedby addition of ß-(1,4)-linked trimer of N-acetyl-D-glucosamine[(NAG)₃] When methylamine or 2-hydroxyethylamine was furtheradded, the extent of the cross-link formation was decreasedand the derivative where the -carboxyl group of Leu¹²⁹ was modifiedwith the amine was newly obtained. On the other hand, when ammoniawas added, the ß-carboxyl group of Asp¹¹⁹ insteadof the -carboxyl group was mainly amidated. From these results,the presence of a salt bridge between Asp¹¹⁹ and Arg¹²⁵ besidesthat between Lys¹³ and Leu¹²⁹ is proposed. Enzymatic activitiesof the derivatives prepared here indicated that the modificationof the -carboxyl group reduced the activity to {small tilde}90% of that of native lysozyme. Des-Leu¹²⁹ lysozyme, which lacksLeu¹²⁹ also showed {small tilde} 90% of the activity of nativelysozyme. Therefore, the salt bridge between Lys¹³ and Leu¹²⁹may play some role in maintaining the active conformation oflysozyine.     

Semi-empirical simulation of Zn/Cd binding site preference in the metal binding domains of mammalian metallothionein

Metallothionein, a two-domain protein, naturally binds sevengram atoms of divalent ions such as Zn and Cd. Four of the metals(Ml, M5, M6 and M7) are found in the -domain and three (M2,M3 and M4) in the ß-domain. Previous studies haveshown that metals in the -domain are more readily exchangeable,and the level of avidity is site specific. By semi-empiricalMNDO modified neglect of diatomic overlap calculations, we foundthe tendency of binding energy for Cd to be M3 > M2 >M4 in the ß-cluster and M5 > M7 > Ml, M6 inthe -cluster. Thus, the replacement of Zn by Cd can be expectedto follow the order M4 M2 M3 in the ß-domain andMS M7 M1 or M6 in the -domain. This is reflected by energydifferences computed with a series of simulated structures derivedfrom either X-ray crystallography or NMR coordinates.     

Study of cysteine residues in the {alpha} subunit of Escherichia coli tryptophan synthase. 2. Role in enzymatic function

To understand the functional roles of Cys residues in the subunitof tryptophan synthase from Escherichia coli, single mutantsof the subunit, in which each of the three Cys residues wassubstituted with Ser, Gly, Ala or Val, were constructed by site-directedmutagenesis. The effects of the substitutions on the functionof tryptophan synthase were investigated by activity measurements,calorimetric measurements of association with the ßsubunit and steadystate kinetic analysis of catalysis. Althoughthe three Cys residues are located away from the apparentlyimportant parts for enzymatic activity, substitutions at position81 by Ser, Ala or Val caused decreases in the intrinsic activityof the subunit. Furthermore, Cys81Ser and Cys81Val reducedstimulation activities in the and ß reactions dueto formation of a complex with the ß subunit. Thelower stimulation activities of the mutant proteins were notcorrelated with their abilities to associate with the ßsubunit but were correlated with decreases in k_cat. The presentresults suggest that position 81 plays an indirectly importantrole in the activity of the subunit itself and the mutual activationmechanism of the complex.     

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.     

Model complexes of tumor necrosis factor-{alpha} with receptors Rl and R2

The biological activities of tumor necrosis factor- (TNF-) aremediated by two different receptors, TNFR1 and TNFR2. To analyzethe receptor binding site(s) of TNF-, molecular models havebeen built of the complexes of TNF- with the extracellular regionsof receptors Rl and R2, based on the known crystal structuresof TNF- and lymphotoxin bound to Rl. The model structure ofR2 from residues 18-160 was built by analogy to the crystalstructure of Rl in complex with lymphotoxin. The amino acidsequences of Rl and R2 show 27.5% identity over this regionand were aligned with five insertions and three deletions. Thereare 18 conserved cysteines that form disulfides. R2 has lostone pair of cysteines compared with Rl, but two new cysteineswere modeled as forming a new disulfide bond. Both symmetricand asymmetric trimers of TNF- were used to model the complexeswith TNFR1 and R2. An analysis of differences in the model complexesshowed good agreement with data on the differential bindingof TNF mutants to its two receptors.     

The net energetic contribution of interhelical electrostatic attractions to coiled-coil stability

The net energetic contribution of interhelical electrostaticattractions to coiled-coil stability has been quantitated usingde novo designed synthetic coiled-coils. The synthesized modelcoiled-coil (EK), denoted by amino acid res-idues in positionse and g, which contains only interhelical ionic interactionswithout any possible (i, i + 3) and (i, i + 4) intrahelicalionic interaction, consists of two identical 35 residue polypeptidechains with a heptad repeat KgLaG-bAcLdEeKf. Three mutant coiled-coilswere prepared where five Glu residues at e positions in EK weremutated to Gin residues (QK); five Lys residues at g positionswere altered to Gin residues (EQ) or these mutations were effectedat both positions e and g (QQ). The stabilities of the fourcoiled-coils were determined by measuring the ellipticitiesat 220 nm as a function of urea concentration at 20C. By usinga double-mutant cycle analysis it was possible to isolate theenergetic contribution of interhelical ionic attractions tocoiled-coil stability from the other contributions such as helicalpreference and hydro-phobicity. The 0.37 0.01 kcal/mol ofenergetic contribution of one interhelical ion pair to the coiled-coilstability was obtained from three independent comparisons. Thisfinding suggests that a large number of weak interhelical electrostaticinteractions on the surface of a protein can make a substantialcontribution to protein stability. In addition, the energeticcontributions of a single mutation E^– Q, K⁺Q, Q E andE^– Ewere also determined (G = 0.22, 0.26, 0.46 and 0.65kcal/mol for the single mutations, respectively). The greatercontribution of a protonated Glu residue to coiled-coil stabilitycompared with an ionized Glu residue (0.65 kcal/mol) can outweighthe relatively smaller contribution of an interhelical ion pair(0.37 kcal/mol), which clearly explains why most coiled-coilsare more stable at acidic pH compared with neutral pH even wheninterhelical salt bridges contribute to the coiled-coil stabilityat neutral pH.     

Expression of goat {alpha}-lactalbumin in Escherichia coli and its refolding to biologically active protein

A cDNA encoding the mature region of goat -lactalbumin and the3'-non-coding region was fused to cDNA of the N-terminal halfof porcine adenylate kinase which had been placed under thecontrol of the tac promoter in an expression vector in Escherichiacoli. In addition, a methionine codon was inserted between thetwo cDNAs. When the plasmid carried the full-length 3'-non-codingregion, little accumulation of the fused protein was observed.However, the deletion of two-thirds of the 3'-non-coding regionproduced significant expression of the fused protein in E.colistrain JM105. Since goat -lactalbumin contains no methionineresidue, the mature goat -lactalbumin was isolated by CNBr digestionof the fused insoluble protein and refolded using thioredoxin.The homogeneous and biologically active goat -lactalbumin waspurified by Ca²⁺ ion-dependent hydrophobic chromatography.     

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.     

{alpha}-Helix folding by Monte Carlo simulated annealing in isolated C-peptide of ribonuclease A

Conformation of the C-peptide fragment of RNase A is calculatedby Monte Carlo simulated annealing. We adopt the total potentialenergy as given by the sum of generic interatomic energies whoseparameters are determined separately for each amino acid withoutreferring to the empirical structure of the C-peptide. The simulationis carried out in a completely unrestricted way without imposingany weight towards given final destinations. Starting from completelyrandom initial conformations and minimizing the total potentialenergy with respect to main-chain dihedral angles and side-chaintorsion angles, we have obtained partial -helix structure witha high probability ({small tilde}40%). The energetically mostfavourable structure exhibits a 2.5-turn -helix at the locationidentical with that of the 3-turn -helix in the native enzymemolecule. Classification of conformations obtained in the simulationinto clusters of similar structure shows that our simulationindeed predicts the -helix structure for the isolated C-peptidewith specific charged residues. The results of simulation withvarious amino acid substitutions are also found to be consistentwith the experimental implication for the importance of intramolecularionic interactions for -helix stability for this peptide.