Progesterone binding to uteroglobin: two alternative orientations of the ligand

Progesterone binding to a homodimer of uteroglobin takes placein a hydrophobic cavity formed by the two subunits. Previousmutational analyses have shown that the tyrosine (21 and 21')and threonine (60 and 60') residues of the uteroglobin dimerare directly involved in progesterone binding. To analyze thecontribution of each of the two tyrosines and threonines inthe dimer, we have constructed a covalently linked uteroglobindimer (UG^cl) by fusing two uteroglobin cDNAs via a syntheticlinker sequence. Escherichia coli expressed UG^cl bound progesteronewith the same affinity as the native dimeric protein. Replacementof both tyrosines by phenylalanines abolished progesterone binding.Replacement of either the C-terminal tyrosine 21 or the N-terminaltyrosine 21' separately, reduced the affinity for progesterone3- to 4-fold, suggesting that both tyrosines participate inprogesterone binding. In contrast, substitutions of the threonineresidues of the C- or Nterminal moities had no effect, whereasthe replacement of both threonines reduced the affinity forprogesterone 2- to 3-fold. These data, together with computermodels, suggest that progesterone docks in the internal bindingpocket of uteroglobin in two different orientations.     

Recombinant rabbit uteroglobin expressed at high levels in E.coli forms stable dimers and binds progesterone

In order to express uteroglobin in Escherichia coli we haveconstructed a DNA coding for complete mature rabbit uteroglobinby fusing genomic sequences from the second exon of the geneto an incomplete cDNA. This DNA was inserted into various positionsof the polylinker cloning region of pDS expression vectors andthe uteroglobin gene was expressed in E.coli by IPTG induction.Four different uteroglobinderived proteins were produced containing1, 3,5 and 7 more N-terminal amino acids than the naturallyoccurring mature protein. The yield of soluble protein stronglyincreased with increasing length of the N-terminal additions.Protein and RNA analysis showed that this variation is mostlikely due to progressively higher translation efficienciesof the larger recombinants. UG7, the most efficiently synthesizedrecombinant protein, carrying seven additional N-terminal aminoacids, was purified and further characterized. Like naturaluteroglobin, UG7 forms a dimer and binds progesterone with anaffinity indistinguishable to the natural protein. This bacteriallyproduced protein can be used for detailed structure–functioninvestigations of uteroglobin.     

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.     

Overproduction of bovine {beta}-casein in Escherichia coli and engineering of its main chymosin cleavage site

A cDNA clone containing the entire coding region for bovineß-casein A³ flanked by 53 base pairs of 5' non-codingand 358 base pairs of 3' non-coding sequences was isolated froma bovine mammary cDNA phagemid library. The coding segment formature ß-casein was subcloned into the T7 expressionsystem, in which the expression of recombinant ß-caseinwas controlled by the T7 gene 10 promoter and ribosome bindingsite. High level expression of Met-ß-casein to 20%of the total soluble proteins was obtained in Escherichia coliwithin 2 h after induction of T7 RNA-polymerase synthesis. Inan attempt to induce secretion the coding segment for matureß-casein was coupled to the ompA translations initiationsignal and signal peptide coding sequence but no secretion ofthe fusion protein and no processing of the signal peptide fromthe fusion protein was observed. Instead, the Met-ß-caseincould be isolated in asoluble form from E.coli cells after anosmotic shock, indicative of a periplasmic location. This proceduredid not lyse the cells. The protein was purified to homogeneityafter a pH 4.8 isoelectric precipitation followed by reversed-phasehigh-performance liquid chromatography. The ß-caseincDNA was altered to change the main chymosin cleavage siteinß-casein at position 192–193 in two ways, namelyfrom Leu–Tyr to Pro–Pro and to Leu–stop. Thesemutations were designed to prevent generation of the bitterpeptide ßcasein(193–209) by chymosin cleavage.The mutant Met-ß-caseins were expressed in E.colito the same level as wild-type Met-ß-casein. Purifiedmutant Met-ß-casein(Prol92– Prol93) was no longerhydrolysed by chymosin at the 192–193 bond.     

Contribution of a disulfide bridge to the stability of 1,3-1,4-P-D-glucan 4-glucanohydrolase from Bacillus licheniformis

Bacillus 1,3-1,4-ß-glucanases possess a highly conserveddisulfide bridge connecting a ß-strand with a solventexposedloop lying on top of the extended binding site cleft The contributionof the disulfide bond and of both individual cysteines (Cys61and Cys90) in the Bacillus licheniformis enzyme to stabilityand activity has been evaluated by protein engineering methods.Reduction of the disulfide bond has no effect on kinetic parameters,has only a minor effect on the activity-temperature profileat high temperatures, and destabilizes the protein by less than0.7 kcal/mol as measured by equilibrium urea denatu ration at37°C. Replacing either of the Cys residues with Ala destabilizesthe protein and lowers the specific activity. C90A retains 70%of wild-type (wt) activity (in terms of Vmax), whereas C61Aand the double mutant C61A–C90A have 10% of wt V_max. Alarger change in free energy of unfolding is seen by equilibriumurea denaturation for the C61A mutation (loop residue, 3.2 kcal/molrelative to reduced wt) as compared with the C90A mutation (ß-strandresidue, 1.8 kcal/mol relative to reduced wt), while the doublemutant C61A–C90A is 0.8 kcal/mol less stable than thesingle C61A mutant. The effects on stability are interpretedas a result of the change in hydrophobic packing that occursupon removal of the sulfur atoms in the Cys to Ala mutations     

Use of site-directed mutagenesis to probe the role of Cys149 in the formation of charge-transfer transition in glyceraldehyde-3-phosphate dehydrogenase

Oligonucleotide-directed mutagenesis was employed to producemutants of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)of Escherichia coli and Bacillus stearothermophilus. Three differentmutant proteins—His¹⁷⁶ — Asn, Cys¹⁴⁹ — Ser,Cys¹⁴⁹ — Gly—were isolated from one or both of theenzymes. The study of the properties of these mutants has shownthat Cys¹⁴⁹ is clearly responsible for the information of acharge-transfer transition, named the Racker band, observedduring the NAD⁺ binding to apoGAPDH. This result excludes asimilarity between the Racker band and the charge-transfer transitionobserved following the alkylation of GAPDH by 3-chloroacetylpyridine-adenine dinucleotide.     

A thermoresistant mutant of ribonuclease T1 having three disulfide bonds

Molecular-dynamic calculations predict that, if Tyr24 and Asn84are each replaced by a Cys residue, it should be possible toform a third disulfide bond in ribonuclease T1 (RNase T1) betweenthese residues, with only minimal conformational changes atthe catalytic site. The gene encoding such a mutant variantof RNase T1 (Tyr24 – Cys24, Asn84 – Cys84) was constructedby the cassette mutagenesis method using a chemically synthesizedgene. In order to reduce the toxic effect of the mutant enzyme(RNase T1S) on an Escherichia coli host, we arranged for theprotein to be secreted into the periplasmic space by using avector that harbors a gene for an alkaline phosphatase signalpeptide under the control of the trp promoter. The nucleolyticactivity of RNase T1S toward pGpC was approximately the sameas that of RNase T1 at 37°C (pH 7.5). Moreover, at 55°C,RNase T1S retained nearly 70% of its activity while the activityof the wild-type enzyme was reduced to <10%. RNase T1S wasalso more resistant to denaturation by urea than the wild-typeenzyme. However, unlike RNase T1, RNase T1S was irreversiblyand almost totally inactivated by boiling at 100°C for 15min.     

Recombinant pro-regions from papain and papaya proteinase IV are selective high affinity inhibitors of the mature papaya enzymes

Proteolytic enzymes require the presence of their proregionsfor correct folding. Of the four proteolytic enzymes from Caricapapaya, papain and papaya proteinase IV (PPIV) have 68% sequenceidentity. We find that their proregions are even more similar,exhibiting 73.6% identity. cDNAs encoding the pro-regions ofthese two proteinases have been expressed in Escherichia coliindependently from their mature enzymes. The recombinant pro-regionsof papain and PPIV have been shown to be high affinity inhibitorsof all four of the mature native papaya cysteine proteinases.Their inhibition constants are in the range 10^–6–;10^–;9M. PPIV was inhibited two to three orders of magnitude lesseffectively than papain, chymopapain and caricain. The pro-regionof PPIV, however, inhibited its own mature enzyme more effectivelythan did the proregion of papain. Alignment of the sequencesof the four papaya enzymes shows that there is a highly variablesection towards the C-terminal of the pro-region. This regionmay therefore confer selectivity to the pro-regions for theindividual proteolytic enzymes.     

Study of cysteine residues in the {alpha} subunit of Escherichia coli tryptophan synthase. 1. Role in conformational stability

To eludicate the role in conformational stability of Cys residuesburied in the interior of a protein, the thermodynamic propertiesof denaturation of mutant subunit of Escherichia coli tryptophansynthase, in which Ser, Ala, Val or Gly was substituted foreach of the three Cys residues, were analyzed using calorimetry.The mutants were less stable than the wild type, indicatingthat Cys residues contribute greatly to the stability of the subunit. In most cases, a large decrease in denaturation enthalpywas observed, compensated for by the denaturation entropy toa major extent. The extent of changes in the denaturation Gibbsenergy and denaturation enthalpy varied greatly depending onboth substituting residues and positions. Of all the mutantproteins, the Cys154Ser mutant showed the greatest decreasein denaturation enthalpy; its denaturation enthalpy was halfthat of the wild type, and was considerably repaired by addinga ligand of the subunit. Because the enthalpy of ligand bindingto Cys154Ser in the native state did not change. it seems thatthe decrease in the denaturation enthalpy of Cys154Ser and itsrecovery by ligand binding are caused by conformational changesin the denatured state due to the mutation.     

Gene synthesis and functional expression of a protein exhibiting monodomain IgG Fc binding

A gene encoding a bacterial IgG Fc binding domain was designedand synthesized. The synthetic DNA fragment was cloned 3' toan inducible trpE promoter such that expression of the genein Escherichia coli produced abundant Fc binding protein fusedto the first seven amino acids of the trpE protein. The recombinantprotein contained a single Fc binding domain and demonstratedefficient binding to'human IgG in Western blot analysis. Thisprotein degraded rapidly following cell lysis in the absenceof protease inhibitors, but could be effectively protected bythe addition of protease inhibitor. After purification of theprotein by IgG affinity chromatography, IgG Fc binding abilitywas retained for at least 24 h at either 23 or 37°C andon heating for 15 min at temperatures up to 65°C. No immunoprecipitationwas observed in interactions between the monodomain Fc bindingprotein and IgG molecules. Unlike staphylococcal protein A,no detectable binding of the monodomain IgG Fc binding proteinwas observed to either IgM or IgA. Truncated proteins, expressedfrom a series of 3' deletions of the synthetic gene, were usedto estimate the minimum portion of a monodomain Fc binding proteinthat retained Fc binding ability.     

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.     

Engineering of the substrate-binding region of the sublilisin-like, cell-envelop proteinase of Lactococcus lactis

The substrate-binding region of the cell-envelope proteinaseof Lactococcus lactis strain SK11 was modelled, based on sequencebomology of the catalytic domain with the serine proteinasessubtilisin and thermitase. Substitutions, deletions and insertionswere introduced, by site-directed and cassette mutagenesfe ofthe prtP gene encoding this enzyme, based on sequence comparisonboth with subtilisin and with the homologous L.lactis strainWg2 proteinase, which has different proteolytic properties.The engineered enzymes were investigated for thermal stability,proteolytic activity and cleavage specificity towards smallchromogenk peptide substrates and the peptide _g1-casein(l–23).Mutations in the subtilisin-like substrate-binding region showedthat Ser433 is the active site residue, and that residues 138and 166 at either side of the binding cleft play an importantrole in substrate specificity, particularly when these residuesand the substrate are oppositely charged. The K748T mutationin a different domain also affected specificity and stability,suggesting that this residue is in close proximity to the subtilisin-likedomain and may form part of the substratebinding site. Severalmutant SK11 proteinases have novel properties not previouslyencountered in natural variants. Replacements of residues 137–139AKTalong one side of the binding cleft produced the 137–139GPPmutant proteinase with reduced activity and narrowed specificity,and the 137–139GLA mutant with increased activity andbroader specificity. Furthermore, the 137–139GDT mutanthad a specificity towards _g1,-casein(l–23) closely resemblingthat of L.lactis Wg2 proteinase. Mutants with an additionalnegative charge in the binding region were more stable towardsautoproteolysis.     

In vivo copper- and cadmium-binding ability of mammalian metallothionein domain

The ß domain of mouse metallothionein 1 (ßMT) wassynthesized in Escherichia coli cells grown in the presenceof copper or cadmium. Homogenous preparations of Cu–ßMTand Cd–ßMT were used to characterize the correspondingin vivo-conformed metal-clusters, and to compare them with thespecies obtained in vitro by metal replacement to a canonicalZn₃–ßMT structure. The copper-containing ßMTclusters formed inside the cells were very stable. In contrast,the nascent ß peptide, although it showed cadmium bindingability, produced a highly unstable species, whose stoichiometrydepended upon culture conditions. The absence of ßMT proteinin E.coli protease-proficient hosts grown in cadmium-supplementedmedium pointed to drastic proteolysis of a poorly folded ßpeptide, somehow enhanced by the presence of cadmium. Possiblefunctional and evolutionary implications of the bioactivityof mammalian ßMT in the presence of monovalent and divalentmetal ions are discussed.     

Molecular mechanics analysis of inhibitor binding to HIV-1 protease

Crystallographic structures of HIV protease with three differentpeptide-mimetic inhibitors were subjected to energy minimizationusing molecular mechanics, the minimized structures analyzedand the inhibitor binding energies calculated. Partial chargeassignment for the hydrogen bonded catalytic aspartk acids,Asp25 and -25', was in good agreement with charge calculationsusing semi-empirical molecular orbital methods. Root mean squaredeviations on minimization were small and similar for both subunitsin the protease dimer. The surface loops, which had the largestB factors, changed most on minimization; the hydrophobic coreand the inhibitor binding site showed little change. The distance-dependentdielectric of D(r) = 4r was found to be preferable to D(r) =r. Distance restraints were applied for the intermolecular hydrogenbonds to maintain the conformation of the inhibitor bindingsite. Using the dielectric of D(r) = 4r, the calculated interactionenergy of the three inhibitors with the protease ranged from–53 to –56 kcal/mol. The groups of the inhibitorswere changed to add or remove a ‘transition state analogue’hydroxyl group, and the loss in energy on the removal of thisgroup was calculated to be 0.9–1.7 kcal/mol. This wouldrepresent 19–36% of the total measured difference in bindingenergy between the inhibitors JG365 and MVT-101.     

Threonine 81 of the trp repressor of Escherichia coli plays an auxiliary role in the formation of the corepressor binding pocket

A mutational study was performed on the corepressor (Ltryptophan)binding site of the trp repressor of Escherichia coli. Threonine81, one of the residues forming the hydrophobic pocket of thebinding site, was replaced with Ser, Cys and Met by cassettemutagenesis. Biochemical characterization showed that all thesemutations caused a moderate decrease in tryptophan binding activity(free energy change 1 kcal/mol). The results suggested thatthe binding pocket is rather flexible in the vicinity of Thr81.On the other hand, the mutations produced a discernible decreasein the repressor activity in vivo, apparently by weakening oreliminating the hydrogen bond between Thr81 and the operatorDNA, as well as by introducing possible side-chain rearrangement.     

Characterization of the apparent negative co-operativity induced in Escherichia coli aspartate aminotransferase by the replacement of Asp222 with alanine. Evidence for an extremely slow conformational change

The strictly conserved active site residue, Asp222, which formsa hydrogen-bonded salt bridge with the pyridine nitrogen atomof the pyridoxal 5' phosphate (PLP) co-factor of aspartate aminotransferase(AATase), was replaced with alanine (D222A) in the Escherichiacoli enzyme. The D222A mutant exhibits non–hyberbolicsaturation behavior with amino acid substrates which appearas apparent negative eooperativity in steady–state kineticanalyses. Single turnover progress curves for D222A are welldescribed by the sum of two exponentials, contrasting with themonophasic kinetics of the wild-type enzyme. An active/inactiveheterodimer containing the D222A mutation retains this biphasickinetic response, proving that the observed eooperativity isnot the result of induced allostery. The anomalous behavioris explained by a hysteretic kinetic model involving two slowlyinterconverting enzyme forms, only one of which is catalyticallycompetent. The slow functional transition between the two formshas a half–life of 10 mins. Preincubation of the mutantwith the dicarboxylk inhibitor maleate shifts the equilibriumpopulation of the enzyme towards the catalytically active form,suggesting that the slow transition is related to the domainclosure known to occur upon association of this inhibitor withthe wild-type enzyme. The importance of Asp222 in the chemicalsteps of transamination is confirmed by the l0⁵fold decreasein catalytic competence in the D222A mutant, and by the largeprimary C–deuterium kinetic isotope effect (6.7 versus2.2 for the wild–type). The transamination activity ofthe D222A mutant is enhanced 4– to 20–fold by reconstltutionwith the co-factor analog N–methylpyridoxal–5–phosphate(N–MePLP), and the C–proton abstraction step isless rate determining, as evidenced by the decrease in the primarykinetic isotope effect from 6.7 to 2.3. These results suggestthat the conserved interaction between the protonated pyridinenitrogen of PLP and the negatively charged carboxylate of Asp222is important not only for efficient C–proton abstraction,but also for conformational transitions concomitant with thetransamination process     

The consequences of engineering an extra disulfide bond in the Penicillium camembertii mono-and diglyceride specific lipase

The extracellular lipase from Penicillium camembertii has uniquesubstrate specificity restricted to mono- and diglycerides.The enzyme is a member of a homologous family of lipases fromfilamentous fungi. Four of these proteins, from the fungi Rhizomucormiehei, Humicola lanuginosa, Rhizopus delemar and P.camembertii,have had their structures elucidated by X-ray crystallography.In spite of pronounced sequence similarities the enzymes exhibitsignificant differences. For example, the thermo-stability ofthe P.camembertii lipase is considerably lower than that ofthe H.lanuginosa enzyme. Since only the P.camembertii enzymelacks the characteristic long disulfide bridge, correspondingto Cys22–Cys268 in the H.lanuginosa lipase, we have engineeredthis disulfide into the former enzyme in the hope of obtaininga significantly more stable fold. The properties of the doublemutant (Y22C and G269C) were assessed by a variety of biophysicaltechniques. The extra disulfide link was found to increase themelting temperature of the protein from 51 to 63°C. However,no difference is observed under reducing conditions, indicatingan intrinsic instability of the new disulfide. The optimal temperaturefor catalytic activity decreased by 10°C and the optimumpH was shifted by 0.7 units to more acidic.     

Introduction of disulfide bonds into Bacillus subtilis neutral protease

The effects of engineered disulfide bonds on autodigestion andthermostability of Bacillus subtilis neutral protease (NP-sub)were studied using site-directed mutagenesis. After modellingstudies two locations that might be capable of forming disulfidebonds, both near previously determined autodigestion sites inNP-sub, were selected for the introduction of cysteines. Analysisof mutant enzymes showed that disulfide bonds were indeed formedin vivo, and that the mutant enzymes were fully active. Theintroduced disulfides did not alter the autodigestion patternof the NP-sub. All mutant NP-subs exhibited decreased thermostability,which, by using reducing agents, was shown to be caused by theintroduction of the cysteines and not by the formation of thedisulfides. Mutants containing one cysteine exhibited intermoleculardisulfide formation at elevated temperatures, which, however,was shown not to be the cause of the decreased thermostability.Combining the present data with literature data, it would seemthat the introduction of disulfide bridges is unsuitable forthe stabilization of proteases. Possible explanations for thisphenomenon are discussed.     

Identification and elimination by site-directed mutagenesis of thermolabile aspartyl bonds in Aspergillus awamori glucoamylase

Both Dative Aspergillus niger glucoamylase and wild-type Aspergillusawamori glucoamylase expressed in Saccharo-myces cerevisiae,which have identical primary structures, undergo hydrolysisat aspartyl bonds at low pH values and elevated temperatures.In native A.niger enzyme the Aspl26–Glyl27 bond was preferentiallycleaved at pH 3.5,while at pH 4.5 cleavage of the Asp257–Pro258and Asp293–Gly294 bonds was dominant. In wild-type A.awamoriglucoamylase, cleavage of the latter was dominant at both pH3.5 and 4.5. Site-directed mutations Aspl26Glu and Glyl27Alain wild-type enzyme decreased specific activities by 60 and30%, respectively, and increased irreversible thermoinactivationrates 3- to 4-fold at pH 4.5. Replacement of Asp257 with Gluand Asp293 with Glu or Gin decreased specific activities by20%, but greatly reduced cleavage of the Asp257–Pro258and Asp293–Gly294 bonds. The Asp257Glu mutant was producedvery slowly and was more thermostable than wild–type glucoamylaseat pH 4.5up to 70°C. Replacement of Asp293 with either Gluor Gln significantly raised protein production and slightlyincreased thermostability at pH 3.5 and 4.5, but not at pH5.