Interchain cysteine bridges control entry of progesterone to the central cavity of the uteroglobin dimer

The progesterone–binding protein uteroglobin has beenexpressed in Escherichia coli in an unfused, soluble form. likemature uteroglobin from rabbit endometrium (UG), the E.coliproduceduteroglobin (UG1) dimerizes in vitro, forms an antiparalleldimer with Cys3–Cys69' and Cys69–Cys3' disulfidebonds and binds progesterone under reducing conditions. In orderto analyze the dimerization and the reduction dependence ofprogesterone binding in more detail, we separately replacedcysteine 3 and cysteine 69 by serines. Under reducing conditions,both uteroglobin variants (UGl–3Ser and UGl–69Ser)bind progesterone with the same affinity as the wild–typesuggesting that both cysteine residues are not directly involvedin progesterone binding. In contrast to the wild–typeprotein, both cysteine variants also bind progesterone withhigh affinity in the absence of reducing agents. In addition,UGl-3Ser and UGl-69Ser both form covalently linked homodimers.Thus, unnatural Cys69–69' and Cys3–3' disulfidebonds exist in UG1–3Ser and UG1–69Ser, respectively.These data together with computer models based on X-ray diffractiondata strongly support the idea that progesterone reaches itsbinding site located in an internal hydrophobic cavity via ahydrophobic tunnel along helices 1 and 4. Under non–reducingconditions the tunnel is closed by two disulfide bridges (Cys3–Cys69'(and Cys69–Cys3') that lie in the most flexible regionof the dimer. Reduction or replacement of a cysteine residueenables conformational changes that open the channel allowingprogesterone to enter.     

Selective cysteine <- serine replacements in p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens allow the unambiguous assignment of Cys211 as the site of modification by spin-labeled p-chloromercuribenzoate

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens containsfive sulfhydryl groups per subunit. Cysteine serine replacementsshow that the thiols are not essential for catalysis. The increaseddissociation constant for FAD in mutant Cysl58Ser suggests thatCysl58 is important for the solvation of the pyrophosphate moietyof the prosthetic group. Wild-type p-hydroxybenzoate hydroxylaseis rapidly inactivated by mercurial compounds. Inactivationby a spin-labeled derivative of p-chloromercuribenzoate is fullyabolished in mutant Cys211Ser. Incorporation of the spin labelin the other Cys Ser mutants strongly impairs substrate bindingwithout affecting the catalytic properties of the FAD. The resultsare discussed with respect to previous tentative assignmentsfrom chemical modification studies and in light of the 3-D structureof the enzyme-substrate complex.     

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.     

Sulfolobus solfataricus protein disulphide oxidoreductase: insight into the roles of its redox sites

Sulfolobus solfataricus protein disulphide oxidoreductase (SsPDO)contains three disulphide bridges linking residues C⁴¹XXC⁴⁴,C¹⁵⁵XXC¹⁵⁸, C¹⁷³XXXXC¹⁷⁸. To get information on the role playedby these cross-links in determining the structural and functionalproperties of the protein, we performed site-directed mutagenesison Cys residues and investigated the changes in folding, stabilityand functional features of the mutants and analysed the resultswith computational analysis. The reductase activity of SsPDOand its mutants was evaluated by insulin and thioredoxin reductaseassays also coupled with peroxiredoxin Bcp1 of S. solfataricus.The three-dimensional model of SsPDO was constructed and correlatedwith circular dichroism data and functional results. Biochemicalanalysis indicated a key function for the redox site constitutedby Cys155 and Cys158. To discriminate between the role of thetwo cysteine residues, each cysteine was mutagenised and thebehaviour of the single mutants was investigated elucidatingthe basis of the electron-shuffling mechanism for SsPDO. Finally,cysteine pK values were calculated and the accessible surfacefor the cysteine side chains in the reduced form was measured,showing higher reactivity and solvent exposure for Cys155.     

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.     

Expression of deletion constructs of bovine {beta}-1, 4-galactosyltransferase in Escherichia coli: importance of Cysl34 for its activity

Bovine ß-1, 4-galactosyltransferase (ß-1,4-GT; EC 2.4.1.90 [EC] ) belongs to the glycosyltransferase familyand as such shares a general topology: an N-terminal cytoplasmictail, a signal anchor followed by a stem region and a catalyticdomain at the C-tenninal end of the protein. cDNA constructsof the N-terminal deleted forms of ß-1, 4-GT wereprepared in pGEX-2T vector and expressed in E.coli as glutathione-S-transferase(GST) fusion proteins. Recombinant proteins accumulated withininclusion bodies as insoluble aggregates that were solubilizedin 5 M guanidine HCl and required an ‘oxido-shuffling’reagent for regeneration of the enzyme activity. The recombinant(ß-1, 4-GT, devoid of the GST domain, has 30–85%of the sp. act. of bovine milk ß-1, 4-GT with apparentK_ms for N-acetylglucosamine and UDP-galactose similar to thoseof milk enzyme. Deletion analysesshow that both (ß-1,4-GT and lactose synthetase activities remain intact even inthe absence of the first 129 residues (pGT-dl29). The activitiesare lost when either deletions extend up to residue 142 (pGT-dl42)or Cysl34 is mutatedto Ser (pGT-dl29C134S). These results suggestthat the formation of a disulfide bond involving Cysl34 holdsthe protein in a conformation that is required for enzymaticactivity.     

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     

Removal of an inter-domain hydrogen bond through site-directed mutagenesis: role of serine 176 in the mechanism of papain

A mutant of papain, where an inter-domain hydrogen bond betweenthe side chain hydroxyl group of a serine residue at position176 and the side chain carbonyl oxygen of a glutamine residueat position 19 has been removed by site-directed mutagenesis,has been produced and characterized kinetically. The mutationof Ser176 to an alanine has only a small effect on the kineticparameters, the k_cat/K_m for hydrolysis of CBZ-Phe-Arg-MCA bythe Serl76Ala enzyme being of 8.1 x 10⁴ /M/s compared with 1.2x 10⁵ /M/s for papain. Serine 176 is therefore not essentialfor the catalytic functioning of papain, even though this residueis conserved in all cysteine proteases sequenced. The pH-activityprofiles were shown to be narrower in the mutant enzyme by upto 1 pH unit at high ionic strength. This result is interpretedto indicate that replacing Ser 176 by an alanine destabilizesthe thiolate—imidazolium form of the catalytic site Cys25-Hisl59residues of papain. Possible explanations for that effect aregiven and the role of a serine residue at position 176 in papainis discussed.     

Effect of Glu-143 and His-231 substitutions on the catalytic activity and secretion of Bacillus subtilis neutral protease

On the basis of the homology with the Bacillus thermoproteolyticuszinc endopeptidase thermotysin, we hypothesized that Glu-143and His-231 are the key residues for the catalytic activityof the Bacillus subtilis neutral protease. To test this possibilityby site-directed mutagenesis, we substituted these two residueswith Ala, Ser, Trp and Arg, and Leu, Val and Cys respectively.All these substitutions dramatically affected the amount ofsecreted mutant proteins, as determined by immunological methods,and their catalytic activities. No appreciable secretion wasobserved with the three Glu mutants Trp, Ser and Arg, whereasthe Glu–Ala mutant enzyme was secreted at a level of afew hundred micrograms per litre of culture. The His mutantswere all secreted at higher levels (in the order of a few milligramsper litre) and their residual catalytic activity could be determinedusing Z-Ala-Leu-Ala as substrate. Our results confirm the keyrole played by Glu-143 and His-231 in catalysis and moreoversuggest the existence of a relationship between the catalyticactivity of the enzyme and the extent of its secretion. In thiscontext, we present data suggesting an autoproteolytic mechanismof cleavage of the precursor form of the enzyme, analogous tothe one previously reported for the B.subtilis subtilisin.     

Analysis of the structure of Pseudomonas glumae lipase

The lipase produced by Pseudomonas glumae is monomeric in thecrystalline state and has a serine protease-like catalytic triad;Ser87-His285-Asp263. The largest domain of the protein resemblesclosely a subset of the frequently observed /ß-hydrolasefold and contains a well-defined calcium site. This paper describesstructural analysis of this protein, focusing on (i) structuralcomparison with the lipase from Geotrichum candidum, (ii) theprobable nature of the conformational change involved in substratebinding and (iii) structural variations amongst the family ofPseudomonas Upases. This analysis reveals similarities betweenP.glumae lipase and G.candidum lipase involving secondary structuralelements of the hydrolase core and the loops carrying the catalyticserine and histidine residues. A possible functional equivalencehas also been identified between parts of the two moleculesthought to be involved in a confonmational change. In addition,determination of the structure of P.glumae lipase has allowedrationalization of previously reported protein engineering experiments,which succeeded in improving the stability of the enzyme withrespect to proteolysis.     

Hydrophobicity engineering of cholera toxin A1 subunit in the strong adjuvant fusion protein CTA1-DD

Protein engineering of the cholera toxin A1 subunit (CTA1) fusedto a dimer of the Ig-binding D-region of Staphylococcus aureusprotein A (DD) was employed to investigate the effect of specificamino acid changes on solubility, stability, enzymatic activityand capacity to act as an adjuvant in vivo. A series of CTA1-DDanalogues were selected by a rational modeling approach, inwhich surface-exposed hydrophobic amino acids of CTA1 were exchangedfor hydrophilic counterparts modeled for best structural fit.Of six different mutants initially produced, two analogues,CTA1Phe132Ser-DD and CTA1Pro185Gln-DD, were demonstrated tohave 50 and 70% increased solubility, respectively, at neutralpH. The double mutant CTA1Phe132Ser/Pro185Gln-DD was at leastthreefold more soluble, demonstrating an additive effect ofthe two mutations. Only the Phe132Ser analogue retained fullbiological activity and stability compared with the native CTA1-DDfusion protein. Two mutants, Pro185Gln and Phe31His mutations,exhibited unaltered ADP-ribosyltransferase activity in vitro,but demonstrated markedly reduced adjuvant function. Since thePro185 and Phe31 amino acids are located in close vicinity onthe distal side of the molecule relative to the enzymaticallyactive cleft, it is conceivable that this region is involvedin mediating a biological function, separate from the enzymaticactivity but intrinsic to the adjuvant activity of CTA1.     

Stabilization of the neutral protease of Bacillus stearothermophilus by removal of a buried water molecule

Using site-directed mutagenesis, Ala166 in the neutral proteaseof Bacillus stearothermophilus was changed into Ser. Model buildingand molecular dynamics simulations of the mutant enzyme indicatedthat the Ser hydroxyl group fits well in a cavity which containsa water molecule in the wild-type enzyme. The Alal66 - Ser mutationwas expected to exert a stabilizing effect because of the gainin entropy resulting from the release of a water molecule fromthe folded protein to the solvent. In addition, the hydrogen-bondingnetwork around residue 166 was improved upon the mutation. Asa result of this mutation the thermostability of the neutralprotease was increased by 1.2 ± 0.1°C.     

Homology modeling of a heme protein, lignin peroxidase, from the crystal structure of cytochrome c peroxidase

A 3-dimensional model of lignin peroxidase (LiP) was constructedbased on its sequence homology with other peroxidases, particularlycytochrome c peroxidase, the only protein with a known crystalstructure in the peroxidase family. The construction of initialconformations of insertions and deletions was assisted by secondarystructure predictions, amphipathic helix predictions, and considerationof the specific protein environment. A succession of moleculardynamics simulations of these regions with surrounding residuesas constraints were carried out to relax the bond lengths andangles. Full protein molecular dynamics simulations with explicitconsideration of bound waters were performed to relax the geometryand to identify dynamically flexible regions of the successivemodels for further refinement. Among the important functionallyrelevant structural features predicted are: (i) four disulfidebonds are predicted to be formed between Cys3 and Cys15, Cys14and Cys285, Cys34 and Cys120 and Cys249 and Cys317; (ii) a glycosyla-tionsite, Asn257, was located on the surface; (iii) Glu40 was predictedto form a salt bridge with Arg43 on the distal side of the hemeand was considered as a possible origin for the pH dependenceof compound I formation; and (iv) two candidate substrate bindingsites with a cluster of surface aromatic residues and flexiblebackbones were found in the refined model, consistent with thenature of known substrates of LiP. Based on these predictedstructural features of the model, further theoretical and experimentalstudies are proposed to continue to elucidate the structureand function of LiP.     

Inactivation of Staphylococcus hyicus lipase by hexadecylsulfonyl fluoride: evidence for an active site serine

The Staphylococcus hyicus lipase is an acyl hydrolase with broadsubstrate specificity including neutral glycerides and phospholipids.To obtain further insight into the mechanism of action of thisenzyme, we tested several sulfonyl fluorides as active site-directedinhibitors. The enzyme is resistant to the well-known serineprotease/esterase inhibitor phenylmethanesulfonyl fluoride (PMSF),but is rapidly inactivated by hexadecylsulfonyl fluoride. Thekinetics of inactivation were studied in Triton X-100 micelles.Inactivation is fast and the rate of inactivation is constantover the pH range where this lipase is active. Metal ions likeCa²⁺ and Sr²⁺ do not appreciably influence the rate of inactivation,although the enzymatic activity is significantly increased,suggesting a structural role for these ions. The S.hyicus lipasecontains a consensus sequence G-H/Y-S-X-G. Substitution by site-directedmutagenesis of this serine (Ser369) by a cysteine resulted ina mutant with only 0.2% residual activity. The activity of thismutant could not be inhibited with water-soluble sulfhydrylreagents either in the presence or absence of Triton X-100 micelles.In the presence of Triton X-100 micelles, inactivation of themutant occurred with 4-nitrophenylhexadecyl disulfide (t_1/2= 125 min) while the wild-type enzyme does not react at all.We conclude that Ser369 is the active site residue and thatin water this residue is inaccessible. Only after interfacialactivation Ser369 (or Cys369) becomes exposed and reacts withirreversible inhibitors.     

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.     

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.     

Mutagenesis and kinetic analysis of the active site Glu177 of ricin A-chain

Ricin A-chain (RTA) is an N-glycosidase which removes a specificadenine residue from the large rRNA of eukaryotic ribosomes.As a consequence, the ribosome is inactivated and protein synthesisis inhibited leading to cell death. This report describes theeffects on enzyme activity of specific mutations of the conservedactive site Glu177. The activity of mutant proteins was initiallyscreened using an in vitro translation system. It was foundthat mutagenesis of Glu177 to Lys led to an apparent total inactivationof the enzyme, Glu177 to Ala had a small effect on activity,whereas the conservative Glu177 to Asp mutation had a significanteffect. The properties of Glu177 to Asp were investigated moreclosely. Mutant protein was purified from an Escherichia coliexpression system and kinetic analysis of the depurination activityassessed using salt-washed yeast ribosomes. It was shown thatthe K, of the mutant protein was unchanged when compared todata of wild type RTA; however, the k_cat was significantly decreased(49-fold compared to wild type RTA). This suggests that Glu177plays a predominant role in the rate-limiting step of the enzymaticmechanism and not in substrate binding. These data are discussedin relation to other reports of ricin Glu177 substitutions.     

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.     

Solution conformation of endothelin determined by means of 1H-NMR spectroscopy and distance geometry calculations

The structure of endothelin-1 (ET-1), an endothelial cell-derivedpeptide with vasoconstricting activity, was determined in anaqueous solution by means of a combination of NMR and distancegeometry calculations. The resulting structure is characterizedby an -helical conformation in the sequence region, Lys9-Cys15.Furthermore, an extended structure and a turn structure existin the Cys1-Ser4 and Ser5-Asp8 regions respectively, and nopreferred conformation was found for the C-terminal part ofthe peptide which was not uniquely constrained by the NMR data.These structural elements, the -helical structure in the sequenceportion, Cys-X-X-X-Cys, and the extended structure in Cys-X-Cys,are homologous to those found commonly in several neurotoxicpeptides.