Cumulative stabilizing effects of hydrophobic interactions on the surface of the neutral protease from Bacillus subtilis

Using genetically engineered mutants of the neutral pro-teasefrom Bacillus stearothermophilus (BsteNP), it had been shownthat the surface-exposed structural motif constituted by Phe63embedded in a four amino acid hydrophobic pocket is criticalfor the thermal stability of the thermophilic neutral proteasesfrom Bacilli. To measure the stabilizing contribution of eachhydrophobic interaction taking place between Phe63 and the hydrophobicpocket, we grafted this structural motif in the neutral proteasefrom the mesophile Bacillus subtilis (BsubNP). This was accomplishedby first creating the Thr63Phe mutant of BsubNP and then generatinga series of mutants in which the four amino acids which in thermolysinsurround Phe63 and form the hydrophobic pocket were added oneafter the other. By analysing the thermal stability of eachmutant it was found that the 2°C destabilizing effect ofthe Thr63Phe substitution was completely suppressed by the additionof the four amino acid hydrophobic pocket, each replacementproviding a stabilizing contribution of approxi mately 0.8–1°C.These results are discussed in the light of the peculiar mechanismof thermal inactivation of proteolytic enzymes.     

Introduction of a stabilizing 10 residue {beta}-hairpin in Bacillus subtilis neutral protease

A 10 residue ß-hairpin, which is characteristic ofthermostable Bacillus neutral proteases, was engineered intothe thermolabile neutral protease of Bacillus subtilis. Therecipient enzyme remained fully active after introduction ofthe loop. However, the mutant protein exhibited autocatalyticnicking and a 0.4°C decrease in thermostability. Two additionalpoint mutations designed to improve the interactions betweenthe enzyme surface and the introduced ß-hairpin resultedin reduced nicking and increased thermostability. After theintroduction of both additional mutations in the loopcontainingmutant, nicking was largely prevented and an increase in thermostabilityof 1.1°C was achieved.     

Contribution of the C-terminal amino acid to the stability of Bacillus subtilis neutral protease

The role of the C-terminal Leu300 in maintaining thermal stabilityof the neutral protease of Bacillus subtilis was investigated.From model building studies based on the three dimensional structureof thermolysin, the neutral protease of B.thermoproteolyticus,it was conduded that this residue is located in a hydrophobicpocket composed of residues located in the C-terminal and themiddle domain. To test the hypothesis that Leu300, by contributingto a stabilizing interaction between these domains, is importantfor enzyme stability, several neutral protease mutants wereconstructed and characterized. The thermostability of the enzymewas lowered by deleting Leu300 or by replacing this residueby a smaller (Ala), a polar (Asn) or a sterically unfavourable(He) amino acid. Thermostabiity was increased upon replacingLeu300 by Phe. These results are in agreement with model-buildingstudies. The effects on thermostability observed after mutatingthe corresponding Val318 in the thermostable neutral proteaseof B.stearothermophilus were less pronounced.     

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.     

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.     

The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease

Cavities in the hydrophobic core of the neutral protease ofBacillus stearothermophilus were analyzed using a threedimensionalmodel that was inferred from the crystal structure of thermolysin,the highly homologous neutral protease of B.thermoproteolyticus(85% sequence identity). Site–directed mutagenesis wasused to fill some of these cavities, thereby improving hydrophobicpacking in the protein interior. The mutations had small effectson the thermostability, even after drastic changes, such asLeu284Trp and Met168Trp. The effects on T50, the temperatureat which 50% of the enzyme is irreversibly inactivated in 30min, ranged from 0.0 to +0.4°C. These results can be explainedby assuming that the mutations have positive and negative structuraleffects of approximately the same magnitude. Alternatively,it could be envisaged that the local unfolding steps, whichrender the enzyme susceptible towards autolysis and which arerate limiting in the process of thermal inactivation, are onlyslightly affected by alterations in the hydrophobic core.     

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.     

Crystal structure of the high-alkaline serine protease PB92 from Bacillus alcalophilus

The crystal structure of a serine protease from the alkalophilicstrain Bacillus alcalophilus PB92 has been determined by X-raydiffraction at 1.75 Â resolution. The structure has beensolved by molecular replacement using the atomic model of subtilisinCarlsberg. The model of the PB92 protease has been refined toan R-factor of 14.0% and contains 1882 protein atoms, two calciumions and 188 water molecules. The overall folding of the polypeptide chain closely resembles that of the subtilisins. Furthermore,almost all of the secondary structure elements found in subtilisinCarlsberg are also present in the PB92 protease. The major differencesbetween the two structures are located around the deletion regions(residues 37 and 158–161 in subtilisin Carlsberg) andin two loops which are known to be the most variable parts ofsubtilisin structures. Flexibility of one of these loops (residues126–130 in the PB92 protease) is believed to account forthe inducedfit mechanism of substrate binding.     

Expression in Escherichia coli of a synthetic gene coding for horse heart myoglobin

A gene for expression of horse heart myoglobin in Escherichiacoli has been constructed in one step from long synthetic oligonucleotides.The synthetic gene contains an efficient translation initiationsignal and used codons that are commonly found in E.coli. Uniquerestriction sites are placed throughout the gene. It has beeninserted in a phagemid vector and is expressed from the lacpromoter in E.coli at high efficiency, the soluble heme proteinrepresenting 10% of soluble protein. Two versions of horse heartmyoglobin were produced with aspartic acid or asparagine atresidue 122. Comparison of chromatographic mobilities of thesetwo proteins with authentic horse heart myoglobin identifiedaspartic acid as the correct residue 122. The availability ofthis gene, which is designed to facilitate oligonucleotide mutagenesisor cassette mutagenesis, will allow systematic structure—functionanalysis of horse heart myoglobin.     

Effect of mutations on the dimer stability and the pH optimum of the human foamy virus protease

To explore the role of residues being close to the catalytic aspartates in the higher pH optimum and in the lower dimer stability of human foamy virus (HFV) protease (PR) in comparison with human immunodeficiency virus type 1 (HIV-1) protease, single (Q8R, H22L, S25T, T28D) and double (Q8R-T28D, H22L-T28D) mutants were created based on sequence alignments and on the molecular model of HFV PR. The wild-type and mutant enzymes were expressed in fusion with maltose binding protein in Escherichia coli and the fusion proteins were purified by affinity chromatography. Specificity constant of most mutants was lower, but the value of Q8R-T28D double mutant enzyme was higher than that of the wild-type HFV PR. Furthermore, urea denaturation at two pH values and pH optimum values showed an increased stability and pH optimum for most mutants. These results suggest that the mutated residues may not be responsible for the higher pH optimum of HFV PR, but they may contribute to the lower dimer stability as compared with that of HIV-1 PR.     

Site-directed mutagenesis to fine-tune enzyme specificity

We have used a combination of a genetic selection and oligonucleotide-directedmutagenesis to introduce a series of amino add replacementsfor a single residue into Escherichia coliglutaminyl-tRNA synthetase.The mutant enzymes mischarge supFtRNA^Tyr, with glutamine, tovarying degrees depending on the polarity of the side chainintroduced but apparently not depending on the size or shapeof the side chain. These results indicate that repulsive charge-chargeinteractions may be important for specific recognition of nucleicacids by proteins and illustrate how a mutant, derived fromgenetic selection, may be further modified in activity by oligonucleotide-directedmutagenesis.     

The effect of engineering surface loops on the thermal stability of Bacillus subtilis neutral protease

Using genetic techniques the contribution of surface loops tothe thermal stability of Bacillus subtUis neutral protease (NPsub)wasstudied. Mutations were designed to make the surface of NP-submore similar to the surface of more thermostable neutral proteasessuch as thermolysin (TLN). The mutations included the replacementof an irregular loop by a shorter variant and the introductionof a ten–residue (3– hairpin. In general, thesedrastic mutations had little effect on the production and activityof NP–sub, indicating the feasibility of major structuralrearrangements at the surface of proteins. In the most stablemutant, exhibiting an increase in thermal stability of 1.1°C, 10% of the surface of NP–sub was modified. Several NP–subvariants carrying multiple mutations were constructed. Non–additiveeffects on thermal stability were observed, which were interpretedon the basis of a model for thermal inactivation, that emphasizesthe importance of local unfolding processes for thermal stability     

Analysis of structural determinants of the stability of thermolysinlike proteases by molecular modelling and site-directed mutagenesis

The thermolysin-like protease (TLP) produced by Bacillus stearothermophilusCU21 (TLP-ste) differs at 43 positions from the more thermallystable thermolysin (containing 316 residues in total). Of thesedifferences, 26 were analysed by studying the effect of replacingresidues in TLP-ste by the corresponding residues in thermolysin.Several stabilizing mutations were identified but, remarkably,considerable destabilizing mutational effects were also found.A Tyr-rich three residue insertion in TLP-ste (the only deletional/insertionaldifference between the two enzymes) appeared to make an importantcontribution to the stability of the enzyme. Mutations withlarge effects on stability were all localized in the ßpleatedN-terminal domain of TLP-ste, confirming observations that thisdomain has a lower intrinsic stability than the largely -helicalC-terminal domain. Rigidifying mutations such as Gly58 Alaand Ala69 Pro were among the most stabilizing ones. Apart fromthis observation, the analyses did not reveal general rulesfor stabilizing proteins. Instead, the results highlight theimportance of context in evaluating the stability effects ofmutations.     

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.     

Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocket

Serine endoproteases such as trypsins and subtilisins are knownto have an extended substrate binding region that interactswith residues P6 to P3' of a substrate. In order to investigatethe structural and functional effects of replacing residuesat the S4 substrate binding pocket, the serine protease fromthe alkalophilic Bacillus strain PB92, which shows homologywith the subtilisins, was mutated at positions 102 and 126–128.Substitution of Val102 by Trp results in a 12–fold increasein activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide(sAAPFpNA). An X-ray structure analysis of the V102W mutantshows that the Trp side chain occupies a hydrophobic pocketat the surface of the molecule leaving a narrow crevice forthe P4 residue of a substrate. Better binding of sAAPFpNA bythe mutant compared with the wild type protein as indicatedby the kinetic data might be due to the hydrophobic interactionof Ala P4 of the substrate with the introduced Trp102 side chain.The observed difference in binding of sAAPFpNA by protease PB92and thermitase, both of which possess a Trp at position 102,is probably related to the amino acid substitutions at positions105 and 126 (in the protease PB92 numbering).Kinetic data forthe variants obtained by random mutation of residues Serl26,Prol27 and Serl28 reveal that the activity towards sAAPFpNAincreases when a hydrophobic residue is introduced at position126. An X-ray diffraction analysis was carried out for the threeprotease PB92 mutants which have residues Serl26-Prol27-Serl28replaced by Met-Ala-Gly(‘MAG’ mutant), Phe-Gln-Ser(‘FQS’ mutant) and Asn-Ser-Ala (‘NSA’mutant). Met 126 and Phel26 in the crystal structures of thecorresponding mutants are fixed in the same hydrophobic environmentas Trp102 in the V102W mutant.In contrast, Asnl26 in the ‘NSA’mutant is completely disordered in both crystal forms for whichthe structure has been determined. According to our kineticmeasurements none of the mutants with Met, Phe, Leu or Val atposition 126 binds sAAPFpNA better than the wild type enzyme.Resultsof the site-directed mutagenesis at position 127 imply thatpossible interaction of this residue with a substrate has almostno effect on activity towards sAAPFpNA and casein.     

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.     

Comparative modelling and analysis of amino acid substitutions suggests that the family of pregnancy-associated glycoproteins includes both active and inactive aspartic proteinases

The pregnancy-associated glycoproteins (PAGs) are secretoryproducts synthesized by the outer epithelial cell layer (chorion)of the placentas of various ungulate species. The amino acidsequences of eight PAGs have been inferred from cloned cDNAof cattle and sheep, as well as of the non-ruminant pig andhorse. We compare the PAG sequences and present results of thethree-dimensional models of boPAG-1 and ovPAG-1 that were constructedon the basis of the crystal structures of homologous porcinepepsin and bovine chymosin using a rule-based comparative modellingapproach. Further, we compare peptide binding subsites definedby interactions with pepstatin and a decapeptide inhibitor (CH-66)modelled on the basis of crystal structures of other asparticproteinases. We have extended our analysis of the peptide bindingsubsites to the other PAG molecules of known sequence by aligningthe PAG sequences to the structural template derived from thepepsin family and by making use of the three-dimensional modelsof the boPAG-1 and ovPAG-1. The residues that are likely toaffect peptide binding in the boPAG-1, ovPAG-1 and other PAGmolecules have been identified. Sequence comparisons revealthat all PAG molecules may have evolved from a pepsin-like progenitormolecule with the equine PAG most closely related to the pepsins.The presence of substitutions at the S₁ and other subsites relativeto pepsin make it unlikely that either bovine, ovine or theporcine PAG-1 have catalytic activity. Only two of the eightPAGs examined (porcine PAG-2 and equine PAG-1) retain featuresof active aspartic proteinases with pepsin-like activity. Ourresults indicate that in the PAGs so far characterized the peptidebinding specificities differ significantly from each other andfrom pepsin, despite their high sequence identities. Analysisof the various peptide binding subsites demonstrates why bothbovine and ovine PAG-1 are capable of binding pepstatin. Thestrong negative charge in the binding cleft of boPAG-1 and ovPAG-1indicates a preference for lysine- or arginine-rich peptides.PAGs represent a family where the possible peptide binding functionmay be retained through their binding specificities, but wherethe catalytic activity may be lost in some cases, such as theboPAG-1, ovPAG-1 and the poPAG-1.     

Improved mutants from directed evolution are biased to orthologous substitutions

We have engineered human epidermal growth factor (EGF) by directed evolution through yeast surface display for significantly enhanced affinity for the EGF receptor (EGFR). Statistical analysis of improved EGF mutants isolated from randomly mutated yeast-displayed libraries indicates that mutations are biased towards substitutions at positions exhibiting significant phylogenetic variation. In particular, mutations in high-affinity EGF mutants are statistically biased towards residues found in orthologous EGF species. This same trend was also observed with other proteins engineered through directed evolution in our laboratory (EGFR, interleukin-2) and in a meta-analysis of reported results for engineered subtilisin. By contrast, reported loss-of-function mutations in EGF were biased towards highly conserved positions. Based on these findings, orthologous mutations were introduced into a yeast-displayed EGF library by a process we term shotgun ortholog scanning mutagenesis (SOSM). EGF mutants with a high frequency of the introduced ortholog mutations were isolated through screening the library for enhanced binding affinity to soluble EGFR ectodomain. These mutants possess a 30-fold increase in binding affinity over wild-type EGF to EGFR-transfected fibroblasts and are among the highest affinity EGF proteins to be engineered to date. Collectively, our findings highlight a general approach for harnessing information present in phylogenetic variability to create useful genetic diversity for directed evolution. Our SOSM method exploits the benefits of library diversity obtained through complementary methods of error-prone PCR and DNA shuffling, while circumventing the need for acquisition of multiple genes for family or synthetic shuffling.     

Contribution of amino acid substitutions at two different interior positions to the conformational stability of human lysozyme

To elucidate correlative relationships between structural changeand thermodynamic stability in proteins, a series of mutanthuman lysozymes modified at two buried positions (Ile56 andIle59) were examined. Their thermodynamic parameters of denaturationand crystal structures were studied by calorimetry and X-raycrystallography. The mutants at positions 56 and 59 exhibiteddifferent responses to a series of amino acid substitutions.The changes in stability due to substitutions showed a linearcorrelation with changes in hydrophobicity of substituted residues,having different slopes at each mutation site. However, thestability of each mutant was found to be represented by a uniqueequation involving physical properties calculated from mutantstructures. By fitting present and previous stability data formutant human lysozymes substituted at various positions to theequation, the magnitudes of the hydrophobicity of a carbon atomand the hydrophobicity of nitrogen and neutral oxygen atomswere found to be 0.178 and –0.013 kJ/mol.Ų, respectively.It was also found that the contribution of a hydrogen bond witha length of 3.0 Å to protein stability was 5.1 kJ/moland the entropy loss of newly introduction of a water moleculeswas 7.8 kJ/mol.     

EcoRV-T94V: a mutant restriction endonuclease with an altered substrate specificity towards modified oligodexynucleotides

Synthetic oligodeoxynucleotides with single methyl phosphonate(mp) substitutions were used for an analysis of the contributionof phosphate contacts to the recognition of the cleavage siteby the restriction endonuclease EcoRV. Only in the last positionwithin the recognition sequence, is the methyl phosphonate substitutiontolerated by the enzyme. The wild-type enzyme cleaves the S_Pdiastereomer of the oligodeoxynucleotide GACGATAT_mpCGTC andthe unmodified sequence with equal rates, whereas the R_P diastereomeris cleaved much more slowly. Inspection of the crystal structureof an EcoRV–DNA complex revealed that the non-bridgingoxygen atoms of the phosphodiester bond between the T and Cbases are in hydrogen bonding distance of the hydroxyl groupof the amino acid Thr94. We therefore tried to engineer a variantof EcoRV that would prefer a methyl phosphonate linkage overa normal phosphodiester bond and produced mutants with aminoacid exchanges at position 94. One of them, Thr94Val, showsa dramatically reduced activity towards the unmodified DNA anddoes not accept the R_p diastereomer, but cleaves the S_P diastereomerwith the same rate as wild-type EcoRV. Its selectivity, i.e.the ratio of cleavage rates determined for the unmodified andmodified substrates, differs by three orders of magnitude fromthat of the wild-type enzyme.