Stability effects associated with the introduction of a partial and a complete Ca2+-binding site into human lysozyme

Two mutants of human lysozyme were synthesized. Mutant A92D,in which Ala92 was substituted by Asp, contains a partial Ca²⁺-bindingsite and mutant M4, in which Ala83, Gm86, Asn88 and Ala92 werereplaced by Lys, Asp, Asp and Asp respectively, contains thecomplete Ca -binding site of bovine a-lactalbumin. The Ca²⁺-bindingconstants of wild type human lysozyme and of mutants A92D andM4, measured at 25C and pH 7.5, were 2(1) x 102 M"1, 8(2)x l^M"1 and 9(0.5) x 10* M"1 respectively. Information gatheredfrom mkrocalorimetrk and CD spectro-scopic measurements indicatesthat the conformational changes of the M4 mutant lysozyme, inducedby Ca²⁺ binding, are smaller than those observed for bovinea-lactalbumin and for the Ca²⁺-binding equine lysozyme. At pH4.5, the thermostability of both the apo and Ca²⁺ forms of theA92D human was decreased in comparison with that of native humanlysozyme. In particular, within the apo form of this mutantan a-helix-containing sequence was destabilized. In contrast,at the same pH the thermostability of the apo and Ca²⁺ formsof the M4 mutant lysozyme was increased. The e-ammonium groupof the Lys83 side chain is assumed to be responsible for thestabilization of the apo form of this mutant.     

3-D structure of the D153G mutant of Escherichia coli alkaline phosphatase: an enzyme with weaker magnesium binding and increased catalytic activity

The substitution of aspartate at position 153 in Escherichiacoli alkaline phosphatase by glycine results in a mutant enzymewith 5-fold higher catalytic activity (k_cat but no change inKm at pH 8.0 in 50 mM Tris-HCl. The increased k_cat is achievedby a faster release of the phosphate product as a result ofthe lower phosphate affinity. The mutation also affects Mg²⁺binding, resulting in an enzyme with lower metal affinity. The3-D X-ray structure of the D153G mutant has been refined at2.5 Å to a crystallographic Rfactor of 16.2%. An analysisof this structure has revealed that the decreased phosphateaffinity is caused by an apparent increase in flexibility ofthe guanidinium side chain of Argl66 involved in phosphate binding.The mutation of Aspl53 to Gly also affects the position of thewater ligands of Mg²⁺, and the loop Glnl52–Thrl55 is shiftedby 0.3 Å away from the active site. The weaker Mg²⁺ bindingof the mutant compared with the wild type is caused by an alteredcoordination sphere in the proximity of the Mg²⁺ ion, and alsoby the loss of an electrostatic interaction (Mg²⁺.COO^-Aspl53)in the mutant Its ligands W454 and W455 and hydroxyl of Thrl55,involved in the octahedral coordination of the Mg²⁺ ion, arefurther apart in the mutant compared with the wild-type     

Dihydrofolate reductase: control of the mode of substrate binding by aspartate 26

The complex of Lactobacillus casei dihydrofolate reductase withthe substrate folate and the coenzyme NADP^* has been shown toexist in solution as a mixture of three slowly interconvertingconformations whose proportions are pH-dependent and which differin the orientation of the pteridine ring of the substrate inthe binding site. The Asp26 – Asn mutant of L. casei dihydrofolatereductase has been prepared by oligonucleotide-directed mutagenesisand studied by one-and two-dimensional ¹H-NMR spectroscopy.NMR studies of the mutant enzyme–folate–NADP^* complexshow that this exists to > 90% in a single conformation overthe pH^* range 5–7.1. The single conformation observedcorresponds to conformation I (the ‘methotrexate-like’conformation) of the wild-type enzyme–folate–NADP^*complex. These observations demonstrate that Asp26 is the ionizablegroup controlling the pH-dependence of the conformational equilibriumseen in the wild-type enzyme.     

Ligand requirements for Ca2+ binding to EGF-like domains

Site-specific mutagenesis studies of the first epidermal growthfactor-like (EGF-like) domain of human clotting factor IX suggestthat the calcium-binding site present in this domain (dissociationconstant K_d=1.8 mM at pH 7.5 and ionic strength I=0.15) involvedthe carboxylate residues Asp47, Asp49 and Asp64. To furthercharacterize the ligands required for calcium binding to EGF-likedomains, two new mutations, Asp47 - Asn and Asp49 - Asn, wereintroduced into the domain by peptide synthesis. ¹H-NMR spectroscopywas used to obtain the dissociation constants for calcium bindingto these mutations. Calcium binding to the Asp49- Asn modifieddomain is only mildly affected (K_d=6 mM, I=0.15), whereas bindingto the Asp47- Asn modified domain is severely reduced (K_d=42mM, I=0.15). From these data, it is proposed that the anionicoxygen atoms of the side chains of residues 47 and 64 are essentialfor calcium binding, whereas the side chain ligand for calciumat residue 49 can be a carboxyamide oxygen. As a control, theintroduction of the modification Glu78- Asp in a region of thedomain not believed to be involved in calcium binding had verylittle effect on the K_d for calcium (K_d=2.6 mM, I=0.15). Finally,the effect of an Asp47- Gly substitution found in the naturalhaemophilia B mutant, factor IX_Alabama, was investigated. Thispeptide has a markedly reduced affinity for calcium (K_d=37 mM,I=0.15), suggesting that the defect in factor IX_Alabama is dueto impaired calcium binding to its first EGF-like domain.     

Free energy simulations of the HyHEL-10/HEL antibody-antigen complex

Free energy simulations are reported for the N31^L-D mutation,both in the HyHEL-10-HEL antibody-lysozyme complex and in theunliganded antibody, using the thermo-dynamic-cycle perturbationmethod. The present study suggests that the mutation would changethe free energy of binding of the complex by –5.6 kcal/mol(unrestrained free energy simulations), by –0.5 kcal/mol(free energy simulations with a restrained backbone) and by1.8 kcal/ mol (Poisson-Boltzmann calculations, which also usea restrained geometry model). A detailed structural analysishelps in estimating the contributions from various residuesand regions of the system. Enhanced recognition of HEL by themutant HyHEL-10 would arise from the combination of thermodynamicallymore favorable conformational changes of the CDR loops uponassociation and subsequent charge pairing with Lys96 in theantigen.     

An unequivocal example of cysteine proteinase activity affected by multiple electrostatic interactions

The role of electrostatic interactions between the ionizableAsp158 and the active site thiolate-imidazolium ion pair ofsome cysteine proteinases has been the subject of controversyfor some time. This study reports the expression of wild typeprocaricain and Asp158Glu, Asp158Asn and Asp158Ala mutants fromEscherichia coli. Purification of autocatalytically maturedenzymes yielded sufficient fully active material for pH (k_cat/K_m)profiles to be obtained. Use of both uncharged and charged substratesallowed the effects of different reactive enzyme species tobe separated from the complications of electrostatic effectsbetween enzyme and substrate. At least three ionizations aredetectable in the acid limb of wild type caricain and the Gluand Asn mutants. Only two pK_a, values, however, are detectablein the acid limb using the Ala mutant. Comparison of pH activityprofiles shows that whilst an ionizable residue at position158 is not essential for the formation of the thiolate-imidazoliumion pair, it does form a substantial part of the electrostaticfield responsible for increased catalytic competence. Changingthe position of this ionizable group in any way reduces activity.Complete removal of the charged group reduces catalytic competenceeven further. This work indicates that hydronations distantto the active site are contributing to the electrostatic effectsleading to multiple active ionization states of the enzyme.     

Extrapolation to water of kinetic and equilibrium data for the unfolding of barnase in urea solutions

Assumptions about the dependence of protein unfolding on theconcentration of urea have been examined by an extensive surveyof the equilibrium unfolding of barnase and many of its mutantsmeasured by urea denaturation and differential scanning calorimetry.The free energy of equilibrium unfolding and the activationenergy for the kinetics of unfolding of proteins are generallyassumed to change linearly with [urea]. A slight downward curvatureis detected, however, in plots of highly precise measurementsof logjtu versus [urea] (where k_u is the observed rate constantfor the unfolding of barnase). The data fit the equation logkk_u= logk_u^H₂O* + mk_u^*.[urea] – 0.014[urea]², where mk_u^*is a variable which depends on the mutation. The constant 0.014 was measured directly on four destabilized mutants and wildtype, and was also determined from a global analysis of data from>60 mutants of barnase. Any equivalent deviations from linearityin the equilibrium unfolding are small and in the same region,as determined from measurements on 166 mutants. The free energyof unfolding of barnase, G_U–F, appears significantly largerby 1.6 kcal mol^–1 when measured by calorimetry than whendetermined by urea denaturation. However, the changes in G_U–Fon mutation, G_U–F, determined by calorimetry and by ureadenaturation are identical. We show analytically how, hi general,the curvature in plots of activation or equilibrium energiesagainst [denaturant] should not affect the changes of thesevalues on mutation provided measurements are made over the sameconcentration ranges of denaturant and the curvature is independentof mutation.     

Shifting the optimal pH of activity for a laccase from the fungus Trametes versicolor by structure-based mutagenesis

Laccases are oxidizing enzymes of interest because of their potential environmental and industrial applications. We performed site-directed mutagenesis of a laccase produced by Trametes versicolor in order to improve its catalytic properties. Considering a strong interaction of the Asp residue in position 206 with the substrate xylidine, we replaced it with Glu, Ala or Asn, expressed the mutant enzymes in the yeast Yarrowia lipolytica and assayed the transformation of phenolic and non-phenolic substrates. The transformation rates remain within the same range whatever the mutation of the laccase and the type of substrate: at most a 3-fold factor increase was obtained for k(cat) between the wild-type and the most efficient mutant Asp206Ala with 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic) acid as a substrate. Nevertheless, the Asn mutation led to a significant shift of the pH (DeltapH = 1.4) for optimal activity against 2,6-dimethoxyphenol. This study also provides a new insight into the binding of the reducing substrate into the active T1 site and induced modifications in catalytic properties of the enzyme.     

Secretion of recombinant human IgE-Fc by mammalian cells and biological activity of glycosylation site mutants

We have constructed an expression vector that leads to secretionof the whole Fc of human immunoglobulin E (hIgE-Fc) from mammaliancells at levels up to 100 mg/l of culture. Two surface glycosylationsites at Asn265 and Asn371 have been changed to glutamine, toobtain a more homogeneous preparation of hIgE-Fc for structuralstudies. Comparison of wild-type and mutant products revealedthat Asn371 is rarely glycosylated in Chinese hamster ovarycells. Both the double mutant and wild-type hIgEFc bind to thehigh-affinity IgE receptor, FcRI, with about the same affinityas myeloma IgE (K_a in the range 10¹⁰–10¹¹ M^–1),and were able to sensitize isolated human basophils for anti-IgEtriggering of histamine release. However, only the double mutanthIgE-Fc approached the affinity of myeloma IgE for the low-affinityreceptor, FcRII (K_a = 7.3x10⁷ M^–1), whereas the wild-type hIgE-Fc bound with a 10-fold lower affinity (K_a = 4.1x10⁶M^–1).     

Function of the fully conserved residues Asp99, Tyr52 and Tyr73 in phospholipase A2

In the active centre of pancreatic phospholipase A₂ His48 isat hydrogen-bonding distance to Asp99. This Asp-His couple isassumed to act together with a water molecule as a catalytictriad. Asp99 is also linked via an extended hydrogen bondingsystem to the side chains of Tyr52 and Tyr73. To probe the functionof the fully conserved Asp99, Tyr52 and Tyr73 residues in phospholipaseA₂, the Asp99 residue was replaced by Asn, and each of the twotyrosines was separately replaced by either a Phe or a Gln.The catalytic and binding properties of the Phe52 and Phe73mutants did not change significantly relative to the wild-typeenzyme. This rules out the possibility that either one of thetwo Tyr residues in the wild-type enzyme can function as anacyl acceptor or proton donor in catalysis. The Gln73 mutantcould not be obtained in any significant amounts probably dueto incorrect folding. The Gln52 mutant was isolated in low yield.This mutant showed a large decrease in catalytic activity whileits substrate binding was nearly unchanged. The results suggesta structural role rather than a catalytic function of Tyr52and Tyr73. Substitution of asparagine for aspartate hardly affectsthe binding constants for both monomeric and micellar substrateanalogues. Kinetic characterization revealed that the Asn99mutant has retained no less than 65% of its enzymatic activityon the monomeric substrate rac 1,2-dihexanoyldithio-propyl-3-phosphocholine,probably due to the fact that during hydrolysis of monomericsubstrate by phospholipase A₂ proton transfer is not the rate-limitingstep. The Asp to Asn substitution decreases the catalytic rateon micellar 1,2-dioctanoyl-sn-glycero-3-phosphocholine 25-fold.To explain this remaining activity we suggest that in the mutantthe Asn99 orients His48 in the same way as Asp99 orients His48in native phospholipase A₂ and that the lowered activity iscaused by a reduced stabilization of the transition state.     

Site-directed mutagenesis study on the roles of evolutionally conserved aspartic acid residues in human glutathione S-transferase P1-1

The evolutionally conserved aspartyl residues (Asp57, Asp98and Asp152) in human glutathione S-transferase P1-1 were replacedwith alanine by site-directed mutagenesis to obtain the mutants(D57A, D98A and D152A). The replacement of Asp98 with alanineresulted in a decrease of the affinity for S-hexyl-GSH-agarose,a 5.5-fold increase of the K_m^GHS and a 2.9-fold increase ofthe I₅₀ of S-hexyl-GSH for GSH–CDNB conjugation. Asp98seems to participate in the binding of GSH through hydrogenbonding with the -carboxylate of the -glutamyl residue of GSH.The k_cat of D98A was 2.6-fold smaller than that of the wild-type,and the pK_a of the thiol group of GSH bound in D98A was {smalltilde}0.8 pK units higher than those in the wild-type. Asp98also seems to contribute to the activation of GSH to some extent.On the other hand, most of the kinetic parameters of D57A andD152A were similar to those of the wild-type. However, the thermostabilitiesof D57A and D152A were significantly lower than that of thewild-type. Asp57 and Asp152 seem to be important for maintainingthe proper conformation of the enzyme.     

Molecular modelling of xylose isomerase catalysis: the role of electrostatics and charge transfer to metals

The two main steps of the mechanism of xylose-xylulose conversioncatalysed by D-xylose isomerase, the ring opening of xyloseand the isomerization of the opened product by hydride transfer,were investigated by molecular mechanical and molecular orbitaltechniques. The activation energies calculated for these reactionsclearly showed that hydrogen transfer is the rate-determiningstep of the enzymatic isomerization and that Mg²⁺ ions activatewhereas Zn²⁺ ions inhibit the reaction, in agreement with theexperiments. The remarkable differences between the net chargesof these ions found by molecular orbital calculations and theinspection of the protein electrostatic potential around thereaction intermediates indicate that the main role of bivalentmetal ions should be the electrostatic stabilization of thesubstrate transition states. In order to propose a more detailedmechanism, an attempt was made to clarify the effects of nearbyresidues (e.g. His54, Asp57, Lysl83, Asp257) in the reaction.Different isomerization mechanisms, such as through an enediolintermediate, were examined and could be excluded, in additionto the charge-relay mechanism during the ring opening.     

Can the stability of protein mutants be predicted by free energy calculations?

The use of free energy simulation techniques in the study ofprotein stability is critically evaluated. Results from twosimulations of the thermostability mutation Asn218 to Ser218in Subtilisin are presented. It is shown that components ofthe free energy change can be highly sensitive to the computationaldetails of the simulation leading to the conclusion that freeenergy calculations cannot currently be used to reliably predictprotein stability. The different factors that undermine thereliability are discussed.     

Application of free energy simulations to the binding of a transition-state-analogue inhibitor to HTV protease

Free energy simulations (slow-change method) have been usedto estimate quantitatively the ratio of the binding constantsof (S) and (R) isomers of a novel HIV protease inhibitor, JG365.As a starting geometry, we used the X-ray crystallographic structureof a complex of HTV protease and JG365 provided by A.Wlodawer.According to our results the (S) configuration, i.e. the formpreviously identified experimentally, binds considerably moretightly to the protease (G° = 2.9 kcal/mol). When the (S)inhibitor is bound, there is a very strong preference for protonationof the Aspl25 (rather than the Asp25) residue of the protease.This study is the first to apply a new method for quantitativelyassessing the precision of free energies calculated by the slow-changemethod     

Effects of substitution of Thr63 by alanine on the structure and function of Lactobacillus casei dihydrofolate reductase

A mutant of Lactobacillus casei dihydrofolate reductase hasbeen constructed in which Thr63, a residue which interacts withthe 2'-phosphate group of the bound coenzyme, is replaced byalanine. This substitution does not affect k_cat, but producesan 800-fold increase in the K_m for NADPH, which reflects dissociationof NADPH from the enzyme-NADPH-tetrahydrofolate complex, anda 625-fold increase (corresponding to 3.8 kcal/mol) in the dissociationconstant for the enzyme-NADPH complex. The difference in magnitudeof these effects indicates a small effect of the substitutionon the negative cooperativity between NADPH and tetrahydrofolate.Stopped-flow studies of the kinetics of NADPH binding show thatthe weaker binding arises predominantly from a decrease in theassociation rate constant. NMR spectroscopy was used to comparethe structures of the mutant and wild-type enzymes in solution,in their complexes with methotrexate and with methotrexate andNADPH. This showed that only minimal structural changes resultfrom the mutation; a total of 47 residues were monitored fromtheir resolved ¹H resonances, and of these nine in the binarycomplex and six in the ternary differed in chemical shift betweenmutant and wild-type enzyme. These affected residues are confinedto the immediate vicinity of residue 63. There is a substantialdifference in the ³¹P chemical shift of the 2'-phosphate ofthe bound coenzyme, reflecting the loss of the interaction withthe side chain of Thr63. The only changes in nuclear Overhausereffects (NOEs) observed were decreases in the intensity of NOEsbetween protons of the adenine ring of the bound coenzyme andthe nearby residues Leu62 and Ile102, showing that the substitutionof Thr63 does cause a change in the position or orientationof the adenine ring in its binding site.     

Molecular dynamics simulation studies on Ca2+ -induced conformational changes of annexin I

Cryo-electron microscopy (EM) and X-ray studies proposed differentmechanisms for annexin-induced membrane aggregation. In thiswork, molecular dynamics (MD) simulation technique was utilizedto gain an insight into the calcium-induced conformational changeson annexin I and their implication in membrane aggregation mechanism.MD simulations were performed on the Ca²⁺-free annexin I withthe N-terminal domain buried inside the core (System 1), theCa²⁺-bound annexin I without N-terminal domain (System 2) andthe Ca²⁺-bound annexin I with the N-terminal domain exposed(System 3). Our results indicated that calcium binding increasesthe flexibility of annexin I core domain residues includingthe calcium coordinating residues. As a result, annexin I wasactivated to interact with the negatively charged membrane.The exposed N-terminal domain was very flexible and graduallylost the secondary structure during MD simulation, suggestingthat the N-terminal may adopt a favorable conformation to binda second membrane and also explaining the failure of attemptsto crystallize the full-length annexin I in the presence ofcalcium ions. The measured dimensions of the averaged simulationstructure of the Ca²⁺-bound annexin I with the N-terminal exposed(System 3) support the proposed membrane aggregation mechanismbased on X-ray studies.     

Site-directed mutagenesis reveals functional contribution of Thr218, Lys220 and Asp304 in chymosin

The functional contributions of amino acid residues Thr218 andAsp304 of chymosin, both of which are highly conserved in theaspartic proteinases, are analysed by means of site-directedmutagenesis. The optimum pH values, milk-clotting (C) and proteolytic(P) activities and kinetic parameters for synthetic oligopeptidesas substrates were examined for the mutant enzymes. The mutationThr2l8Ser caused a marked increase in the C/P ratio, which seemedto be due to a change in substrate recognition. Although thenegative charge of Asp304 had been expected to play a role inlowering the optimum pH values in the aspartic proteinases,this turned out not to be the case in chymosin because boththe mutations Asp304Ala and Asp304Glu caused a similar shiftof the optimum pH towards the acidic side. In addition, themutation Lys220Leu, which we generated previously, was foundto cause a decrease in the C/P ratio, mainly due to the increasein the proteolytic activity.     

Functional roles and subsite locations of Leu177, Trp178 and Asn182 of Aspergillus awamori glucoamylase determined by site-directed mutagenesis

Fungal glucoamylases contain four conserved regions. One regionfrom the Aspergillus niger enzyme contains three key carboxylicacid residues, the general acid catalytic group, Glu179, alongwith Asp176 and Glu180. Three site-directed mutations, Leu177– His, Trp178 – Arg and Asn182 – Ala, wereconstructed near these acidic groups to reveal the functionof other conserved residues in this region. Leu177 and Trp178are strictly conserved among fungal glucoamylases, while anamide, predominantly Asn, always occurs at position 182. Substitutionsof Leu177 or Trp178 cause significant decreases in k_cat withthe substrates tested. Similar increases in activation energiesobtained with Leu177 – His with both -(1,4)- and -(1,6)-linkedsubstrates indicate Leu177 is located in subsite 1. K_M valuesobtained with the Trp178 – Arg mutation increase for an-(1,6)-linked substrate, but not for -(1,4)-linked substrates.Calculated differences in activation energy between substratesindicate Trp178 interacts specifically with subsite 2. The Asn182 Ala mutation did not change k_cat or K_M values, indicating thatAsn182 is not crucial for activity. These results support amechanism for glucoamylase catalytic activity consisting ofa fast substrate binding step followed by a conformational changeat subsite 1 to stabilize the transition state complex.     

Involvement of aspartic and glutamic residues in kringle-2 of tissue-type plasminogen activator in lysine binding, fibrin binding and stimulation of activity as revealed by chemical modification and oligonucleotide-directed mutagenesis

Modification of glutamic and aspartic acid residues of tissue-typeplasminogen activator (t-PA) with 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimideleads to a decrease in affinity for lysine and fibrin, to adecrease of plasminogen activation activity in the presenceof a fibrin mimic, but leaves amidolytic activity and plasminogenactivation without fibrin mimic unaffected. Experiments withkringle-2 ligands and a deletion mutant of t-PA (K₂P) suggeststhat glutamic or aspartic acid residues in K₂ of t-PA are involvedin stimulation of activity, lysine binding and fibrin binding.Mutant t-PA molecules were constructed by site-directed mutagenesisin which one or two of the five aspartic or glutamic acid residuesin K₂ were changed to asparagine or glutamine respectively.Mutation of Asp236 and/or Asp238 leads to t-PA molecules with3- to 4-fold lower specific activity in the presence of fibrinmimic and having no detectable affinity for lysine analogs.However, fibrin binding was not influenced. Mutation of Glu254also leads to a 3- to 4-fold lower activity, but to a much smallerreduction of lysine or fibrin binding. Residues Asp236 and Asp238are both essential for binding to lysine derivatives, whileGlu254 might be involved but is not essential. Residues Asp236,Asp238 and Glu254 are all three involved in stimulation of activity.Remarkably, mutation of residues Asp236 and/or Asp238 appearsnot to influence fibrin binding of t-PA whereas that of Glu254does.