Structure-oriented rational design of chymotrypsin inhibitor models

Three peptides modelling a highly potent, 35-residue chymotrypsininhibitor (Schistocerca gregaria chymotrypsin inhibitor) weredesigned and synthesized by convergent peptide synthesis. Foreach model peptide, the inhibitory constant (K_i) on chymotrypsinand the solution structure were determined. In addition, moleculardynamics calculations were performed for all of them. Two modelscontaining approximately half of the parent inhibitor (17 of35 residues) were designed and subsequently found to have nosubstantial inhibitory activity (K_i values in the mM range).The third model composed of 24 amino acid residues proved tobe an effective (K_i 10^–7) inhibitor of bovine chymotrypsin.Both the solution structure properties determined by NMR spectroscopyand the dynamic behaviour of the latter model system are comparableto the native inhibitor. In contrast, the structure and dynamicsof the first two related model peptides show characteristicdifferences. We suggest that the conformation and flexibilityof the modelled protease inhibitor are crucial for its biologicalefficiency. Moreover, the structural and dynamic features ofthe binding loop (28–33) and those of the rest of themolecule appear to be interdependent. Most importantly, thesestructural characteristics can be rationally modified, at leastpartially, by peptide design. Received March 7, 2003; revised August 25, 2003; accepted August 26, 2003.     

Catalytic mechanism of fungal glucoamylase as defined by mutagenesis of Asp176, Glu179 and Glu180 in the enzyme from Aspergillus awamori

Asp176, Glu179 and Glu180 of Aspergillus awamori glucoamylaseappeared by differential labeling to be in the active site.To test their functions, they were replaced by mutagenesis withAsn, Gln and Gln respectively, and kinetic parameters and pHdependencies of all enzyme forms were determined. Glu179 –Gln glucoamylase was not active on maltose or isomaltose, whilethe k_cat for maltoheptaose hydrolysis decreased almost 2000-foldand the K_M was essentially unchanged from wild-type glucoamylase.The Glu180 – Gln mutation drastically increased the K_Mand moderately decreased the k_cat with maltose and maltoheptaose,but affected isomaltose hydrolysis less. Differences in substrateactivation energies between Glu180 – Gln and wild-typeglucoamylases indicate that Glu180 binds D-glucosyl residuesin subsite 2. The Asp176 – Asn substitution gave moderateincreases and decreases in K_M and k_cat respectively, and thereforesimilar increases in activation energies for the three substrates.This and the differences in subsite binding energies betweenAsp176 – Asn and wild-type glucoamylases suggest thatAsp176 is near subsite 1, where it stabilizes the transitionstate and interacts with Trp120 at subsite 4. Glu179 and Asp176are thus proposed as the general catalytic acid and base ofpK_a 5.9 and 2.7 respectively. The charged Glu180 contributesto the high pK_a value of Glul79. Received May 25, 1989; accepted October 19, 1989.     

The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase

We generated replacement sets for three highly conserved residues,Pro196, Pro197 and His199, that flank the catalytic nucleophile,Cys198. Pro196 and Pro197 have restricted mobility that couldbe important for the structural transitions known to be essentialfor activity. To test this hypothesis we obtained and characterized13 amino acid substitutions for Pro196, 14 for Pro197 and 14for His199. All of the Pro196 and Pro197 variants, except P197R,and four of the His199 variants complemented TS-deficient Escherichiacoli cells, indicating they had at least 1% of wild-type activity.For all His199 mutations, k_cat/K_m for substrate and cofactordecreased more than 40-fold, suggesting that the conserved hydrogenbond network co-ordinated by His199 is important for catalysis.Pro196 can be substituted with small hydrophilic residues withlittle loss in k_cat, but 15- to 23-fold increases in K_m^dUMP.Small hydrophobic substitutions for Pro197 were most active,and the most conservative mutant, P197A, had only a 5-fold lowerk_cat/K_m^dUMP than wild-type TS. Several Pro196 and Pro197 variantswere temperature sensitive. The small effects of Pro196 or Pro197mutations on enzyme kinetics suggest that the conformationalrestrictions encoded by the Pro–Pro sequence are largelymaintained when either member of the pair is mutated. Received February 24, 2003; revised June 19, 2003; accepted June 20, 2003.     

The active site of carboxypeptidase Taq possesses the active-site motif His-Glu-X-X-His of zinc-dependent endopeptidases and aminopeptidases

Carboxypeptidase (CPase) Taq possesses the His–Glu–X–X–Hissequence, which is the consensus sequence in the active siteof zinc-dependent endopeptidases and amino-peptidases, at positions276–280. Amino acid replacement of the conserved His andGlu drastically diminished the activity of CPase Taq, and thezinc content of the enzyme was also greatly reduced when eitherof the two His residues was replaced with Arg or Tyr. The resultsindicate that this sequence actually functions as the activesite in CPase Taq, showing that CPase Taq is a novel type ofzinc-dependent CPase that possesses the His–Glu–X–X–Hisactive-site motif.     

Improvement of binding of Puumala virus neutralization site resembling peptide with a second-generation phage library

We have previously selected a peptide insert FPCDRLSGYWERGIPSPCVRrecognizing the Puumala virus (PUUV) G2-glycoprotein-specificneutralizing monoclonal antibody (MAb) 1C9 with K_d of 2.85x10^–8from a random peptide library X₂CX₁₄CX₂ expressed on the pIIIprotein of the filamentous phage fd-tet. We have now createda second-generation phage-displayed peptide library in whicheach amino acid of the peptide was mutated randomly to anotherwith a certain probability. Peptides were selected for higheraffinity for MAb 1C9 and for a common binding motif for MAb4G2 having an overlapping epitope with MAb 1C9 in G2 glycoprotein.The resulting peptides were synthesized as spots on cellulosemembrane. Amino acid changes which improved the reactivity ofthe peptides to MAb 1C9 were combined in the peptide ATCDKLFGYYERGIPLPCALwith K_d of 1.49x10^–9 in biosensor measurements. Our resultsshow that the binding properties of peptides, the affinity andthe specificity can be improved and the binding specificitydetermining amino acids and structural factors can be analyzedby combining binding assays with synthetic peptides on membranewith the use of second-generation phage display libraries. Received October 18, 2002; revised April 16, 2003; accepted May 26, 2003.     

Effect of mutations involving charged residues on the stability of staphylococcal nuclease: a continuum electrostatics study

A continuum electrostatics model is used to calculate the relativestabilities of 117 mutants of staphylococcal nuclease (SNase)involving the mutation of a charged residue to an unchargedresidue. The calculations are based on the crystallographicstructure of the wild-type protein and attempt to take implicitlyinto account the effect of the mutations in the denatured stateby assuming a linear relationship between the free energy changescaused by the mutation in the native and denatured states. Agood correlation (linear correlation coefficient of 0.8) isfound with published experimental relative stabilities of thesemutants. The results suggest that in the case of SNase (i) chargedresidues contribute to the stability of the native state mainlythrough electrostatic interactions, and (ii) native-like electrostaticinteractions may persist in the denatured state. The continuumelectrostatics method is only moderately sensitive to modelparameters and leads to quasi-predictive results for the relativemutant stabilities (error of 2–3 kJ mol^–1 or ofthe order of k_BT), except for mutants in which a charged residueis mutated to glycine. Received March 24, 2003; revised August 11, 2003; accepted September 12, 2003.     

Module shuffling of a family F/10 xylanase: replacement of modules M4 and M5 of the FXYN of Streptomyces olivaceoviridis E-86 with those of the Cex of Cellulomonas fimi

To facilitate an understanding of structure–function relationships,chimeric xylanases were constructed by module shuffling betweenthe catalytic domains of the FXYN from Streptomyces olivaceoviridisE-86 and the Cex from Cellulomonas fimi. In the family F/10xylanases, the modules M4 and M5 relate to substrate bindingso that modules M4 and M5 of the FXYN were replaced with thoseof the Cex and the chimeric enzymes denoted FCF-C4, FCF-C5 andFCF-C4,5 were constructed. The k_cat value of FCF-C5 for p-nitrophenyl-ß-D-cellobiosidewas similar to that of the FXYN (2.2 s^–1); however, thek_cat value of FCF-C4 for p-nitrophenyl-ß-D-cellobiosidewas significantly higher (7.0 s^–1). The loss of the hydrogenbond between E46 and S22 or the presence of the I49W mutationwould be expected to change the position of Q88, which playsa pivotal role in discriminating between glucose and xylose,resulting in the increased k_cat value observed for FCF-C4 actingon p-nitrophenyl-ß-D-cellobioside since module M4directly interacts with Q88. To investigate the synergisticeffects of the different modules, module M10 of the FCF-C4 chimerawas replaced with that of the Cex. The effects of replacementof module M4 and M10 were almost additive with regard to theK_m and k_cat values.     

Computational and experimental studies of the catalytic mechanism of Thermobifida fusca cellulase Cel6A (E2)

Mutagenesis experiments suggest that Asp79 in cellulase Cel6A(E2) from Thermobifida fusca has a catalytic role, in spiteof the fact that this residue is more than 13 Å from thescissile bond in models of the enzyme–substrate complexbuilt upon the crystal structure of the protein. This suggeststhat there is a substantial conformational shift in the proteinupon substrate binding. Molecular mechanics simulations wereused to investigate possible alternate conformations of theprotein bound to a tetrasaccharide substrate, primarily involvingshifts of the loop containing Asp79, and to model the role ofwater in the active site complex for both the native conformationand alternative low-energy conformations. Several alternativeconformations of reasonable energy have been identified, includingone in which the overall energy of the enzyme–substratecomplex in solution is lower than that of the conformation inthe crystal structure. This conformation was found to be stablein molecular dynamics simulations with a cellotetraose substrateand water. In simulations of the substrate complexed with thenative protein conformation, the sugar ring in the –1binding site was observed to make a spontaneous transition fromthe ⁴C₁ conformation to a twist-boat conformer, consistent withgenerally accepted glycosidase mechanisms. Also, from thesesimulations Tyr73 and Arg78 were found to have important rolesin the active site. Based on the results of these various MDsimulations, a new catalytic mechanism is proposed. Using thismechanism, predictions about the effects of changes in Arg78were made which were confirmed by site-directed mutagenesis.     

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.     

Role of tryptophan 121 in the soluble CuA domain of cytochrome c oxidase: structure and electron transfer studies

To investigate the contribution of tryptophan-121 (Trp121) residueto the structure and function of soluble Cu_A domain of cytochromec oxidase, three mutant proteins, Trp121Tyr, Trp121Leu and Trp121-deletedmutant of the soluble domain of Paracoccus versutus cytochromec oxidase, were constructed and expressed in Escherichia coliBL21 (DE3). Optical spectral studies showed that both the coordinationstructure of the Cu_A center and the secondary structure of theprotein were changed significantly in the Leu substitution anddeletion mutants of Trp121. Their electron transfer activitywith cytochrome c was inhibited severely, as shown in stopped-flowkinetic studies. However, the Cu_A center can be reconstructedin the Trp121Tyr mutant although its stability decreases comparedwith the wild-type protein. This mutant keeps the same secondarystructure as the wild-type protein, but can only transfer electronswith cytochrome c at a rate of one-seventh-fold. Based on theinformation on the structure, we also investigated and analyzedthe possible factors that affect electron transfer. It appearsthat the aromatic ring, the size of the side chain and the hydrogenbonding ability of the Trp121 are crucial to the structure andfunction of the soluble Cu_A domain. Received September 11, 2002; revised February 26, 2003; accepted May 27, 2003.     

Stabilization of a chitinase from Serratia marcescens by Gly->Ala and Xxx->Pro mutations

This paper describes attempts to increase the kinetic stabilityof chitinase B from Serratia marcescens (ChiB) by the introductionof semi-automatically designed rigidifying mutations of theGlyAla and XxxPro type. Of 15 single mutants, several displayedsignificant increases in thermal stability, whereas most mutantsshowed minor effects. All mutations with non-marginal effectson stability clustered in a limited, surface-exposed regionof the enzyme, indicating that this region is involved in apartial unfolding process that triggers irreversible thermalinactivation (aggregation). A double mutant containing two stabilizingmutations in this region (G188A, A234P) displayed a 10-foldincrease in half-life at 57°C and a 4.2°C increase inapparent T_m. These results show that entropic stabilizationworks well for ChiB and they pinpoint a region whose unfoldingmay be crucial for the kinetic stability of this enzyme. Received June 18, 2003; revised September 3, 2003; accepted September 12, 2003.     

Replacement set mutagenesis of the four phosphate-binding arginine residues of thymidylate synthase

Arginines R23, R178, R179 and R218 in thymidylate synthase (TS,EC 2.1.1.45) are hydrogen bond donors to the phosphate moietyof the substrate, dUMP. In order to investigate how these argininescontribute to enzyme function, we prepared complete replacementsets of mutants at each of the four sites in Lactobacillus caseiTS. Mutations of R23 increase K_m for dUMP 2–20-fold, increaseK_m for cofactor 8–40-fold and decrease k_cat 9–20-fold,reflecting the direct role of the R23 side chain in bindingand orienting the cofactor in ternary complexes of the enzyme.Mutations of R178 increase K_m for dUMP 40–2000-fold, increaseK_m for cofactor 3–20-fold and do not significantly affectk_cat. These results are consistent with the fact that this residueis an integral part of the dUMP-binding wall and contributesto the orientation and ordering of several other dUMP bindingresidues. Kinetic parameters for all R179 mutations except R179Pwere not significantly different from wild-type values, reflectingthe fact that this external arginine does not directly contactthe cofactor or other ligand-binding residues. R218 is essentialfor the structure of the catalytic site and all mutations ofthis arginine except R218K were inactive.     

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.     

Vicinal disulfide turns

The formation of a disulfide bond between adjacent cysteineresidues is accompanied by the formation of a tight turn ofthe protein backbone. In nearly 90% of the structures analyzeda type VIII turn was found. The peptide bond between the twocysteines is in a distorted trans conformation, the omega torsionangle ranges from 159 to –133°, with an average valueof 171°. The constrained nature of the vicinal disulfideturn and the pronounced difference observed between the oxidizedand reduced states, suggests that vicinal disulfides may beemployed as a ‘redox-activated’ conformational switch. Received December 16, 2002; revised June 30, 2003; accepted July 30, 2003.     

VL position 34 is a key determinant for the engineering of stable antibodies with fast dissociation rates

Predictive engineering of antibodies exhibiting fast kineticproperties could provide reagents for biotechnological applicationssuch as continuous monitoring of compounds or affinity chromatography.Based on covariance analysis of murine germline antibody variabledomains, we selected position L34 (Kabat numbering) for mutationalstudies. This position is located at the VL/VH interface, atthe base of the paratope but with limited antigen contacts,thus making it an attractive position for mild alterations ofantigen binding properties. We introduced a serine at positionL34 in two different antibodies: Fab (fragment antigen binding)57P (Asn34Ser) and scFv (single chain fragment variable) 1F4(Gln34Ser), that recognize peptides derived from the coat proteinof tobacco mosaic virus and the oncoprotein E6, respectively.Both mutated antibodies exhibited similar properties: (i) expressionlevels of active fragments in Escherichia coli were markedlyimproved; (ii) thermostability was enhanced; and (iii) dissociationrate parameters (k_off) were increased by 2- and at least 57-foldfor scFv1F4 and Fab57P, respectively, while their associationrate parameters (k_on) remained unchanged. The L34 Ala and Thrmutants of both antibody fragments did not possess these properties.This first demontration of similar effects observed in two antibodieswith different specificities may open the way to the predictivedesign of molecules with enhanced stability and fast dissociationrates. Received October 3, 2002; revised March 7, 2003; accepted April 4, 2003.     

Prediction of protein secondary structure with a reliability score estimated by local sequence clustering

Most algorithms for protein secondary structure prediction arebased on machine learning techniques, e.g. neural networks.Good architectures and learning methods have improved the performancecontinuously. The introduction of profile methods, e.g. PSI-BLAST,has been a major breakthrough in increasing the prediction accuracyto close to 80%. In this paper, a brute-force algorithm is proposedand the reliability of each prediction is estimated by a z-scorebased on local sequence clustering. This algorithm is intendedto perform well for those secondary structures in a proteinwhose formation is mainly dominated by the neighboring sequencesand short-range interactions. A reliability z-score has beendefined to estimate the goodness of a putative cluster foundfor a query sequence in a database. The database for predictionwas constructed by experimentally determined, non-redundantprotein structures with <25% sequence homology, a list maintainedby PDBSELECT. Our test results have shown that this new algorithm,belonging to what is known as nearest neighbor methods, performedvery well within the expectation of previous methods and thatthe reliability z-score as defined was correlated with the reliabilityof prediction. This led to the possibility of making very accuratepredictions for a few selected residues in a protein with anaccuracy measure of Q₃ > 80%. The further development ofthis algorithm, and a nucleation mechanism for protein foldingare suggested. Received March 27, 2003; revised June 30, 2003; accepted August 22, 2003.     

Complex chimeras to map ligand binding sites of GPCRs

Family 1a GPCRs are thought to bind small molecule ligands ina pocket comprising sequences from non-contiguous transmembranehelices. In this study, receptor–ligand binding determinantswere defined by building a series of complex chimeras wheremultiple sequences were exchanged between related G-proteincoupled receptors. Regions of P2Y₁, P2Y₂ and BLT₁ predictedto interact with nucleotide and leukotriene ligands were identifiedand receptors were engineered within their transmembrane helicesto transpose the ligand binding site of one receptor on to anotherreceptor. Ligand-induced activation of chimeras was comparedwith wild-type receptor activation in a yeast reporter geneassay. Binding of ligand to a P2Y₂/BLT₁ chimera confirmed thatthe ligand binding determinants of BLT₁ are located in the upperregions of the helices and extracellular loops of this receptorand that they had been successfully transferred to a receptorthat normally binds unrelated ligands. Received April 20, 2002; revised March 31, 2003; accepted April 8, 2003.     

Simple consensus procedures are effective and sufficient in secondary structure prediction

We have analyzed the performance of majority voting on minimalcombination sets of three state-of-the-art secondary structureprediction methods in order to obtain a consensus prediction.Using three large benchmark sets from the EVA server, our resultsshow a significant improvement in the average Q₃ predictionaccuracy of up to 1.5 percentage points by consensus formation.The application of an additional trivial filtering procedurefor predicted secondary structure elements that are too short,does not significantly affect the prediction accuracy. Our analysisalso provides valuable insight into the similarity of the resultsof the prediction methods that we combine as well as the higherconfidence in consistently predicted secondary structure. Received March 7, 2003; revised May 24, 2003; accepted June 6, 2003.     

Increased thermal stability against irreversible inactivation of 3-isopropylmalate dehydrogenase induced by decreased van der Waals volume at the subunit interface

We have investigated factors affecting stability at the subunit–subunitinterface of the dimeric enzyme 3-isopropylmalate dehydrogenase(IPMDH) from Bacillus subtilis. Site-directed mutagenesis wasused to replace methionine 256, a key residue in the subunitinteraction, with other amino acids. Thermal stability againstirreversible inactivation of the mutated enzymes was examinedby analyzing the residual activity after heat treatment. Themutations M256V and M256A increased thermostability by 2.0 and6.0°C, respectively, whereas the mutations M256L and M256Ihad no effect. Thermostability of the M256F mutated enzyme was4.0°C lower than that of the wild-type enzyme. To our surprise,increasing the hydrophobicity of residue 256 within the hydrophobiccore of the enzyme resulted in a lower thermal stability. Themutated enzymes showed an inverse correlation between thermostabilityand the volume of the side chain at position 256. Based on theX-ray crystallographic structure of Escherichia coli IPMDH,the environment around M256 in the B.subtilis homolog is predictedto be sterically crowded. These results suggest that Met256prevents favorable packing. Introduction of a smaller aminoacid at position 256 improves the packing and stabilizes thedimeric structure of IPMDH. The van der Waals volume of theamino acid residue at the hydrophobic subunit interface is animportant factor for maintaining the stability of the subunit–subunitinterface and is not always optimized in the mesophilic IPMDHenzyme. Received September 3, 2002; revised June 13, 2003; accepted June 20, 2003.