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.     

Double-site ricin B chain mutants retain galactose binding

Three distinct double-site and two single-site ricin B chain(RTB) mutants were expressed in Spodoptera frugiperda insectcells and purified from infected cell supernatants. The yieldsof recombinant proteins were 0.01–0.2 mg/1. The purityafter monoclonal antibody affinity chromatography was 1–20%.The mutant proteins were soluble, immuno-reactive with monoclonalantibodies and polyclonal antibodies to RTB and demonstratedmolecular weights of 32 kDa, similar to plant RTB. All threedouble-site and both single-site mutants bound asialofetuinand mammalian cell surfaces based on an asialofetuin ELISA andcell binding immunofluorescence assay. While one double-sitemutant, W37S/Y248S, had a 1 log drop in sugar binding, the othertwo double-site mutants W37S/Y248H and D22E/D234E had 2 logreductions in sugar binding. Each mutant reassociated efficiently(25–75%) with plant ricin A chain (RTA) to form cytotoxicheterodimers. The concentration of protein required to reduceprotein synthesis 50% (ID₅₀) was 1 log higher than plant ricinfor W37S/Y248S-RTA and the single-site mutant heterodimers,Q35N-RTA and D22E-RTA and 2 logs higher than plant ricin forthe other two double-site mutant heterodimers. The results suggestamino acid residues in both the 1 and 2 subdomains of RTB participatein sugar binding. However, other subdomains must contributeto the avidity of ricin for cell surface oligosaccharides.     

Contribution of a salt bridge to binding affinity and dUMP orientation to catalytic rate: mutation of a substrate-binding arginine in thymidylate synthase

Invariant arginine 179, one of four arginines that are conservedin all thymidylate synthases (TS) and that bind the phosphatemoiety of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP),can be altered even to a negatively charged glutainic acid withlittle effect on k_cat. In the mutant structures, ordered wateror the other phosphate binding arginines compensate for thehydrogen bonds made by Arg179 in the wild-type enzyme and thereis almost no change in the conformation or binding site of dUMP.Correlation of dUMP K_ds for TS R179A and TS R179K with the structuresof their binary complexes shows that the positive charge onArg179 contributes significantly to dUMP binding affinity. k_cat/Kmfor dUMP measures the rate of dUMP binding to TS during theordered bi-substrate reaction, and in the ternary complex dUMPprovides a binding surface for the cofactor. k_cat/Km reflectsthe ability of the enzyme to accept a properly oriented dUMPfor catalysis and is less sensitive than is K_d to the changesin electrostatics at the phosphate binding site.     

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

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

A combinatorial library of an {alpha}-helical bacterial receptor domain

The construction and characterization of a combinatorial libraryof a solvent-exposed surface of an -helical domain derived froma bacterial receptor is described. Using a novel solid-phaseapproach, the library was assembled in a directed and successivemanner utilizing single-stranded oligonucleotides containingmultiple random substitutions for the variegated segments ofthe gene fragment The simultaneous substitution of 13 residuesto all 20 possible amino acids was carried out in a region spanning81 nucleotides. The randomization was made in codons for aminoacids that were modelled to be solvent accessible at a surfacemade up from two of the three a-helices of a monovalent Fc-bindingdomain of staphylococcal protein A. After cloning of the PCR-amplifiedlibrary into a phagemid vector adapted for phage display ofthe mutants, DNA sequencing analysis suggested a random distributionof codons in the mutagenized positions. Four members of thelibrary with multiple substitutions were produced in Escherichiacoli as fusions to an albumin-binding affinity tag derived fromstreptococcal protein G. The fusion proteins were purified byhuman serum albumin affinity chromatography and subsequentlycharacterized by SDSelectrophoresis, CD spectroscopy and biosensoranalysis. The analyses showed that the mutant protein A derivativescould all be secreted as soluble full-length proteins. Furthermore,the CD analysis showed that all mutants, except one with a prolineintroduced into helix 2, have secondary structures in closeagreement with the wild-type domain. These results proved thatmembers of this -helical receptor library with multiple substitutionsin the solvent-exposed surface remain stable and soluble inE.coli. The possibility of using this library for a phenotypicselection strategy to obtain artificial antibodies with novelfunctions is discussed.     

High-level bacterial expression, purification and characterization of human calreticulin

To investigate its cellular function and role in autoimmunedisease pathogenesis, we have bacterlally expressed human calreticulin,a major calcium-binding protein in the endoplasmic reticulumand a human autoantigen. This is the First report describingthe heterologous expression of calreticulin from any source.The recombinant calreticulin constituted {small tilde}32% ofthe soluble Escherichia coli proteins, and was purified to apparenthomogeneity by ion exchange and hydrophobic liquid chromatography.As does the bona fide protein, the recombinant calreticulinbinds calcium and undergoes changes in its conformation uponZn²⁺ binding. We take this as a strong indication that the foldingof the E.coli-expressed calreticulin is very similar, if notidentical, to that of the authentic protein. Moreover, the bacteriallyexpressed calreticulin readily reacted with anti-human and anti-rabbitantibodies, and the anti-recombinant calreticulin antibodiesimmunoreacted with HeLa calreticulin. The availability of thisexpression system will allow us to carry out site-specific anddeletion mutagenesis analysis in structure-function studiesof calreticulin.     

Progesterone binding to uteroglobin: two alternative orientations of the ligand

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

pH-sensitive interactions between IgG and a mutated IgG-binding protein based upon two B domains of Protein A from Staphylococcus aureus

A fusion protein, consisting of the N-terminal 81 amino acidsfrom an inactive bovine DNase I (Q38,E39–E38,Q39) andtwo sequential synthetic IgG-binding domains based upon domainB of Protein A from Staphylococcus aureus has been shown tobind to porcine IgG with a similar affinity and pH profile toProtein A. The same residue in each B domain (Tyr111 and Tyr169)has been mutated by cassette mutagenesis to Ser, Glu, His, Lysor Arg and the effect of the mutation on binding interactionswith porcine IgG investigated. The evidence presented suggeststhat the interactions at the B domain are highly sensitive tothe presence of a charged residue.     

The 2 A crystal structure of subtilisin E with PMSF inhibitor

Using enzyme prepared by the DNA recombination technique, subtilisinE from Bacillus subtilis was crystallizedin space group P2₁2₁2₁with two molecules in an asymmetric unit. The crystal structureof PMSF-inhibited subtilisin E was solved by molecular replacementfollowed by refinement with the X-PLOR program. This resultedin the 2.0 Å structure of subtilisin E with an R-factorof 0.191 for 8–2 Å data and r.m.s. deviations fromideal values of 0.021 Å and 2.294° for bond lengthsand bond angles respectively. The PMSF group covalently boundto Ser221 appeared very clearly in the electron density map.Except for the active site disturbed by PMSF binding, the structuralfeatures of subtilisin E are almost the same as in other subtilisins.The calcium-binding sites are different in detail in the twoindependent molecules of subtilisin E. Based on the structure,the remarkably enhanced heat stability of mutant N118S of subtilisinE is discussed. It is very likely that there is an additionalwater molecule in the mutant structure, which is hydrogen bondedto side chains of Serll8 and its neighbouring residues Lys27and Asp 120.     

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     

Engineering ribonuclease A: production, purification and characterization of wild-type enzyme and mutants at Gln11

Bovine pancreatic ribonuclease A (RNase A) has been the objectof much landmark work in biological chemistry. Yet the applicationof the techniques of protein engineering to RNase A has beenlimited by problems inherent in the isolation and heterologousexpression of its gene. A cDNA library was prepared from cowpancreas, and from this library the cDNA that codes for RNaseA was isolated. This cDNA was inserted into expression plasmidsthat then directed the production of RNase A in Saccharomycescerevisiae (fused to a modified -factor leader sequence) orEscherichia coli (fused to the pelB signal sequence). RNaseA secreted into the medium by S.cerevisiae was an active buthighly glycosylated enzyme that was recoverable at 1 mg/l ofculture. RNase A produced by E.coli was in an insoluble fractionof the cell lysate. Oxidation of the reduced and denatured proteinproduced active enzyme which was isolated at 50 mg/l of culture.The bacterial expression system is ideal for the large-scaleproduction of mutants of RNase A. This system was used to substitutealanine, asparagine or histidine for Gln11, a conserved residuethat donates a hydrogen bond to the reactive phosphoryl groupof bound substrate. Analysis of the binding and turnover ofnatural and synthetic substrates by the wild-type and mutantenzymes shows that the primary role of Gln11 is to prevent thenon-productive binding of substrate.     

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.     

FK506-binding protein mutational analysis: defining the active-site residue contributions to catalysis and the stability of ligand complexes

The 12 kDa FK506-binding protein FKBP12 is a cis-trans peptidyl-prolylisomerase that binds the macrolides FK506 and rapamycin. Wehave examined the role of the binding pocket residues of FKBP12in protein–ligand interactions by making conservativesubstitutions of 12 of these residues by site-directed mutagenesis.For each mutant FKBP12, we measured the affinity for FK506 andrapamycin and the catalytic efficiency in the cis–transpeptidyl-prolyl isomerase reaction. The mutation of Trp59 orPhe99 generates an FKBP12 with a significantly lower affinityfor FK506 than wild-type protein. Tyr26 and Tyr82 mutants areenzymatically active, demonstrating that hydrogen bonding bythese residues is not required for catalysis of the cis–transpeptidyl-prolyl isomerase reaction, although these mutationsalter the substrate specificity of the enzyme. We conclude thathydrophobic interactions in the active site dominate in thestabilization of FKBP12 binding to macrolide ligands and tothe twisted-amide peptidyl-prolyl substrate intermediate.     

The influence of Glu44 and Glu56 of cytochrome b5 on the protein structure and interaction with cytochrome c

The gene encoding trypsin-solubilized bovine liver microsomalcytochrome b₅ (82 residues in length) has been mutated, in whichthe codons of Glu44 and Glu56 were changed to those of Ala.The mutated genes were expressed in Escherichia coli successfullyand three mutant proteins (E44A, E56A and E44/56A) were obtained.The UV-visible, CD and ¹H NMR spectra of proteins have beenstudied. The results show that the mutagenesis at surface residuesdoes not alter the secondary and tertiary structures of cytochromeb₅ significantly. The interactions between recombinant cytochromeb₅ and its mutants with cytochrome c were studied by using opticaldifference spectra. The results demonstrated that both Glu44and Glu56 of cytochrome b₅ participate in the formation of acomplex between cytochrome b₅ and cytochrome c.     

Fluorescent properties of the Escherichia coli D-xylose isomerase active site

The consequences of active site mutations of the Escherichiacoli D-xylose isomerase (E.C. 5.3.1.5 [EC] ) on substrate bindingwere examined by fluorescence spectroscopy. Site-directed mutagenesisof conserved tryptophan residues in the E.coli enzyme (Trp49and Trpl88) reveals that fluorescence quenching of these residuesoccurs during the binding of xylose by the wild-type enzyme.The fluorescent properties of additional active site substitutionsat His101 were also examined. Substitutions of His101 whichinactivate the enzyme were shown to have altered spectral characteristics,which preclude detection of substrate binding. In the case ofH101S, a mutant protein with measurable isomerizing activity,substrate binding with novel fluorescent properties was observed,possibly the bound pyranose form of xylose under steady-stateconditions.     

Effects of humanization by variable domain resurfacing on the antiviral activity of a single-chain antibody against respiratory syncytial virus

HNK20 is a mouse monoclonal IgA that binds to the F glycoproteinof respiratory syncytial virus (RSV) and neutralizes the virus,both in vitro and in vivo. The single-chain antibody fragment(scFv) derived from HNK20 is equally active and has allowedus to assess rapidly the effect of mutations on affinity andantiviral activity. Humanization by variable domain resurfacingrequires that surface residues not normally found in a humanFv be mutated to the expected human amino acid, thereby eliminatingpotentially immunogenic sites. We describe the constructionand characterization of two humanized scFvs, hu7 and hu10, bearing7 and 10 mutations, respectively. Both molecules show unalteredbinding affinities to the RSV antigen (purified F protein) asdetermined by ELISA and surface plasmon resonance measurementsof binding kinetics (K_a 1x10⁹ M^–1). A competition ELISAusing captured whole virus confirmed that the binding affinitiesof the parental scFv and also of hu7 and hu10 scFvs were identical.However, when compared with the original scFv, hu10 scFv wasshown to have significantly decreased antiviral activity bothin vitro and in a mouse model. Our observations suggest thatbinding of the scFv to the viral antigen is not sufficient forneutralization. We speculate that neutralization may involvethe inhibition or induction of conformational changes in thebound antigen, thereby interfering with the F protein-mediatedfusion of virus and cell membranes in the initial steps of infection.     

Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling

The possibility of increasing the affinity of a Taq DNA polymerasespecific binding protein (affibody) was investigated by an -helixshuffling strategy. The primary affibody was from a naive combinatoriallibrary of the three-helix bundle Z domain derived from staphylococcalprotein A. A hierarchical library was constructed through selectivere-randomization of six amino acid positions in one of the two-helices of the domain, making up the Taq DNA polymerase bindingsurface. After selections using monovalent phage display technology,second generation variants were identified having affinities(K_D) for Taq DNA polymerase in the range of 30–50 nM asdetermined by biosensor technology. Analysis of binding dataindicated that the increases in affinity were predominantlydue to decreased dissociation rate kinetics. Interestingly,the affinities observed for the second generation Taq DNA polymerasespecific affibodies are of similar strength as the affinitybetween the original protein A domain and the Fc domain of humanimmunoglobulin G. Further, the possibilities of increasing theapparent affinity through multimerization of affibodies wasdemonstrated for a dimeric version of one of the second generationaffibodies, constructed by head-to-tail gene fusion. As comparedwith its monomeric counterpart, the binding to sensor chip immobilizedTaq DNA polymerase was characterized by a threefold higher apparentaffinity, due to slower off-rate kinetics. The results showthat the binding specificity of the protein A domain can bere-directed to an entirely different target, without loss ofbinding strength.     

Activation of E350A mutant maltodextrin phosphorylase by exogenously added acetate

Site-directed mutagenesis of E350 to alanine in Escherichiacoli maltodextrin phosphorylase reduced both enzyme activity(100-fold) and apparent binding of the oligosaccharide substrate(10-fold), suggesting a participation of this residue in bindingof the substrate in the ground and transition states. The E350Amutant enzyme was found to be activated up to 20-fold by exogenousacetate ions which substitute for the deleted side chain. Incontrast, apparent binding was not affected by acetate ions,indicating a dual role for the carboxylic group of this residuein catalysis and binding. Formate also appears to activate theE350A mutant enzyme, but this effect is obscured by the stronginhibitory effect of formate on the wild-type enzyme. For propionateions, a weak 2-fold activation was noticed, while other compoundslike trifluoroacetate and acetamide had no effects on the catalyticproperties of either the E350A mutant enzyme or wild-type enzyme.If E350 was substituted by a glutamine, no activation was observedupon the addition of acetate ions. However, a weak activationby formate was found, confirming that activation by acetateis caused by specific binding at the mutated site.     

X-ray structures of two single-residue mutants of DNase I: H134Q and Y76A

The structures of the single-residue mutants H134Q and Y76Aof bovine pancreatic DNase I have been determined and refinedincluding data to 2.3 and 2.4 Å resolution respectively,by X-ray crystallography. H134 is an essential catalytic residue,while Y76 contributes to the binding of DNA by providing a largevan der Waals contact area that stabilizes the wide minor grooveseen in DNase I-DNA complexes. The mutant proteins, which showstrongly reduced activities of 0.001% (H134Q) and 0.3% (Y76A),were expressed in E.coli and both crystallize in space-groupC2 with almost identical unit cells. The crystal packing schemeis different from that found in wild type crystals grown undervery similar conditions, presumably due to the absence of thecarbohydrate moiety. In both mutants the conformation of theprotein is nearly identical to that of the wild type enzymeand changes are confined to surface loops involved in packing.The disruption of the hydrogen bonds between H134, E78 and Y76in both mutants leads to an increased mobility and positionalshifts in the DNA-binding loop, mainly around residue Y76. Thisin turn may further reduce DNA-binding affinity and, thus, contributeto the low activity. In contrast, symmetry contacts involvingresidues 97–108 lead to a stabilization of the flexibleloop compared to wild type DNase I.     

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

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