Conserved sequence and structure association motifs in antibody-protein and antibody-hapten complexes

In this paper, we present the association requirements across a wide variety of antibody-antigen complexes. Phylogenetic analysis clearly indicates the representative nature of our structural dataset. Antigen molecules range from small-molecule haptens to complete protein structures. Common association motifs identified include five conserved tyrosine residues and a single conserved arginine residue from CDR-H3. Further, specificity is refined by a diverse array of antibody-antigen electrostatic interactions that maximize complex specificity. Through analysis of calculated pKa shifts on antigen binding, we find that these interactions are conserved at 23 alignment 'hot-spot' positions. Despite consistent roles in defining substrate specificity, 16 hot-spot positions are conserved less than 50% of the time. On the other hand, because of the conserved functional role of these positions, mutant screening at hot-spots is more likely to result in increased antigen specificity than elsewhere. Therefore, we believe these results should facilitate subsequent antibody design experimentation.     

Molecular modelling and site-directed mutagenesis on a bovine anti-testosterone monoclonal antibody

A three-dimensional (3D) molecular model of the antigen-combiningsite of a bovine anti-testosterone monoclonal antibody has beenconstructed. In the model, the CDRs, and a single heavy chainframework region residue (Trp47), associate to form a hydrophobiccavity large enough to accommodate a single molecule of testosterone.Tyr97 of CDR-H3 lies at the bottom of the cavity with its hydroxylgroup exposed to solvent. Using the model and data from bindingstudies, we predicted that the cavity forms the antibody's paratopeand on binding testosterone a hydrogen bond is formed betweenTyr97 of CDR-H3 and the hydroxyl group on the D-ring of testosterone.This prediction has subsequently been tested by site-directedmutagenesis. An antibody with phenylalanine in place of tyrosineat position 97 in CDR-H3 has its affinity reduced by {smalltilde}800 fold. The reduction in binding energy associated withthe reduced affinity has been calculated to be 3.9 kcal/molwhich is within the range (0.5–4.0 kcal/mol) expectedfor the loss of a single hydrogen bond. The model has been usedto suggest ways of increasing the antibody's affinity for testosterone.     

Structure and function prediction of the Brucella abortus P39 protein by comparative modeling with marginal sequence similarities

A methodology is proposed to solve a difficult modeling problemrelated to the recently sequenced P39 protein. This sequenceshares no similarity with any known 3D structure, but a foldis proposed by several threading tools. The difficulty in aligningthe target sequence on one of the proposed template structuresis overcome by combining the results of several available predictionmethods and by refining a rational consensus between them. Insilico validation of the obtained model and a preliminary cross-checkwith experimental features allow us to state that this borderlineprediction is at least reasonable. This model raises relevanthypotheses on the main structural features of the protein andallows the design of site-directed mutations. Knowing the geneticcontext of the P39 reading frame, we are now able to suggesta function for the P39 protein: it would act as a periplasmicsubstrate-binding protein.     

Modeling the uncleaved serpin antichymotrypsin and its chymotrypsin complex

In order to provide a structural reference for protein engineeringexperiments involving the serpin ₁-antichymotrypsin (ACT) andits complexes with chymotrypsin and DNA, a homology model ofACT has been constructed based on the 3-D structure of the relatedprotein ovalbumin [29% identical and 44% similar; see Stein,P., Leslie, A., Finch, J., Turnell, W., McLaughlin, P. and Carrell,R. (1990) Nature, 347, 99–102]. After mapping the aminoacid sequence of ACT onto the peptide backbone of ovalbumin,the resulting model was subjected to simulated annealing andenergy minimization. Overall, the final ACT model is structurallysimilar to ovalbumin, although the 2.4 Å root mean squaredeviation of corresponding C atoms reflects the presence ofregions exhibiting notable structural differences. The hydrogenbond stereochemistry of the ACT model is consistent with patternsfound in high resolution protein structures and 92% of its backboneatoms have acceptable conformations when evaluated in a Ramachandrananalysis. Significantly, the homology model serves as a structuralreference for protein engineering experiments aimed at redesigningthe functional properties of ACT, particularly with regard toits protease-bound conformation. Additionally, the homologymodel may be useful as a probe for solving the crystal structuresof certain ACT variants (e.g. Thr345 Arg) by molecular replacementmethods. Ultimately, the homology approach may be applied towardthe construction of other serpin models starting with an experimentallydetermined structure of uncleaved ACT as a template.     

Humanization of a mouse monoclonal antibody by CDR-grafting: the importance of framework residues on loop conformation

A mouse monoclonal antibody (mAb 425) with therapeutic potentialwas ‘humanized’ in two ways. Firstly the mouse variableregions from mAb 425 were spliced onto human constant regionsto create a chimeric 425 antibody. Secondly, the mouse complementarity–determiningregions (CDRs) from mAb 425 were grafted into human variableregions, which were then joined to human constant regions, tocreate a reshaped human 425 antibody. Using a molecular modelof the mouse mAb 425 variable regions, framework residues (FRs)that might be critical for antigen-binding were identified.To test the importance of these residues, nine versions of thereshaped human 425 heavy chain variable (VH) regions and twoversions of the reshaped human 425 light chain variable (VJregions were designed and constructed. The recombinant DNAscoding for the chimeric and reshaped human light and heavy chainswere coexpressed transiently in COS cells. In antigen-bindingassays and competition-binding assays, the reshaped human antibodieswere compared with mouse 425 antibody and to chimeric 425 antibody.The different versions of 425–reshaped human antibodyshowed a wide range of avidities for antigen, indicating thatsubstitutions at certain positions in the human FRs significantlyinfluenced binding to antigen. Why certain individual FR residuesinfluence antigen-binding is discussed. One version of reshapedhuman 425 antibody bound to antigen with an avidity approachingthat of the mouse 425 antibody.     

A mini-protein designed by removing a module from barnase: molecular modeling and NMR measurements of the conformation

A globular domain can be decomposed into compact modules consistingof contiguous 10–30 amino acid residues. The correlationbetween modules and exons observed in different proteins suggeststhat each module was encoded by an ancestral exon and that moduleswere combined into globular domains by exon fusion. Barnaseis a single domain RNase consisting of 110 amino acid residuesand was decomposed into six modules. We designed a mini-proteinby removing the second module, M2, from barnase in order togain an insight into the structural and functional roles ofthe module. In the molecular modeling of the mini-protein, weevaluated thermodynamic stability and aqueous solubility togetherwith mechanical stability of the model. We chemically synthesizeda mini-barnase with ¹⁵N-labeling at 10 residues, whose correspondingresidues in barnase are all found in the region around the hydrophobiccore. Circular dichroism and NMR measurements revealed thatmini-barnase takes a non-random specific conformation that hasa similar hydrophobic core structure to that of barnase. Thisresult, that a module could be deleted without altering thestructure of core region of barnase, supports the view thatmodules act as the building blocks of protein design.     

Engineering of protein epitopes: a single deletion in a snake toxin generates full binding capacity to a previously unrecognized antibody

Structural features associated with the ability of a monoclonalantibody (mAb) to discriminate between protein variants areidentified and engineered. The variants are the curaremimetictoxin from Naja nigricollis and erabutoxin a or b from Laticaudasemifasciata which differ from each other by 16 substitutionsand one insertion. The neutralizing mAb M1 recognizes with highaffinity a topographical epitope on the surface of toxin , butfails to recognize the erabutoxins although they possess mostof the residues forming the presumed epitope. Examinations ofthe toxin and erabutoxin 3-D structures and molecular dynamicssimulations reveal several differences between the variants.In particular, the region involving the ß-turn 17–24is organized differently. Analysis of the differences foundin this region suggests that the insertion (or deletion) atposition 18 of the variant amino add sequences is particularlyimportant in determining the differential cross-reactivity.To test this proposal, residue 18 was deleted in one erabutoxinusing sitedirected mutagenesis, and the biological propertiesof the resulting mutant were examined. We found that full antigenicitywas restored in the previously unrecognized variant. The implicationsof this finding are discussed.     

Molecular modeling of the interaction of polyproline-based peptides with the Abl-SH3 domain: rational modification of the interaction

A molecular model of the interaction of polyproline-rich peptideswith the Abl-SH3 domain is proposed, based on docking calculationswith the DOCK program coupled with molecular dynamics simulations.Two distinct binding modes of the peptide to the same aromatic-richregion (TyrlO, Phel2, Trp39, Trp50, Tyr55) of the domain wereobtained. It is proposed that these two models could representdifferent binding modes of proline-rich peptides to Src homologyregion 3 domains. Several peptide mutants were designed to determinewhether the two orientations were possible. Analysis of theKd values and fluorescence emission of these peptides indicatethat one of the orientations is more plausible and that residuesat position 4 of the peptide interact with the RT loop, beingimportant in modulating the peptide affinity for the Abl-SH3domain     

Association and dissociation kinetics of bobwhite quail lysozyme with monoclonal antibody HyHEL-5

The anti-hen egg lysozyme monoclonal antibody HyHEL-5 and itscomplexes with various species-variant and mutant lysozymeshave been the subject of considerable experimental and theoreticalinvestigation. The affinity of HyHEL-5 for bobwhite quail lysozyme(BWQL) is over 1000-fold lower than its affinity for the originalantigen, hen egg lysozyme (HEL). This difference is believedto arise almost entirely from the replacement in BWQL of thestructural and energetic epitope residue Arg68 by lysine. Inthis study, the association and dissociation kinetics of BWQLwith HyHEL-5 were investigated under a variety of conditionsand compared with previous results for HEL. HyHEL-5–BWQLassociation follows a bimolecular mechanism and the dissociationof the antibody–antigen complex is a first-order process.Changes in ionic strength (from 27 to 500 mM) and pH (from 6.0to 10.0) produced about a 2-fold change in the association anddissociation rates. The effect of viscosity modifiers on theassociation reaction was also studied. The large differencein the HEL and BWQL affinities for HyHEL-5 is essentially dueto differences in the dissociation rate constant.     

Interaction between a Fab fragment against gp41 of human immunodeficiency virus 1 and its peptide epitope: characterization using a peptide epitope library and molecular modeling

The molecular interaction of the Fab fragment of the human monoclonalantibody 3D6, directed against the transmembrane protein gp41of human immunodeficiency virus (HTV) 1, with its peptide epitopeis characterized by a panel of overlapping peptides, a peptideepitope library and molecular modeling techniques. The sequenceCSGKLICTTAVPW, corresponding to amino acids 605–617 ofgp41, was identified as the best binding peptide (K_D = 1x10^-8mol/1). This peptide served as a starting point to prepare acellulose-bound peptide epitope library in which each residueof the epitope is substituted by all L- and D-amino acids, resultingin 494 epitope peptide variants which were subsequently analyzedfor binding 3D6. The library was synthesized to identify residuescritical for binding and to obtain information about the molecularenvironment of the epitope peptide bound to 3D6. Both cysteineresidues, as well as isoleucine 6, threonine 8 and proline 12,of the epitope were highly sensitive to substitution. Usingthe data obtained from the epitope characterization, as wellas a low-resolution electron density map of a 3D6 Fab-peptidecomplex, a 3-D model of the Fab-peptide complex was generatedby molecular modeling. The modeling experiments predict bindingof the peptide, which is cyclized via the two cysteine residues,to a pocket formed dominantly by the hypervariable loops complementaritydetermining regions CDR3L, CDR2H and CDR3H.     

Cloning, sequencing and expression of the Fab fragment of a monoclonal antibody to the herbicide atrazine

The Fab region of an IgG2b antibody (AM7B2.1) reactive to theherbicide atrazine was cloned into a plasmid vector using thepolymerase chain reaction and two sets of degenerate oligonucleotideprimers designed to mimic the amino acid variation at the N-terminiof _L-chains and TH-chains. These primers also provide a secretionsignal fused precisely to the antibody gene sequence for secretionof the mature antibody. A further set of universal oligonucleotideprimers was developed for the direct sequencing of the V_H andC_m regions of _B-chains and the V_L and C_L regions of _L-chainswithout subcloning and were used to determine the sequence ofthis antibody. The _L-chain was found to not possess a conservedCys residue at position 23 and the implications of this observationare discussed. The cloned genes were expressed in Escherichiacoli using a commercially available T7 RNA polymerase-basedplasmid. The clones were also expressed in a 17 RNA polymerasebasedsystem containing an attenuated version of the T7 RNA polymerasepromoter, plus a lac promoter placed in an antisense orientation,to enhance plasmid stability. The expressed products were confirmedas atrazine reactive by binding to an atrazine derivative conjugatedwith alkaline phosphatase.     

Efficient generation of a reshaped human mAb specific for the {alpha} toxin of Clostridium perfringens

We have used the technique of antibody reshaping to producea humanized antibody specific for the a toxin of Clostridiumperfringens. The starting antibody was from a mouse hybridomafrom which variable (V) region nucleo-tide sequences were determined.The complementarity-determining regions (CDRs) from these Vregions were then inserted into human heavy and light chainV region genes with human constant region gene fragments subsequentlyadded. The insertion of CDRs alone into human frameworks didnot produce a functional reshaped antibody and modificationsto the V region framework were required. With minor frameworkmodifications, the affinity of the original murine mAb was restoredand even exceeded. Where affinity was increased, an alteredbinding profile to overlapping peptides was observed. Computermodelling of the reshaped heavy chain V regions suggested thatamino acids adjacent to CDRs can either contribute to, or distort,CDR loop conformation and must be adjusted to achieve high bindingaffinity.     

Molecular modeling of the amyloid-{beta}-peptide using the homology to a fragment of triosephosphate isomerase that forms amyloid in vitro

The main component of the amyloid senile plaques found in Alzheimer'sbrain is the amyloid-ß-peptide (Aß), a proteolyticproduct of a membrane precursor protein. Previous structuralstudies have found different conformations for the Aßpeptide depending on the solvent and pH used. In general, theyhave suggested an -helix conformation at the N-terminal domainand a ß-sheet conformation for the C-terminal domain.The structure of the complete Aß peptide (residues 1–40)solved by NMR has revealed that only helical structure is presentin Aß. However, this result cannot explain the large ß-sheetAß aggregates known to form amyloid under physiologicalconditions. Therefore, we investigated the structure of Aßby molecular modeling based on extensive homology using theSmith and Waterman algorithm implemented in the MPsrch program(Blitz server). The results showed a mean value of 23% identitywith selected sequences. Since these values do not allow a clearhomology to be established with a reference structure in orderto perform molecular modeling studies, we searched for detailedhomology. A 28% identity with an /ß segment of a triosephosphateisomerase (TIM) from Culex tarralis with an unsolved three-dimensionalstructure was obtained. Then, multiple sequence alignment wasperformed considering Aß, TIM from C.tarralis and anotherfive TIM sequences with known three-dimensional structures.We found a TIM segment with secondary structure elements inagreement with previous experimental data for Aß. Moreover,when a synthetic peptide from this TIM segment was studied invitro, it was able to aggregate and to form amyloid fibrils,as established by Congo red binding and electron microscopy.The Aß model obtained was optimized by molecular dynamicsconsidering ionizable side chains in order to simulate Aßin a neutral pH environment. We report here the structural implicationsof this study.     

Humanization of a mouse anti-human IgE antibody: a potential therapeutic for IgE-mediated allergies

Mouse mAb TES-C21(C21) recognizes an epitope on human IgE and,therefore, has potential as a therapeutic agent in patientswith IgE-mediated allergies such as hay fever, food and drugallergies and extrinsic asthma. The clinical usefulness of mouseantibodies is limited, however, due to their immunogenidty inhumans. Mouse C21 antibody was humanized by complementaritydetermining region (CDR) grafting with the aim of developingan effective and safe therapeutic for the treatment of IgE-mediatedallergies. The CDR-grafted, or reshaped human, C21 variableregions were carefully designed using a specially constructedmolecular model of the mouse C21 variable regions. A key stepin the design of reshaped human variable regions is the selectionof the human framework regions (FRs) to serve as the backbonesof the reshaped human variable regions. Two approaches to theselection of human FRs were tested: (i) selection from humanconsensus sequences and (ii) selection from individual humanantibodies. The reshaped human and mouse C21 antibodies weretested and compared using a biosensor to measure the kineticsof binding to human IgE. Surprisingly, a few of the reshapedhuman C21 antibodies exhibited patterns of binding and affinitiesthat were essentially identical to those of mouse C21 antibody.     

Molecular modeling of single polypeptide chain of calcium-binding protein p26olf from dimeric S100B()

P26olf from olfactory tissue of frog, which may be involvedin olfactory transduction or adaptation, is a Ca²⁺-binding proteinwith 217 amino acids. The p26olf molecule contains two homologousparts consisting of the N-terminal half with amino acids 1–109and the C-terminal half with amino acids 110–217. Eachhalf resembles S100 protein with about 100 amino acids and containstwo helix–loop–helix Ca²⁺-binding structural motifsknown as EF-hands: a normal EF-hand at the C-terminus and apseudo EF-hand at the N-terminus. Multiple alignment of thetwo S100-like domains of p26olf with 18 S100 proteins indicatedthat the C-terminal putative EF-hand of each domain containsa four-residue insertion when compared with the typical EF-handmotifs in the S100 protein, while the N-terminal EF-hand ishomologous to its pseudo EF-hand. We constructed a three-dimensionalmodel of the p26olf molecule based on results of the multiplealignment and NMR structures of dimeric S100B(ßß)in the Ca²⁺-free state. The predicted structure of the p26olfsingle polypeptide chain satisfactorily adopts a folding patternremarkably similar to dimeric S100B(ßß). Each domainof p26olf consists of a unicornate-type four-helix bundle andthey interact with each other in an antiparallel manner formingan X-type four-helix bundle between the two domains. The twoS100-like domains of p26olf are linked by a loop with no sterichindrance, suggesting that this loop might play an importantrole in the function of p26olf. The circular dichroism spectraldata support the predicted structure of p26olf and indicatethat Ca²⁺-dependent conformational changes occur. Since theC-terminal putative EF-hand of each domain fully keeps the helix–loop–helixmotif having a longer Ca²⁺-binding loop, regardless of the four-residueinsertion, we propose that it is a new, novel EF-hand, althoughit is unclear whether this EF-hand binds Ca²⁺. P26olf is a newmember of the S100 protein family.     

脱氧雪腐镰刀菌烯醇免疫分析核心试剂特异性单克隆抗体的研制与特性研究

采用1,1’-羰基二咪唑(CDI)活化法成功将脱氧雪腐镰刀菌烯醇分子上C-3和C-15位羟基与载体蛋白赖氨酸的氨基共价偶联得到免疫原DON-BSA和包被原DON-OVA,并用三硝基苯磺酸法对合成结果进行鉴定。以人工抗原免疫Balb/c小鼠,取小鼠脾细胞与SP2/O鼠骨髓瘤细胞融合,经筛选和多次亚克隆,得到了1株能稳定分泌脱氧雪腐镰刀菌烯醇抗体的单克隆细胞株1D5,并制备单克隆抗体腹水。经检测该抗体亚型为IgG1,亲和力常数Ka为8.33×107L/mol,交叉反应的试验结果显示该抗体与其他真菌毒素无交叉反应率,稳定性良好,为粮油食品中脱氧雪腐镰刀菌烯醇免疫快速检测技术建立及产品研发提供了关键试剂材料。     

Structure and humanization of a rat monoclonal Fab to human interleukin-5

The X-ray crystal structure of a rat monoclonal Fab JES1-39D10,raised against recombinant human interleukin-5, has been determinedwith the use of molecular replacement techniques and refinedat 2.7 Å resolution by simulated annealing. The overallstructure is similar to a murine Fab HyHEL-10 that is specificfor hen egg white lysozyme. An interesting feature of the structureis the presence of leucine residues to support the H1 complementarity-determiningregion (CDR) loop. To our knowledge this is the first Fab crystalstructure containing this unusual HI loop support pattern. Theactivity of three humanized versions of 39D10 is explained byanalysis of Fv interface residues and H1 support patterns of39D10 and the human template HLL.     

scFv multimers of the anti-neuraminidase antibody NC10: length of the linker between VH and VL domains dictates precisely the transition between diabodies and triabodies

Single-chain Fv antibody fragments (scFvs) incorporate a polypeptidelinker to tether the V_H and V_L domains together. An scFv moleculewith a linker 5–12 residues long cannot fold into a functionalFv domain and instead associates with a second scFv moleculeto form a bivalent dimer (diabody). Direct ligation of V_H andV_L domains further restricts association and forces three scFvmolecules to associate into a trivalent trimer (triabody). Wehave defined the effect of linker length on scFv associationby constructing a series of scFvs from anti-neuraminidase antibodyNC10 in which the linker varied from one to four glycine residues.NC10 scFv molecules containing linkers of three and four residuesshowed a strong preference for dimer formation (diabodies),whereas a linker length of one or two glycine residues preventedthe formation of diabodies and directed scFv association intotrimers (triabodies). The data suggest a relatively strict transitionfrom dimer (diabody) to trimer (triabody) upon reduction ofthe linker length from three to two glycine residues. Modellingstudies are consistent with three residues as the minimum linkerlength compatible with diabody formation. Electron microscopeimages of complexes formed between the NC10 scFv multimers andan anti-idiotype Fab' showed that the dimer was bivalent forantigen binding and the trimer was trivalent.     

Synthesis and expression in Escherichia coli of DNA encoding the murine {lambda}1 chain of a monoclonal antibody specific for Salmonella serotype B O-antigen

A 658 bp DNA sequence corresponding to the murine ₁ chain ofa monoclonal antibody, Se 155-4, specific for the Salmonellaserotype B O-antigen, was designed using Escherichia coli preferredcodons and chemically synthesized by ligation of synthetic fragmentsinto a linearized plasmid followed by transformation into E.coli.A synthetic signal peptide (ompA) was fused to express the Lchain as a free polypeptide into the periplasm of E.coli cells.After isolation and purification, heterologous recombinationof the E.coli L chain with mouse H chain gave an active antigen-bindingprotein. The activity was 15–20% when compared to proteincreated by an equivalent association of isolated natural mouseL and H chains as measured by a direct EIA assay. In inhibitionexperiments with the polysaccharide antigen, the two proteinsshowed identical titration curves and 50% inhibition points,indicating comparable K_A values.     

Synthesis, cloning and expression of the single-chain Fv gene of the HPr-specific monoclonal antibody, Jel42. Determination of binding constants with wild-type and mutant HPrs

The monoclonal antibody Jel42 is specific for the Escherichiacoli histidine-containing protein, HPr, which is an 85 aminoacid phosphocarrier protein of the phosphoenolpyruvate:sugarphosphotransferase system. The binding domain (Fv) has beenproduced as a single chain Fv (scFv). The scFv gene was synthesizedin vitro and coded for pelB leader peptide–heavy chain–linker–lightchain–(His)₅ tail. The linker is three repeats from theC-terminal repetitive sequence of eukaryotic RNA polymeraseII. This linker acts as a tag; it is the antigen for the monoclonalantibody Jel352. The codon usage was maximized for E.coli expression,and many unique restriction endonuclease sites were incorporated.The scFv gene incorporated into pT7-7 was highly expressed,yielding 10–30% of the cell protein as the scFv, whichwas found in inclusion bodies with the leader peptide cleaved.Jel42 scFv was purified by denaturation/renaturation yieldingpreparations with K_d values from 20 to 175 nM. However, basedupon an assessment of the amount of active refolded scFv, thebinding dissociation constant was estimated to be 2.7 ±2.0 nM compared with 2.8 ± 1.6 and 3.7 ± 0.3 nMpreviously determined for the Jel42 antibody and Fab fragmentrespectively. The effect of mutation of the antigen HPr on thebinding constant of the scFv was very similar to the propertiesdetermined for the antibody and the Fab fragment. It was concludedthat the small percentage (~6%) of refolded scFv is a true mimicof the Jel42 binding domain and that the incorrectly foldedscFv cannot be detected in the binding assay.