NMR-derived model for a peptide-antibody complex

The TE34 monoclonal antibody against cholera toxin peptide 3 (CTP3; VEVPGSQHIDSQKKA) was sequenced and investigated by two-dimensional transferred NOE difference spectroscopy and molecular modeling. The VH sequence of TE34, which does not bind cholera toxin, shares remarkable homology to that of TE32 and TE33, which are both anti-CTP3 antibodies that bind the toxin. However, due to a shortened heavy chain CDR3, TE34 assumes a radically different combining site structure. The assignment of the combining site interactions to specific peptide residues was completed by use of AcIDSQRKA, a truncated peptide analogue in which lysine-13 was substituted by arginine, specific deuteration of individual polypeptide chains of the antibody, and a computer model for the Fv fragment of TE34. NMR-derived distance restraints were then applied to the calculated model of the Fv to generate a three-dimensional structure of the TE34/CTP3 complex. The combining site was found to be a very hydrophobic cavity composed of seven aromatic residues. Charged residues are found in the periphery of the combining site. The peptide residues HIDSQKKA form a beta-turn inside the combining site. The contact area between the peptide and the TE34 antibody is 388 A2, about half of the contact area observed in protein-antibody complexes.     

Antibodies against a peptide of cholera toxin differing in cross-reactivity with the toxin differ in their specific interactions with the peptide as observed by 1H NMR spectroscopy.

The interactions between the aromatic residues of the monoclonal antibody TE34, and its peptide antigen CTP3, have been studied by 2D TRNOE difference spectroscopy. The sequence of CTP3 corresponds to residues 50-64 of the B subunit of cholera toxin (VEVPGSQHIDSQKKA). Unlike two previously studied anti-CTP3 antibodies (TE32 and TE33), the TE34 antibody does not bind the toxin. The off-rate of CTP3 from TE34 was found to be too slow to measure strong TRNOE cross-peaks between the antibody and the peptide. Much faster off-rates, resulting in a strong TRNOE, were obtained for two peptide analogues: (a) CTP3 with an amide in the C-terminus (VEVPGSQHIDSQKKA-NH2) and (b) a truncated version of the peptide (N-acetyl-IDSQKKA). These modifications do not interfere significantly either with the interactions of the unmodified part of the peptide with the antibody or with intramolecular interactions occurring in the epitope recognized by the antibody. The combined use of these peptides allows us to study the interactions between the antibody and the whole peptide. Two tyrosine residues and one or more tryptophan and phenylalanine residues have been found to interact with histidine-8, isoleucine-9, aspartate-10, lysine-13 and/or lysine-14, and alanine-15 of the peptide. In the bound peptide, we observe interactions of a lysine residue with aspartate-10 beta protons. While the peptide epitope recognized by TE34 is between histidine-8 and the negatively charged C-terminus, that recognized by TE32 and TE33 is between residues 3 and 10 of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR study of the complexes between a synthetic peptide derived from the B subunit of cholera toxin and three monoclonal antibodies against it

The contact interactions between a synthetic peptide and three different anti-peptide monoclonal antibodies have been studied by nuclear magnetic resonance (NMR). The synthetic peptide is CTP3 (residues 50-64 of the B subunit of cholera toxin) suggested as a possible epitope for synthetic vaccine against cholera. The hybridoma cell lines TE33 and TE32 derived after immunization with CTP3 produce antibodies cross-reactive with the native toxin. The cell line TE34 produces anti-CTP3 antibodies that do not bind the toxin. Selective deuteriation of the antibodies has been used to simplify the proton NMR spectra and to assign resonances to specific types of amino acids. The difference spectra between the proton NMR spectrum of the peptide-Fab complex and that of Fab indicate that the combining site structures of TE32 and TE33 are very similar but differ considerably from the combining site structure of TE34. By magnetization transfer experiments with selectively deuteriated Fab fragment of the antibody, we have found that in TE32 and TE33 the histidine residue of the peptide is buried in a hydrophobic pocket of the antibody combining site, formed by a tryptophan and two tyrosine residues. The hydrophobic nature of the pocket is further demonstrated by the lack of any pH titration effect on the chemical shift of the C4H of the bound peptide histidine. In contrast, for TE34 we have found only one tyrosine residue in contact with the histidine of the peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Probing antibody diversity by 2D NMR: comparison of amino acid sequences, predicted structures, and observed antibody-antigen interactions in complexes of two antipeptide antibodies

The interactions between the aromatic amino acids of two monoclonal antibodies (TE32 and TE33) with specific amino acid residues of a peptide of cholera toxin (CTP3) have been determined by two-dimensional (2D) transferred NOE difference spectroscopy. Aromatic amino acids are found to play an important role in peptide binding. In both antibodies two tryptophan and two tyrosine residues and one histidine residue interact with the peptide. In TE33 there is an additional phenylalanine residue that also interacts with the peptide. The residues of the CTP3 peptide that have been found to interact with the antibody are val 3, pro 4, gly 5, gln 7, his 8, and asp 10. We have determined the amino acid sequences of the two antibodies by direct mRNA sequencing. Computerized molecular modeling has been used to build detailed all-atom models of both antibodies from the known conformations of other antibodies. These models allow unambiguous assignment of most of the antibody residues that interact with the peptide. A comparison of the amino acid sequences of the two anti-CTP3 antibodies with other antibodies from the same gene family reveals that the majority of the aromatic residues involved in the binding of CTP3 are conserved although these antibodies have different specificities. This similarity suggests that these aromatic residues create a general hydrophobic pocket and that other residues in the complementarity-determining regions (CDRs) modulate the shape and the polarity of the combining site to fit the specific antigens.     

Use of 2D NMR, protein engineering, and molecular modeling to study the hapten-binding site of an antibody Fv fragment against 2-phenyloxazolone

Two-dimensional (2D) 1H NMR spectroscopy was used to study the hapten-binding site of a recombinant antibody Fv fragment expressed in Escherichia coli. Point mutations of residues in the CDR loops of the Fv fragment were designed in order to investigate their influence on hapten binding and to make site-specific assignments of aromatic NMR proton signals. Two tyrosines giving NOEs to the ligand 2-phenyloxazolone were identified, residue 33 in CDR1 of the heavy chain and residue 32 in CDR1 of the light chain. The benzyl portion of 2-phenyloxazolone is located between these two residues. The binding site is close to the surface of the Fv fragment. Comparison with a different anti-2-phenyloxazolone antibody, the crystal structure of which has recently been solved, shows that the general location of the hapten-binding site in both antibodies is similar. However, in the crystallographically solved antibody, the hapten is bound farther from the surface in a pocket created by a short CDR3 loop of the heavy chain. In the binding site identified in the Fv fragment studied in this report, this space is probably filled by the extra seven residues of the CDR3.     

Structure, function and properties of antibody binding sites

Do antibody combining sites possess general properties that enable them to bind different antigens with varying affinities and to bind novel antigens? Here, we address this question by examining the physical and chemical characteristics most favourable for residues involved in antigen accommodation and binding. Amphipathic amino acids could readily tolerate the change of environment from hydrophilic to hydrophobic that occurs upon antibody-antigen complex formation. Residues that are large and can participate in a wide variety of van der Waals' and electrostatic interactions would permit binding to a range of antigens. Amino acids with flexible side-chains could generate a structurally plastic region, i.e. a binding site possessing the ability to mould itself around the antigen to improve complementarity of the interacting surfaces. Hence, antibodies could bind to an array of novel antigens using a limited set of residues interspersed with more unique residues to which greater binding specificity can be attributed. An individual antibody molecule could thus be cross-reactive and have the capacity to bind structurally similar ligands. The accommodation of variations in antigenic structure by modest combining site flexibility could make an important contribution to immune defence by allowing antibody binding to distinct but closely related pathogens. Tyr and Trp most readily fulfil these catholic physicochemical requirements and thus would be expected to be common in combining sites on theoretical grounds. Experimental support for this comes from three sources, (1) the high frequency of participation by these amino acids in the antigen binding observed in six crystallographically determined antibody-antigen complexes, (2) their frequent occurrence in the putative binding regions of antibodies as determined from structural and sequence data and (3) the potential for movement of their side-chains in known antibody binding sites and model systems. The six bound antigens comprise two small different haptens, non-overlapping regions of the same large protein and a 19 amino acid residue peptide. Out of a total of 85 complementarity determining region positions, only 37 locations (plus 3 framework) are directly involved in antigen interaction. Of these, light chain residue 91 is utilized by all the complexes examined, whilst light chain 32, light chain 96 and heavy chain 33 are employed by five out of the six. The binding sites in known antibody-antigen complexes as well as the postulated combining sites in free Fab fragments show similar characteristics with regard to the types of amino acids present. The possible role of other amino acids is also assessed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Two-Dimensional nmr studies of the interactions between a peptide of cholera toxin and monoclonal antibodies

To increase our understanding of the molecular basis for antibody specificity and for the cross-reactivity of antipeptide antibodies with native proteins, it is important to study the three-dimensional structure of antibody complexes with their peptide antigens. For this purpose it may not be necessary to solve the structure of the whole antibody complex but rather to concentrate on elucidating the combining site structure, the interactions of the antibody with its antigen, and the bound peptide conformation. To extract the information about antibody–peptide interactions and intramolecular interactions in the bound ligand from the complicated and unresolved spectrum of the Fab–peptide complex (Fab: antibody fragment made of Fv—the antibody fragment composed of the variable regions of the light and heavy chains forming a single combining site for the antigen—the light chain, and the first heavy chain constant regions), an nmr methodology based on measurements of two-dimensional transferred nuclear Overhauser effect (NOE) difference spectra was developed. Using this methodology the interactions of three monoclonal antibodies with a cholera toxin peptide were studied. The observed interactions were assigned to the antibody protons involved by specific deuteration of aromatic amino acids and specific chain labeling, and by using a predicted model for the structure of the antibody combining site. The assigned NOE interactions were translated to restraints on interproton distances in the complex that were used to dock the peptide into calculated models for the antibodies combining sites. Comparison of the interactions of three antibodies against a cholera toxin peptide (CTP3). which differ in their cross-reactivity with the toxin, yields information about the size and conformation of antigenic determinants recognized by the antibodies, the structure of their combining sites, and relationships between antibodies' primary structure and their interactions with peptide antigens. © 1994 John Wiley & Sons, Inc.     

Dissecting contact potentials for proteins: relative contributions of individual amino acids

Mimotopes mimic the three-dimensional topology of an antigen epitope, and are frequently recognized by antibodies with affinities comparable to those obtained for the original antibody-antigen interaction. Peptides and anti-idiotypic antibodies are two classes of protein mimotopes that mimic the topology (but not necessarily the sequence) of the parental antigen. In this study, we combine these two classes by selecting mimotopes based on single domain IgNAR antibodies, which display exceptionally long CDR3 loop regions (analogous to a constrained peptide library) presented in the context of an immunoglobulin framework with adjacent and supporting CDR1 loops. By screening an in vitro phage-display library of IgNAR variable domains (V(NAR)s) against the target antigen monoclonal antibody MAb5G8, we obtained four potential mimotopes. MAb5G8 targets a linear tripeptide epitope (AYP) in the flexible signal sequence of the Plasmodium falciparum Apical Membrane Antigen-1 (AMA1), and this or similar motifs were detected in the CDR loops of all four V(NAR)s. The V(NAR)s, 1-A-2, -7, -11, and -14, were demonstrated to bind specifically to this paratope by competition studies with an artificial peptide and all showed enhanced affinities (3-46 nM) compared to the parental antigen (175 nM). Crystallographic studies of recombinant proteins 1-A-7 and 1-A-11 showed that the SYP motifs on these V(NAR)s presented at the tip of the exposed CDR3 loops, ideally positioned within bulge-like structures to make contact with the MAb5G8 antibody. These loops, in particular in 1-A-11, were further stabilized by inter- and intra- loop disulphide bridges, hydrogen bonds, electrostatic interactions, and aromatic residue packing. We rationalize the higher affinity of the V(NAR)s compared to the parental antigen by suggesting that adjacent CDR1 and framework residues contribute to binding affinity, through interactions with other CDR regions on the antibody, though of course definitive support of this hypothesis will rely on co-crystallographic studies. Alternatively, the selection of mimotopes from a large (<4 x 10(8)) constrained library may have allowed selection of variants with even more favorable epitope topologies than present in the original antigenic structure, illustrating the power of in vivo selection of mimotopes from phage-displayed molecular libraries.     

A central core structure in an antibody variable domain determines antigen specificity

Antibody binding sites provide an adaptable surface capable of interacting with essentially any molecular target. Using CDR shuffling, residues important for the assembly of mucin-1 specific paratopes were defined by random recombination of the complementarity determining regions derived from a set of mucin-1 specific clones, previously selected from an antibody fragment library. It was found that positions 33 and 50 in the heavy chain and 32, 34, 90, 91 and 96 in the light chain were conserved in many of the clones. These particular residues seem to be located centrally in the binding site as indicated by a structure model analysis. The importance of several of these conserved residues was supported by their presence in a mouse monoclonal antibody with a known structure and the same epitope specificity. Several of these corresponding residues in the mouse monoclonal antibody are known to interact with the antigen. In conclusion, critical residues important for maintaining a human antigen-specific binding site during the process of in vitro antibody evolution were defined. Furthermore, an explanation for the observed restricted germline gene usage in certain antibody responses against protein epitopes is provided.     

A 1H NMR method for the analysis of antigen-antibody interactions: binding of a peptide fragment of lysozyme to anti-lysozyme monoclonal antibody

A proton nuclear magnetic resonance (NMR) study is reported of the molecular structural basis of antigen-antibody interactions. An immunologically reactive proteolytic fragment corresponding to one of the antigenic regions on hen egg-white lysozyme (HEL) was used in combination with a monoclonal antibody that recognizes this site. Using spin diffusion, we prepared an antibody in which the magnetization of the antigen binding site was saturated by non-specific nuclear Overhauser effect. Under these conditions the effect of the saturation of the antibody was observed to spread over the peptide fragment through the antigen binding site. On the basis of the results obtained for the intermolecular nuclear Overhauser effect, we discuss how the peptide fragment interacts with the antibody. The side chains of aromatic residues, Trp, Tyr, and His, and of ionic residues, especially Arg, Lys, and Glu, are suggested to be important in the antigen-antibody interaction.     

Interactions of antibody aromatic residues with a peptide of cholera toxin observed by two-dimensional transferred nuclear Overhauser effect difference spectroscopy

The interactions between a peptide of cholera toxin and the aromatic amino acids of the TE33 antipeptide antibody, cross-reactive with the toxin, have been studied by NOESY difference spectroscopy. The 2D difference between the NOESY spectrum of the Fab with a 4-fold excess of the peptide and that of the peptide-saturated Fab reveals cross-peaks growing with excess of the peptide. These cross-peaks are due to magnetization transfer between the Fab and neighboring bound peptide protons, and a further transfer to the free peptide protons by exchange between bound and free peptide (transferred NOE). Additional cross-peaks appearing in the difference spectrum are due to a combination of intramolecular interactions between bound peptide protons and exchange between bound and free peptide. Assignment of cross-peaks is attained by specific deuteration of antibody aromatic amino acids using also the resonance assignment of the free peptide, deduced from the COSY spectrum of the peptide solution. The antibody combining site is found to be highly aromatic. We have identified one or two histidine, two tyrosine, and two tryptophan residues and one phenylalanine residue of the antibody interacting with valine-3, proline-4, glycine-5, glutamine-7, histidine-8, and aspartate-10 of the peptide. The 2D TRNOE difference spectroscopy can be used to study protein-ligand interactions, given that the ligand off rate is fast relative to the spin-lattice relaxation time of the protein and ligand protons (about 1 s). The resolution obtained in the difference spectra implies that the technique is equally applicable for studying proteins having a molecular weight larger than 50,000.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antigen-antibody interaction. Synthetic peptides define linear antigenic determinants recognized by monoclonal antibodies directed to the cytoplasmic carboxyl terminus of rhodopsin

The specificities of four monoclonal antibodies rho 1D4, 1C5, 3A6, and 3D6 prepared by immunization of rod outer segments containing rhodopsin have been defined using synthetic peptides. All of these antibodies interact within the 18 residues at the COOH terminus of rhodopsin and recognize linear antigenic determinants of 4-11 residues. Twenty-seven synthetic peptide analogs of varying lengths of native sequence or containing single amino acid substitutions at each position of the COOH-terminal 18 residues have provided some insight into the mechanism of antigen-antibody binding. Our results clearly demonstrate that antibodies can be highly specific at key positions as shown by the loss of binding on single amino acid substitutions in the binding site. In contrast single amino acid substitutions at other positions in the binding site only affect affinity for some antibodies. Ionic interactions can dominate immunogenic determinants. Immunogenic determinants are not restricted to highly charged hydrophilic regions on the surface of a protein and may be dominated by hydrophobic interactions. Although certain side chains can dominate the interaction of the antigen with antibody, our results are in agreement with the interpretation that the free energies of all the contact points are additive and a certain free energy must be present to achieve binding. Antibodies with different specificities directed to the same region of the protein antigen can be produced in an immune response. Peptide antigens representing regions of a protein antigen bind best to the anti-protein antibody when the sequence is shortened to contain only those residues binding to the specificity site in the antibody. Cross-reactivity between protein antigens can be explained by conservation of the critical residues in the combining site.     

A model of a gp120 V3 peptide in complex with an HIV-neutralizing antibody based on NMR and mutant cycle-derived constraints.

The 0.5beta monoclonal antibody is a very potent strain-specific HIV-neutralizing antibody raised against gp120, the envelope glycoprotein of HIV-1. This antibody recognizes the V3 loop of gp120, which is a major neutralizing determinant of the virus. The antibody-peptide interactions, involving aromatic and negatively charged residues of the antibody 0.5beta, were studied by NMR and double-mutant cycles. A deuterated V3 peptide and a Fab containing deuterated aromatic amino acids were used to assign these interactions to specific V3 residues and to the amino acid type and specific chain of the antibody by NOE difference spectroscopy. Electrostatic interactions between negatively charged residues of the antibody Fv and peptide residues were studied by mutagenesis of both antibody and peptide residues and double-mutant cycles. Several interactions could be assigned unambiguously: F96(L) of the antibody interacts with Pro13 of the peptide, H52(H) interacts with Ile7, Ile9 and Gln10 and D56(H) interacts with Arg11. The interactions of the light-chain tyrosines with Pro13 and Gly14 could be assigned to either Y30a(L) and Y32(L), respectively, or Y32(L) and Y49(L), respectively. Three heavy-chain tyrosines interact with Ile7, Ile20 and Phe17. Several combinations of assignments involving Y32(H), Y53(H), Y96(H) and Y100a(H) may satisfy the NMR and mutagenesis constraints, and therefore at this stage the interactions of the heavy-chain tyrosines were not taken into account. The unambiguous assignments [F96(L), H52(H) and D56(H)] and the two possible assignments of the light-chain tyrosines were used to dock the peptide into the antibody-combining site. The peptide converges to a unique position within the binding site, with the RGPG loop pointing into the center of the groove formed by the antibody complementary determining regions while retaining the beta-hairpin conformation and the type-VI RGPG turn [Tugarinov, V., Zvi, A., Levy, R. & Anglister, J. (1999) Nat. Struct. Biol. 6, 331-335].     

Structural diversity in a conserved cholera toxin epitope involved in ganglioside binding.

Cholera is a widespread disease for which there is no efficient vaccine. A better understanding of the conformational rearrangements at the epitope might be very helpful for the development of a good vaccine. Cholera toxin (CT) as well as the closely related heat-labile toxin from Escherichia coli (LT) are composed of two subunits, A and B, which form an oligomeric assembly AB5. Residues 50-64 on the surface of the B subunits comprise a conserved loop (CTP3), which is involved in saccharide binding to the receptor on epithelial cells. This loop exhibits remarkable conformational plasticity induced by environmental constraints. The crystal structure of this loop is compared in the free and receptor-bound toxins as well as in the crystal and solution structures of a complex with TE33, a monoclonal antibody elicited against CTP3. In the toxins this loop forms an irregular structure connecting a beta-strand to the central alpha-helix. Ser 55 and Gln 56 exhibit considerable conformational variability in the five subunits of the unliganded toxins. Saccharide binding induces a change primarily in Ser 55 and Gln 56 to a conformation identical in all five copies. Thus, saccharide binding confers rigidity upon the loop. The conformation of CTP3 in complex with TE33 is quite different. The amino-terminal part of CTP3 forms a beta-turn that fits snugly into a deep binding pocket on TE33, in both the crystal and NMR-derived solution structure. Only 8 and 12 residues out of 15 are seen in the NMR and crystal structures, respectively. Despite these conformational differences, TE33 is cross-reactive with intact CT, albeit with a thousandfold decrease in affinity. This suggests a different interaction of TE33 with intact CT.     

NMR studies of carbohydrates and carbohydrate-mimetic peptides recognized by an anti-group B Streptococcus antibody

As part of a program to investigate the origins of peptide-carbohydrate mimicry, the conformational preferences of peptides that mimic the group B streptococcal type III capsular polysaccharide have been investigated by NMR spectroscopy. Detailed studies of a dodecapeptide, FDTGAFDPDWPA, a molecular mimic of the polysaccharide antigen, and two new analogs, indicated a propensity for beta-turn formation. Different beta-turn types were found to be present in the trans and cis (Trp-10-Pro-11) isomers of the peptide: the trans isomer favored a type I beta-turn from residues Asp-7-Trp-10, whereas the cis isomer exhibited a type VI beta-turn from residues Asp-9-Ala-12. The interaction of the dodecapeptide FDTGAFDPDWPA with a protective anti-group B Streptococcus monoclonal antibody has also been investigated, by transferred nuclear Overhauser effect NMR spectroscopy and saturation-transfer difference NMR spectroscopy (STD-NMR). The peptide was found to adopt a type I beta-turn conformation on binding to the antibody; the peptide residues (Asp-7-Trp-10) forming this turn are recognized by the antibody, as demonstrated by STD-NMR experiments. STD-NMR studies of the interactions of oligosaccharide fragments of the capsular polysaccharide have also been performed and provide evidence for the existence of a conformational epitope.     

Binding of complement by complexes between antibodies to nucleic acid constituents and short oligodeoxyribonucleotides

Oligodeoxyribonucleotides act as inhibitors of the complement fixation caused by complexes between antibodies to defined oligodeoxyribonucleotides and denatured DNA. At concentrations higher than 50 micrograms oligodeoxyribonucleotide/ml complement fixation occurred in the absence of antigen. The extent of complement binding depends on the specificity of the antibodies as well as on the composition of the oligodeoxyribonucleotides. Complement fixation is observed most strongly with antisera to oligodeoxyribonucleotides and to denatured DNA, which belong predominantly to the IgM class. With two LE-sera, containing antibodies to denatured and to native DNA, no complement fixation was found. It is supposed that specific interactions of the oligodeoxyribonucleotides with amino acid residues closely neighbored to the antibody combining site lead to conformational changes in the antibody molecules and to an activation of the complement binding site.     

Structural insights into parallel strategies for germline antibody recognition of lipopolysaccharide from Chlamydia

The structure of the antigen-binding fragment from the monoclonal antibody S64-4 in complex with a pentasaccharide bisphosphate fragment from chlamydial lipopolysaccharide has been determined by x-ray diffraction to 2.6 ? resolution. Like the well-characterized antibody S25-2, S64-4 displays a pocket formed by the residues of germline sequence corresponding to the heavy and light chain V gene segments that binds the terminal Kdo residue of the antigen; however, although S64-4 shares the same heavy chain V gene segment as S25-2, it has a different light chain V gene segment. The new light chain V gene segment codes for a combining site that displays greater affinity, different specificity, and allows a novel antigen conformation that brings a greater number of antigen residues into the combining site than possible in S25-2. Further, while antibodies in the S25-2 family use complementarity determining region (CDR) H3 to discriminate among antigens, S64-4 achieves its specificity via the new light chain V gene segment and resulting change in antigen conformation. These structures reveal an intriguing parallel strategy where two different combinations of germline-coded V gene segments can act as starting points for the generation of germline antibodies against chlamydial antigens and show how anti-carbohydrate antibodies can exploit the conformational flexibility of this class of antigens to achieve high affinity and specificity independently of CDR H3.     

A peptide mimetic of an anti-CD4 monoclonal antibody by rational design

The development of rational methods to design 'continuous' sequence mimetics of discontinuous regions of protein sequence has, to now, been only marginally successful. This has been largely due to the difficulty of constraining the recognition elements of a mimetic structure to the relative conformational and spatial orientations present in the parent molecule. Using peptide mapping to determine 'active' antigen recognition residues, molecular modeling, and a molecular dynamics trajectory analysis, we have developed a peptide mimic of an anti-CD4 antibody, containing antigen contact residues from multiple CDRs. The design described is a 27-residue peptide formed by juxtaposition of residues from 5 CDR regions. It displays an affinity for the antigen (CD4) of 0.9nM, compared to 2nM for the parent antibody ST40. Nevertheless, the mimetic shows low biological activity in an anti-retroviral assay.     

Humanization of a murine monoclonal antibody by simultaneous optimization of framework and CDR residues.

Optimal protein function often depends on co-operative interactions between amino acid residues distant in the protein primary sequence yet spatially near one another following protein folding. For example, antibody affinity is influenced by interactions of framework residues with complementarity-determining region (CDR) residues. However, despite the abundance of antibody structural information and computational tools the humanization of rodent antibodies for clinical use often results in a significant loss of affinity. To date, antibody engineering efforts have focused either on optimizing CDR residues involved in antigen binding or on optimizing antibody framework residues that serve critical roles in preserving the conformation of CDRs. In the present study a new approach which permits the rapid identification of co-operatively interacting framework and CDR residues was used to simultaneously humanize and optimize a murine antibody directed against CD40. Specifically, a combinatorial library that examined eight potentially important framework positions concomitantly with focused CDR libraries consisting of variants containing random single amino acid mutations in the third CDR of the heavy and light chains was expressed. Multiple anti-CD40 Fab variants containing as few as one murine framework residue and displaying up to approximately 500-fold higher affinity than the initial chimeric Fab were identified. The higher affinity humanized variants demonstrated a co-operative interaction between light chain framework residue Y49 and heavy chain CDR3 residue R/K101 (coupling energy, DeltaGI=0.9 kcal/mol). Screening of combinatorial framework-CDR libraries permits identification of monoclonal antibodies (mAb) with structures optimized for function, including instances in which the antigen induces conformational changes in the mAb. Moreover, the enhanced humanized variants contain fewer murine framework residues and could not be identified by sequential in vitro humanization and affinity muturation strategies. This approach to identifying co-operatively interacting residues is not restricted to antibody-antigen interactions and consequently, may be used broadly to gain insight into protein structure-function relationships, including proteins that serve as catalysts.     

Structural Basis for the Recognition of Human Cytomegalovirus Glycoprotein B by a Neutralizing Human Antibody

Human cytomegalovirus (HCMV) infections are life-threating to people with a compromised or immature immune system. Upon adhesion, fusion of the virus envelope with the host cell is initiated. In this step, the viral glycoprotein gB is considered to represent the major fusogen. Here, we present for the first time structural data on the binding of an anti-herpes virus antibody and describe the atomic interactions between the antigenic domain Dom-II of HCMV gB and the Fab fragment of the human antibody SM5-1. The crystal structure shows that SM5-1 binds Dom-II almost exclusively via only two CDRs, namely light chain CDR L1 and a 22-residue-long heavy chain CDR H3. Two contiguous segments of Dom-II are targeted by SM5-1, and the combining site includes a hydrophobic pocket on the Dom-II surface that is only partially filled by CDR H3 residues. SM5-1 belongs to a series of sequence-homologous anti-HCMV gB monoclonal antibodies that were isolated from the same donor at a single time point and that represent different maturation states. Analysis of amino acid substitutions in these antibodies in combination with molecular dynamics simulations show that key contributors to the picomolar affinity of SM5-1 do not directly interact with the antigen but significantly reduce the flexibility of CDR H3 in the bound and unbound state of SM5-1 through intramolecular side chain interactions. Thus, these residues most likely alleviate unfavorable binding entropies associated with extra-long CDR H3s, and this might represent a common strategy during antibody maturation. Models of entire HCMV gB in different conformational states hint that SM5-1 neutralizes HCMV either by blocking the pre- to postfusion transition of gB or by precluding the interaction with additional effectors such as the gH/gL complex.