Crystal structures of two mutant neuraminidase-antibody complexes with amino acid substitutions in the interface.

The site on influenza virus N9 neuraminidase recognized by NC41 monoclonal antibody comprises 19 amino acid residues that are in direct contact with 17 residues on the antibody. Single sequence changes in some of the neuraminidase residues in the site markedly reduce antibody binding. However, two mutants have been found within the site, Ile368 to Arg and Asn329 to Asp selected by antibodies other than NC41, and these mutants bind NC41 antibody with only slightly reduced affinity. The three-dimensional structures of the two mutant N9-NC41 antibody complexes as derived from the wild-type complex are presented. Both structures show that some amino acid substitutions can be accommodated within an antigen-antibody interface by local structural rearrangements around the mutation site. In the Ile368 to Arg mutant complex, the side-chain of Arg368 is shifted by 2.9 A from its position in the uncomplexed mutant and a shift of 1.3 A in the position of the light chain residue HisL55 with respect to the wild-type complex is also observed. In the other mutant, the side-chain of Asp329 appears rotated by 150 degrees around C alpha-C beta with respect to the uncomplexed mutant, so that the carboxylate group is moved to the periphery of the antigen-antibody interface. The results provide a basis for understanding some of the potential structural effects of somatic hypermutation on antigen-antibody binding in those cases where the mutation in the antibody occurs at antigen-contacting residues, and demonstrate again the importance of structural context in evaluating the effect of amino acid substitutions on protein structure and function.     

Structural basis for antibody catalysis of a cationic cyclization reaction

Antibody 4C6 efficiently catalyzes a cationic cyclization reaction. Crystal structures of the antibody 4C6 Fab in complex with benzoic acid and in complex with its eliciting hapten were determined to 1.30A and 2.45A resolution, respectively. These crystal structures, together with computational analysis, have elucidated a possible mechanism for the monocyclization reaction. The hapten complex revealed a combining site pocket with high shape complementarity to the hapten. This active site cleft is dominated by aromatic residues that shield the highly reactive carbocation intermediates from solvent and stabilize the carbocation intermediates through cation-pi interactions. Modeling of an acyclic olefinic sulfonate ester substrate and the transition state (TS) structures shows that the chair-like transition state is favored, and trapping by water directly produces trans-2-(dimethylphenylsilyl)-cyclohexanol, whereas the less favored boat-like transition state leads to cyclohexene. The only significant change observed upon hapten binding is a side-chain rotation of Trp(L89), which reorients to form the base of the combining site. Intriguingly, a benzoic acid molecule was sequestered in the combining site of the unliganded antibody. The 4C6 active site was compared to that observed in a previously reported tandem cyclization antibody 19A4 hapten complex. These cationic cyclization antibodies exhibit convergent structural features with terpenoid cyclases that appear to be important for catalysis.     

Elbow flexibility and ligand-induced domain rearrangements in antibody Fab NC6.8: large effects of a small hapten

Four 700-ps molecular dynamics simulations were carried out to analyze the structural dynamics of the antigen-binding antibody fragment NC6.8, which is known to exhibit large structural changes upon complexation. The first simulation was started from the x-ray structure of the uncomplexed Fab and produced trajectory averages that closely match the crystallographic results. It allowed assessment of the flexibility of the Fab, revealing an elbow motion of the variable domains with respect to the constant domains. The second simulation was started from the uncomplexed x-ray structure after insertion of the ligand into the binding site. This perturbation resulted in a significantly altered trajectory, with quaternary structural changes corresponding in many aspects to the experimental differences between complexed and uncomplexed state. The observed trend toward a smaller elbow angle and a higher flexion of the H-chain could also be seen in the third simulation, which was started from the x-ray structure of the complex. The changes were revealed to be a clear consequence of the complexation with the ligand because in the fourth simulation (started from the experimental complex structure after removal of the hapten) the Fab remained close to its initial structure. Analyses of the quaternary structure and the binding site of Fab NC6.8 are presented for all four simulations, and possible interpretations are discussed.     

Refined structures of Bobwhite quail lysozyme uncomplexed and complexed with the HyHEL-5 Fab fragment

The HyHEL-5 antibody has more than a thousandfold lower affinity for bobwhite quail lysozyme (BWQL) than for hen egg-white lysozyme (HEL). Four sequence differences exist between BWQL and HEL, of which only one is involved in the interface with the Fab. The structure of bobwhite quail lysozyme has been determined in the uncomplexed state in two different crystal forms and in the complexed state with HyHEL-5, an anti-hen egg-white lysozyme Fab. Similar backbone conformations are observed in the three molecules of the two crystal forms of uncomplexed BWQL, although they show considerable variability in side-chain conformation. A relatively mobile segment in uncomplexed BWQL is observed to be part of the HyHEL-5 epitope. No major backbone conformational differences are observed in the lysozyme upon complex formation, but side-chain conformational differences are seen in surface residues that are involved in the interface with the antibody. The hydrogen bonding in the interface between BWQL and HyHEL-5 is similar to that in previously determined lysozyme-HyHEL-5 complexes. © 1996 Wiley-Liss, Inc.     

Refined crystal structure of the influenza virus N9 neuraminidase-NC41 Fab complex.

The crystal structure of the complex between neuraminidase from influenza virus (subtype N9 and isolated from an avian source) and the antigen-binding fragment (Fab) of monoclonal antibody NC41 has been refined by both least-squares and simulated annealing methods to an R-factor of 0.191 using 31,846 diffraction data in the resolution range 8.0 to 2.5 A. The resulting model has a root-mean-square deviation from ideal bond-length of 0.016 A. One fourth of the tetrameric complex comprises the crystallographic model, which has 6577 non-hydrogen atoms and consists of 389 protein residues and eight carbohydrate residues in the neuraminidase, 214 residues in the Fab light chain, and 221 residues in the heavy chain. One putative Ca ion buried in the neuraminidase, and 73 water molecules, are also included. A remarkable shape complementarity exists between the interacting surfaces of the antigen and the antibody, although the packing density of atoms at the interface is somewhat looser than in the interior of a protein. Similarly, there is a high degree of chemical complementarity between the antigen and antibody, mediated by one buried salt-link, two solvated salt-links and 12 hydrogen bonds. The antibody-binding site on neuraminidase is discontinuous and comprises five chain segments and 19 residues in contact, whilst 33 neuraminidase residues in eight segments have 899 A2 of surface area buried by the interaction (to a 1.7 A probe), including two hexose units. Seventeen residues in NC41 Fab lying in five of the six complementarity determining regions (CDRs) make contact with the neuraminidase and 36 antibody residues in seven segments have 916 A2 of buried surface area. The interface is more extensive than those of the three lysozyme-Fab complexes whose crystal structures have been determined, as judged by buried surface area and numbers of contact residues. There are only small differences (less than 1.5 A) between the complexed and uncomplexed neuraminidase structures and, at this resolution and accuracy, those differences are not unequivocal. The main-chain conformations of five of the CDRs follow the predicted canonical structures. The interface between the variable domains of the light and heavy chains is not as extensive as in other Fabs, due to less CDR-CDR interaction in NC41. The first CDR on the NC41 Fab light chain is positioned so that it could sterically hinder the approach of small as well as large substrates to the neuraminidase active-site pocket, suggesting a possible mechanism for the observed inhibition of enzyme activity by the antibody.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Crystal structure of an anti-interleukin-2 monoclonal antibody Fab complexed with an antigenic nonapeptide

The three-dimensional structure of the Fab fragment of a monoclonal antibody (LNKB-2) to human interleukin-2 (IL-2) complexed with a synthetic antigenic nonapeptide, Ac-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-OMe, has been determined at 3.0 A resolution. In the structure, four out of the six hypervariable loops of the Fab (complementarity determining regions [CDRs] L1, H1, H2, and H3) are involved in peptide association through hydrogen bonding, salt bridge formation, and hydrophobic interactions. The Tyr residues in the Fab antigen binding site play a major role in antigen-antibody recognition. The structures of the complexed and uncomplexed Fab were compared. In the antigen binding site the CDR-L1 loop of the antibody shows the largest structural changes upon peptide binding. The peptide adopts a mostly alpha-helical conformation similar to that in the epitope fragment 64-72 of the IL-2 antigen. The side chains of residues Leu 66, Val 69, and Leu 70, which are shielded internally in the IL-2 structure, are involved in interactions with the Fab in the complex studied. This indicates that antibody-antigen complexation involves a significant rearrangement of the epitope-containing region of the IL-2 with retention of the alpha-helical character of the epitope fragment.     

Revisiting the structural flexibility of the complex p21(ras)-GTP: the catalytic conformation of the molecular switch II.

The hydrolysis of GTP in p21(ras) triggers conformational changes that regulate the ras/ERK signaling pathway. An important active site residue is Gln61, which has been found to be mutated in 30% of human tumors. The dynamics of the active site conformation is studied by using molecular dynamics simulation of two independent structures of the GTP-bound uncomplexed enzyme. Two distinct conformations of the enzyme are observed, in which the side-chain residue Gln61 is in different orientations. Essential dynamics analysis is used to describe the essential motions in the transition between the two conformations. Results are compared with earlier simulations of p21(ras) and its complex with GTPase activating protein p21-GAP.     

Molecular modeling of antibody-antigen complexes between the Brucella abortus O-chain polysaccharide and a specific monoclonal antibody.

A molecular model of the binding site of an anti-carbohydrate antibody (YsT9.1) has been developed using computer-assisted modeling techniques and molecular dynamics calculations. Sequence homologies among YsT9.1 and the Fv regions of McPC603, J539 and human Bence--Jones protein REI, all of which have solved crystal structures, provided the basis for the modeling. The groove-type combining site model had a topography which was complementary to low energy conformers of the polysaccharide, a Brucella O-antigen, and the site could be almost completely filled by a pentasaccharide epitope in either of two docking modes. Putative interactions between this epitope and the antibody are consistent with the known structural requirements for binding and lead to the design of oligosaccharide inhibitors that probe the veracity of the modeled docked complex. Ultimately both the Fv model and the docked complex will be compared with independent crystal structures of YsT9.1 Fab with and without pentasaccharide inhibitor, currently at the stage of refinement.     

Crystal structure of a human rhinovirus neutralizing antibody complexed with a peptide derived from viral capsid protein VP2.

The three-dimensional structure of the complex between the Fab fragment of an anti-human rhinovirus neutralizing antibody (8F5) and a cross-reactive synthetic peptide from the viral capsid protein VP2 has been determined at 2.5 A resolution by crystallographic methods. The refinement is presently at an R factor of 0.18 and the antigen-binding site and viral peptide are well defined. The peptide antigen adopts a compact fold by two tight turns and interacts through hydrogen bonds, some with ionic character, and van der Waals contacts with antibody residues from the six hypervariable loops as well as several framework amino acids. The conformation adopted by the peptide is closely related to the corresponding region of the viral protein VP2 on the surface of human rhinovirus 1A whose three-dimensional structure is known. Implications for the cross-reactivity between peptides and the viral capsid are discussed. The peptide-antibody interactions, together with the analysis of mutant viruses that escape neutralization by 8F5 suggest two different mechanisms for viral escape. The comparison between the complexed and uncomplexed antibody structures shows important conformational rearrangements, especially in the hypervariable loops of the heavy chain. Thus, it constitutes a clear example of the 'induced fit' molecular recognition mechanism.     

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)     

Structural modeling of the complex between an acetylcholine receptor-mimicking antibody and its snake toxin antigen

The antibody Mα2-3 neutralizes the functional, acetylcholine receptor binding activity of its antigen, neurotoxin α, and exhibits several other properties in common with the receptor itself. We present here the results of calculations examining the three-dimensional structure of the toxin α:Mα2-3 complex. The antigen structure, determined by nuclear magnetic resonance spectroscopy,¹ was docked to models of the variable fragment of the antibody combining site² by using a method based on surface complementarity and maximization of buried surface area^3,4 while taking into account the possibility of conformational change on complexation. Extensive experimental information on the location of the functional epitope was incorporated into the analysis and used to screen candidate geometries of the complex resulting from the modeling. Eight plausible structures that are in accord with the experimental data were derived. Common structural features of the models are discussed, and residues of the antibody-combining site that are expected to play important roles in complexation are identified. In particular, three epitope residues that, according to mutagenesis experiments, make particularly strong contributions to the binding, interact excentrically and do not make contact with the central loops of the combining site, L3 and H3. Proteins 30:249–263, 1998. © 1998 Wiley-Liss, Inc.     

Protein-protein binding is often associated with changes in protonation state

pK(a) values of ionizable residues have been calculated using the PROPKA method and structures of 75 protein-protein complexes and their corresponding free forms. These pK(a) values were used to compute changes in protonation state of individual residues, net changes in protonation state of the complex relative to the uncomplexed proteins, and the correction to a binding energy calculated assuming standard protonation states at pH 7. For each complex, two different structures for the uncomplexed form of the proteins were used: the X-ray structures determined for the proteins in the absence of the other protein and the individual protein structures taken from the structure of the complex (referred to as unbound and bound structures, respectively). In 28 and 77% of the cases considered here, protein-protein binding is accompanied by a complete (>95%) or significant (>50%) change in protonation state of at least one residue using unbound structures. Furthermore, in 36 and 61% of the cases, protein-protein binding is accompanied by a complete or significant net change in protonation state of the complex relative to the separated monomers. Using bound structures, the corresponding values are 12, 51, 20, and 48%. Comparison to experimental data suggest that using unbound and bound structures lead to over- and underestimation of binding-induced protonation state changes, respectively. Thus, we conclude that protein-protein binding is often associated with changes in protonation state of amino acid residues and with changes in the net protonation state of the proteins. The pH-dependent correction to the binding energy contributes at least one order of magnitude to the binding constant in 45 and 23%, using unbound and bound structures, respectively.     

Three-dimensional structures of the free and antigen-bound Fab from monoclonal antilysozyme antibody HyHEL-63(,)

Antigen-antibody complexes provide useful models for studying the structure and energetics of protein-protein interactions. We report the cloning, bacterial expression, and crystallization of the antigen-binding fragment (Fab) of the anti-hen egg white lysozyme (HEL) antibody HyHEL-63 in both free and antigen-bound forms. The three-dimensional structure of Fab HyHEL-63 complexed with HEL was determined to 2.0 A resolution, while the structure of the unbound antibody was determined in two crystal forms, to 1.8 and 2.1 A resolution. In the complex, 19 HyHEL-63 residues from all six complementarity-determining regions (CDRs) of the antibody contact 21 HEL residues from three discontinuous polypeptide segments of the antigen. The interface also includes 11 bound water molecules, 3 of which are completely buried in the complex. Comparison of the structures of free and bound Fab HyHEL-63 reveals that several of the ordered water molecules in the free antibody-combining site are retained and that additional waters are added upon complex formation. The interface waters serve to increase shape and chemical complementarity by filling cavities between the interacting surfaces and by contributing to the hydrogen bonding network linking the antigen and antibody. Complementarity is further enhanced by small (<3 A) movements in the polypeptide backbones of certain antibody CDR loops, by rearrangements of side chains in the interface, and by a slight shift in the relative orientation of the V(L) and V(H) domains. The combining site residues of complexed Fab HyHEL-63 exhibit reduced temperature factors compared with those of the free Fab, suggesting a loss in conformational entropy upon binding. To probe the relative contribution of individual antigen residues to complex stabilization, single alanine substitutions were introduced in the epitope of HEL recognized by HyHEL-63, and their effects on antibody affinity were measured using surface plasmon resonance. In agreement with the crystal structure, HEL residues at the center of the interface that are buried in the complex contribute most to the binding energetics (DeltaG(mutant) - DeltaG(wild type) > 3.0 kcal/mol), whereas the apparent contributions of solvent-accessible residues at the periphery are much less pronounced (<1.5 kcal/mol). In the latter case, the mutations may be partially compensated by local rearrangements in solvent structure that help preserve shape complementarity and the interface hydrogen bonding network.     

Unexpected active-site flexibility in the structure of human neutrophil elastase in complex with a new dihydropyrimidone inhibitor

Human neutrophil elastase (HNE), a trypsin-type serine protease, is of pivotal importance in the onset and progression of chronic obstructive pulmonary disease (COPD). COPD encompasses a group of slowly progressive respiratory disorders and is a major medical problem and the fifth leading cause of death worldwide. HNE is a major target for the development of compounds that inhibit the progression of long-term lung function decline in COPD patients.Here, we present the three-dimensional structure of a potent dihydropyrimidone inhibitor (DHPI) non-covalently bound to HNE at a resolution of 2.0 Å. The inhibitor binds to the active site in a unique orientation addressing S1 and S2 subsites of the protease. To facilitate further analysis of this binding mode, we determined the structure of the uncomplexed enzyme at a resolution of 1.86 Å. Detailed comparisons of the HNE:DHPI complex with the uncomplexed HNE structure and published structures of other elastase:inhibitor complexes revealed that binding of DHPI leads to large conformational changes in residues located in the S2 subsite. The rearrangement of residues Asp95-Leu99B creates a deep, well-defined cavity, which is filled by the P2 moiety of the inhibitor molecule to almost perfect shape complementarity. The shape of the S2 subsite in complex with DHPI clearly differs from all other observed HNE structures. The observed structural flexibility of the S2 subsite is a key feature for the understanding of the binding mode of DHPIs in general and the development of new HNE selective inhibitors.     

Modelling of the combining sites of three anti-lysozyme monoclonal antibodies and of the complex between one of the antibodies and its epitope.

Models of the antigen combining sites of three monoclonal antibodies, which recognise different but overlapping epitopes within the 'loop' region of hen egg lysozyme (HEL), have been generated from the cDNA sequences of their Fv regions (the VL and VH domains) and the known crystal structures of immunoglobulin fragments. The alpha-carbon backbone of the structurally conserved framework region has been derived from the IgG myeloma protein NEW, and models for the hypervariable loop regions have been selected on the basis of length and maximum sequence homology. The model structures have been refined by energy minimisation. Both the size and chemical nature of the predicted combining site models correlate broadly with the epitope boundaries previously determined by affinity studies. A model of the complex formed between one antibody and the corresponding lysozyme epitope is described, and contact residues are identified for subsequent testing by oligonucleotide-directed site-specific mutagenesis.     

MD simulations of anthrax edema factor: calmodulin complexes with mutations in the edema factor "switch a" region and docking of 3'-deoxy ATP into the adenylyl cyclase active site of wild-type and mutant edema factor variants

Bacillus anthracis, a spore-forming infectious bacterium, produces an exotoxin, called the edema factor (EF), that functions in part by disrupting internal signalling pathways. When complexed with human host cell calmodulin (CaM), EF becomes an active adenylyl cyclase, producing the internal signal substance cyclic-AMP in an uncontrolled fashion. Recently, the crystal structures for uncomplexed EF and EF:CaM complexes in the presence and absence of a substrate analog (3'-deoxy-ATP), were reported. EF mutational studies have implicated a number of residues important in CaM binding and/or in the generation of the adenylyl cyclase active site, formed by the movements of the EF switch A, B and C regions upon CaM binding. Here we report on the results of molecular dynamics (MD) simulations on two EF:CaM complexes, one containing wild-type EF and the other containing EF in which a cluster of residues in the switch A region (L523, K525, Q526 and V529) have been mutated to alanine. The switch A mutations cause a large increase in the flexibility of the switch C region, the rupture of a number of EF-CaM interactions, an expansion of the carboxyl-terminal domain of CaM, and a change in the Ca(2+) ion binding abilities of the CaM that is in complex with EF. The results indicate the importance of the mutated switch A residues in maintaining a compact EF:CaM complex that appears to be a prerequisite for the generation of a fully-functional adenylyl cyclase active site. The effects of mutating key residues (K346, K353, H577, E588, D590 and N639) in the active site region of EF (to alanine) on the ability of EF to bind the 3'-deoxy-ATP substrate analog were also examined. Active-site residue substitutions at positions 583 (N583A) and 577 (H577A) were found to be particularly disruptive for the placement of the adenine ring moiety into the position found in the x-ray crystal structure of the ligand-protein complex.     

A Similar Pattern of Interaction for Different Antibodies with a Major Antigenic Site of Foot-and-Mouth Disease Virus: Implications for Intratypic Antigenic Variation

The three-dimensional structures of the Fab fragment of a neutralizing antibody raised against a foot-and-mouth disease virus (FMDV) of serotype C₁, alone and complexed to an antigenic peptide representing the major antigenic site A (G-H loop of VP1), have been determined. As previously seen in a complex of the same antigen with another antibody which recognizes a different epitope within antigenic site A, the receptor recognition motif Arg-Gly-Asp and some residues from an adjacent helix participate directly in the interaction with the complementarity-determining regions of the antibody. Remarkably, the structures of the two antibodies become more similar upon binding the peptide, and both undergo considerable induced fit to accommodate the peptide with a similar array of interactions. Furthermore, the pattern of reactivities of five additional antibodies with versions of the antigenic peptide bearing amino acid replacements suggests a similar pattern of interaction of antibodies raised against widely different antigens of serotype C. The results reinforce the occurrence of a defined antigenic structure at this mobile, exposed antigenic site and imply that intratypic antigenic variation of FMDV of serotype C is due to subtle structural differences that affect antibody recognition while preserving a functional structure for the receptor 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.