Crystal structure of a 3B3 variant—A broadly neutralizing HIV‐1 scFv antibody

We present the crystal structure determination of an anti‐HIV‐1 gp120 single‐chain variable fragment antibody variant, 3B3, at 2.5 Å resolution. This 3B3 variant was derived from the b12 antibody, using phage display and site‐directed mutagenesis of the variable heavy chain (V_H) complementary‐determining regions (CDRs). 3B3 exhibits enhanced binding affinity and neutralization activity against several cross‐clade primary isolates of HIV‐1 by interaction with the recessed CD4‐binding site on the gp120 envelope protein. Comparison with the structures of the unbound and bound forms of b12, the 3B3 structure closely resembles these structures with minimal differences with two notable exceptions. First, there is a reorientation of the CDR‐H3 of the V_H domain where the primary sequences evolved from b12 to 3B3. The structural changes in CDR‐H3 of 3B3, in light of the b12‐gp120 complex structure, allow for positioning an additional Trp side chain in the binding interface with gp120. Finally, the second region of structural change involves two peptide bond flips in CDR‐L3 of the variable light (V_L) domain triggered by a point mutation in CDR‐H3 of Q100eY resulting in changes in the intramolecular hydrogen bonding patterning between the V_L and V_H domains. Thus, the enhanced binding affinities and neutralization capabilities of 3B3 relative to b12 probably result from higher hydrophobic driving potential by burying more aromatic residues at the 3B3‐gp120 interface and by indirect stabilization of intramolecular contacts of the core framework residues between the V_L and V_H domains possibly through more favorable entropic effect through the expulsion of water.     

Unraveling the effect of changes in conformation and compactness at the antibody VL‐VH interface upon antigen binding

We have analyzed conformational changes that occur at the interface between the light (V_L) and heavy (V_H) chains in antibody variable fragments upon binding to antigens. We wrote and applied the Tiny Probe program that computes the buried atomic contact surface area of three‐dimensional structures to evaluate changes in compactness of the V_L–V_H interface between bound and unbound antibodies. We found three categories of these changes, which correlated with the size of the antigen. Upon binding, medium‐sized nonprotein antigens cause an opening of the V_L–V_H interface (less compact), small antigens or haptens cause a closure of the interface (more compact), whereas large protein antigens have little effect on the compactness of the V_L–V_H interface. The largest changes in the atomic buried contact surface area at the V_L–V_H interface occur in residue pairs providing two ‘shock absorbers’ between the edge β‐strands of the V_L and V_H β‐sheets forming the antibody binding site. Importantly, the correlation between the size of antigens and conformational changes indicates that the V_L–V_H interface in antibodies plays a significant role in the antigen binding process. Furthermore, as the energy involved in such a motion is significant (up to 3 kcal/mol), these results provide a general mechanism for how residues distant from the combining site can significantly alter the affinity of an antibody for its antigen. Thus, mutations introduced at the V_L–V_H interface can be used to change antibody binding affinity with antigens. Due to the tightly packed V_L–V_H interface, the introduction of random mutations is not advisable. Rather our analysis suggests that concerted mutations of residues preceding CDRL2 and following CDRH3 or residues preceding CDRH2 and at the end of CDRL3 are most likely to alter or improve antigen binding affinity. Copyright © 1999 John Wiley & Sons, Ltd.     

Evaluation of a noncanonical Cys40‐Cys55 disulfide linkage for stabilization of single‐domain antibodies

Incorporation of noncanonical disulfide linkages into single‐domain antibodies (sdAbs) has been shown to enhance thermostability and other properties. Here, we evaluated the effects of introducing a novel disulfide linkage formed between Cys residues at IMGT positions 40 and 55 on the melting temperatures (T _ms), reversibility of thermal unfolding, solubility, and antigen‐binding affinities of three types of sdAbs (V_HH, V_H, and V_L domains). The Cys40‐Cys55 disulfide linkage was tolerated by 9/9 V_HHs, 12/12 V_Hs, and 2/11 V_Ls tested and its formation was confirmed by mass spectrometry. Using circular dichroism, we found that the Cys40‐Cys55 disulfide linkage increased sdAb T _m by an average of 10.0°C (range: 0–21.8°C). However, enhanced thermostability came at the cost of a partial loss of refolding ability upon thermal denaturation as well as, for some sdAbs, significantly decreased solubility and antigen‐binding affinity. Thus, Cys40/Cys55 can be added to the panel of known locations for introducing stabilizing noncanonical disulfide linkages into antibody variable domains, although its effects should be tested empirically for individual sdAbs.     

Energy-Based Analysis and Prediction of the Orientation between Light- and Heavy-Chain Antibody Variable Domains

Diversity in antibody structure is crucial to the ability of the adaptive immune system to recognize the tremendously diverse set of potential antigens. The diversity in structure is most apparent in the six hypervariable loops of the complementarity-determining regions. However, given that these loops occur at the interface of the heavy- and light-chain variable domains and form the antigen-binding site, the relative orientation of the heavy- and light-chain variable domains can create another source of structural diversity leading to changes in antigen binding. Here, we first reexamine the diversity of V_L:V_H orientations in existing antibody crystal structures using 153 nonredundant sequences, demonstrating that the variation in V_L:V_H orientation is greater than that expected from effects of crystal packing, antigen binding, or the presence of antibody constant regions and increases, on average, as sequence similarity decreases for residues in the interface between the domains. We developed a tool for predicting the relative orientations of the heavy- and light-chain variable domains using side-chain rotamer sampling in the interface and molecular-mechanics-based energy calculations. When using variable domain backbones from the crystal structures, the predicted orientation is very close (< 1 Å RMSD) to the crystallographically observed orientation in most cases, confirming that the V_L:V_H orientation is determined by the antibody sequence and suggesting an approach to predicting the relative orientation of the variable domains when building homology models of antibodies. When applied to antibody homology models generated from templates with 55-75% sequence identity, we predict the V_L:V_H orientation of 20 antibodies with an average/median RMSD of 2.1/1.6 Å to the crystal structures.     

Folding and aggregation of a multi-domain engineered immunotoxin

Engineered immunotoxins with specific targeting mechanisms have potential applications for the treatment of cancer and other diseases; however, their folding behavior is often poorly understood and this presents challenges during process development, manufacturing, and formulation. Folding thermodynamics of an antibody variable domain (V_H/V_L) genetically fused to a biological toxin payload were characterized at pH 6.0 and pH 8.0 in order to assess the relative domain stabilities, along with time scales on which they fold, and the competition between aggregation and folding. The toxin and V_H/V_L domains had considerably different unfolding free energies (ΔG_UNF), leading to a thermodynamically-distinct intermediate species, with the toxin domain unfolded and the V_H/V_L folded. The intermediate is the majority species over a range of denaturant concentrations (∼4–6 M urea; ∼2–4 M guanidine HCl). Thermal unfolding resulted in reversible unfolding of the toxin domain at pH 8, but at pH 6 thermal unfolding was convoluted with aggregation due to irreversible unfolding and aggregation for the V_H/V_L domain. Chemical unfolding of both domains was more easily reversible, provided that the refold was done stepwise, allowing the antibody domain to fold first at intermediate denaturant concentration, as folding of the V_H/V_L domain played a key role in aggregation of this antibody fusion protein.     

scFv-based fluorogen activating proteins and variable domain inhibitors as fluorescent biosensor platforms

Single chain antibodies (scFvs) are engineered proteins composed of IgG variable heavy (V_H) and variable light (V_L) domains tethered together by a flexible peptide linker. We have characterized the individual V_H or V_L domain activities of several scFvs isolated from a yeast surface-display library for their ability to bind environmentally sensitive fluorogenic dyes causing them to fluoresce. For many of the scFvs, both V_H and V_L domains are required for dye binding and fluorescence. The analysis of other scFvs, however, revealed that either the V_H or the V_L domain alone is sufficient to cause the fluorogenic dye activation. Furthermore, the inactive complementary domains in the original scFvs either contribute nothing to, or actually inhibit the activity of these active single domains. We have explored the interactions between active variable domains and inactive complementary domains by extensive variable domain swapping through in vitro gene manipulations to create hybrid scFvs. In this study, we demonstrate that significant alteration of the fluorogenic dye activation by the active V_H or V_L domains can occur by partnering with different V_H or V_L complementary domains in the scFv format. Hybrid scFvs can be generated that have fluorogen-activating domains that are completely inhibited by interactions with other domains. Such hybrid scFvs are excellent platforms for the development of several types of genetically encoded, fluorescence-generating biosensors.     

The intervening removable affinity tag (iRAT) production system facilitates Fv antibody fragment‐mediated crystallography

Fv antibody fragments have been used as co‐crystallization partners in structural biology, particularly in membrane protein crystallography. However, there are inherent technical issues associated with the large‐scale production of soluble, functional Fv fragments through conventional methods in various expression systems. To circumvent these problems, we developed a new method, in which a single synthetic polyprotein consisting of a variable light (V_L) domain, an intervening removable affinity tag (iRAT), and a variable heavy (V_H) domain is expressed by a Gram‐positive bacterial secretion system. This method ensures stoichiometric expression of V_L and V_H from the monocistronic construct followed by proper folding and assembly of the two variable domains. The iRAT segment can be removed by a site‐specific protease during the purification process to yield tag‐free Fv fragments suitable for crystallization trials. In vitro refolding step is not required to obtain correctly folded Fv fragments. As a proof of concept, we tested the iRAT‐based production of multiple Fv fragments, including a crystallization chaperone for a mammalian membrane protein as well as FDA‐approved therapeutic antibodies. The resulting Fv fragments were functionally active and crystallized in complex with the target proteins. The iRAT system is a reliable, rapid and broadly applicable means of producing milligram quantities of Fv fragments for structural and biochemical studies.     

A Residue-specific Shift in Stability and Amyloidogenicity of Antibody Variable Domains

Variable (V) domains of antibodies are essential for antigen recognition by our adaptive immune system. However, some variants of the light chain V domains (V_L) form pathogenic amyloid fibrils in patients. It is so far unclear which residues play a key role in governing these processes. Here, we show that the conserved residue 2 of V_L domains is crucial for controlling its thermodynamic stability and fibril formation. Hydrophobic side chains at position 2 stabilize the domain, whereas charged residues destabilize and lead to amyloid fibril formation. NMR experiments identified several segments within the core of the V_L domain to be affected by changes in residue 2. Furthermore, molecular dynamic simulations showed that hydrophobic side chains at position 2 remain buried in a hydrophobic pocket, and charged side chains show a high flexibility. This results in a predicted difference in the dissociation free energy of ∼10 kJ mol⁻¹, which is in excellent agreement with our experimental values. Interestingly, this switch point is found only in V_L domains of the κ family and not in V_Lλ or in V_H domains, despite a highly similar domain architecture. Our results reveal novel insight into the architecture of variable domains and the prerequisites for formation of amyloid fibrils. This might also contribute to the rational design of stable variable antibody domains.     

Bispecific antibodies with Fab-arms featuring exchanged antigen-binding constant domains

Monoclonal antibodies can acquire the property of engagement of a second antigen via fusion methods or modification of their CDR loops, but also by modification of their constant domains, such as in the mAb² format where a set of mutated amino acid residues in the C_H3 domains enables a high-affinity specific interaction with the second antigen. We tested the possibility of introducing multiple binding sites for the second antigen by replacing the Fab C_H1/C_L domain pair with a pair of antigen-binding C_H3 domains in a model scaffold with trastuzumab variable domains and VEGF-binding C_H3 domains. Such bispecific molecules were produced in a “Fab-like” format and in a full-length antibody format. Novel constructs were of expected molecular composition using mass spectrometry. They were expressed at a high level in standard laboratory conditions, purified as monomers with Protein A and gel filtration and were of high thermostability. Their high-affinity binding to both target antigens was retained. Finally, the Her2/VEGF binding domain-exchanged bispecific antibody was able to mediate a potentiated surface Her2-internalization effect on the Her2-overexpressing cell line SK-BR-3 due to improved level of cross-linking with the endogenously secreted cytokine. To conclude, bispecific antibodies with Fabs featuring exchanged antigen-binding C_H3 domains offer an alternative solution in positioning and valency of antigen binding sites.     

Phylogenetic analysis of condensation domains in NRPS sheds light on their functional evolution

Background  

Non-ribosomal peptide synthetases (NRPSs) are large multimodular enzymes that synthesize a wide range of biologically active natural peptide compounds, of which many are pharmacologically important. Peptide bond formation is catalyzed by the Condensation (C) domain. Various functional subtypes of the C domain exist: An^LC_L domain catalyzes a peptide bond between two L-amino acids, a^DC_L domain links an L-amino acid to a growing peptide ending with a D-amino acid, a Starter C domain (first denominated and classified as a separate subtype here) acylates the first amino acid with a β -hydroxy-carboxylic acid (typically a β -hydroxyl fatty acid), and Heterocyclization (Cyc) domains catalyze both peptide bond formation and subsequent cyclization of cysteine, serine or threonine residues. The homologous Epimerization (E) domain flips the chirality of the last amino acid in the growing peptide; Dual E/C domains catalyze both epimerization and condensation.     

The Antibody Light-Chain Linker Regulates Domain Orientation and Amyloidogenicity

The antibody light chain (LC) consists of two domains and is essential for antigen binding in mature immunoglobulins. The two domains are connected by a highly conserved linker that comprises the structurally important Arg108 residue. In antibody light chain (AL) amyloidosis, a severe protein amyloid disease, the LC and its N-terminal variable domain (V_L) convert to fibrils deposited in the tissues causing organ failure. Understanding the factors shaping the architecture of the LC is important for basic science, biotechnology and for deciphering the principles that lead to fibril formation. In this study, we examined the structure and properties of LC variants with a mutated or extended linker. We show that under destabilizing conditions, the linker modulates the amyloidogenicity of the LC. The fibril formation propensity of LC linker variants and their susceptibility to proteolysis directly correlate implying an interplay between the two LC domains. Using NMR and residual dipolar coupling-based simulations, we found that the linker residue Arg108 is a key factor regulating the relative orientation of the V_L and C_L domains, keeping them in a bent and dense, but still flexible conformation. Thus, inter-domain contacts and the relative orientation of V_L and C_L to each other are of major importance for maintaining the structural integrity of the full-length LC.     

ELPylated anti‐human TNF therapeutic single‐domain antibodies for prevention of lethal septic shock

Tumour necrosis factor (TNF) is a major pro‐inflammatory cytokine involved in multiple inflammatory diseases. The detrimental activity of TNF can be blocked by various antagonists, and commercial therapeutics based upon this principle have been approved for treatment of diseases including rheumatoid arthritis, Crohn’s disease and psoriasis. In a search for new, improved anti‐inflammatory therapeutics we have designed a single‐domain monoclonal antibody (V_HH), which recognizes TNF. The antibody component (TNF‐V_HH) is based upon an anti‐human TNF Camelidae heavy‐chain monoclonal antibody, which was linked to an elastin‐like polypeptide (ELP). We demonstrate that ELP fusion to the TNF‐V_HH enhances accumulation of the fusion protein during biomanufacturing in transgenic tobacco plants. With this study, we show for the first time that this plant‐derived anti‐human TNF‐V_HH antibody was biologically active in vivo. Therefore, therapeutic application of TNF‐V_HH‐ELP fusion protein was tested in humanized TNF mice and was shown to be effective in preventing death caused by septic shock. The in vivo persistence of the ELPylated antibody was ～24 fold longer than that of non‐ELPylated TNF‐V_HH.     

Antibody light chain variable domains and their biophysically improved versions for human immunotherapy

We set out to gain deeper insight into the potential of antibody light chain variable domains (V_Ls) as immunotherapeutics. To this end, we generated a naïve human V_L phage display library and, by using a method previously shown to select for non-aggregating antibody heavy chain variable domains (V_Hs), we isolated a diversity of V_L domains by panning the library against B cell super-antigen protein L. Eight domains representing different germline origins were shown to be non-aggregating at concentrations as high as 450 µM, indicating V_L repertoires are a rich source of non-aggregating domains. In addition, the V_Ls demonstrated high expression yields in E. coli, protein L binding and high reversibility of thermal unfolding. A side-by-side comparison with a set of non-aggregating human V_Hs revealed that the V_Ls had similar overall profiles with respect to melting temperature (T_m), reversibility of thermal unfolding and resistance to gastrointestinal proteases. Successful engineering of a non-canonical disulfide linkage in the core of V_Ls did not compromise the non-aggregation state or protein L binding properties. Furthermore, the introduced disulfide bond significantly increased their T_ms, by 5.5–17.5 °C, and pepsin resistance, although it somewhat reduced expression yields and subtly changed the structure of V_Ls. Human V_Ls and engineered versions may make suitable therapeutics due to their desirable biophysical features. The disulfide linkage-engineered V_Ls may be the preferred therapeutic format because of their higher stability, especially for oral therapy applications that necessitate high resistance to the stomach’s acidic pH and pepsin.     

Growth control of hybridoma cells with an artificially induced EpoR-gp130 heterodimer

IL-6 has been known to modulate the growth of many hybridoma cells and also promote resultant antibody productivity. However, IL-6 is so expensive that the use of IL-6-dependent hybridomas for industrial antibody production is not practical. In this study, we aimed at designing antibody/gp130 and antibody/EpoR chimeras which could tightly control cell growth in response to more affordable cognate antigen. Retroviral vectors encoding V_H or V_L region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 tethered to a pair of extracellular D2/transmembrane domains of erythropoietin receptor (EpoR) and cytoplasmic domains of either EpoR or gp130, were constructed, and a homodimeric or a heterodimeric pair of chimeric receptor combinations (V_H-gp130 and V_L-gp130 or V_H-gp130 and V_L-EpoR) were expressed in an IL-6-dependent hybridoma 7TD1. The chimeric receptor-derived growth signal was observed in both combinations, while some residual growth signal was observed in the absence of HEL. To reduce interchain interaction between the two receptor chains, we introduced mutations to the transmembrane domain of both chimera combinations. Consequently, the heterodimeric combination of V_H-gp130 and V_L-EpoR showed clear HEL-dependent cell growth, while the homodimeric combination of V_H-gp130 and V_L-gp130 showed reduced cell growth in the absence of HEL. This is the first report that an EpoR-gp130 cytoplasmic domain heterodimer could transduce a growth signal in hybridoma cells, indicating tight and economical growth control of hybridoma cells via our chimeric receptors.     

Noncollagenous region of the streptococcal collagen‐like protein is a trimerization domain that supports refolding of adjacent homologous and heterologous collagenous domains

Proper folding of the (Gly‐Xaa‐Yaa)_n sequence of animal collagens requires adjacent N‐ or C‐terminal noncollagenous trimerization domains which often contain coiled‐coil or beta sheet structure. Collagen‐like proteins have been found recently in a number of bacteria, but little is known about their folding mechanism. The Scl2 collagen‐like protein from Streptococcus pyogenes has an N‐terminal globular domain, designated V_sp, adjacent to its triple‐helix domain. The V_sp domain is required for proper refolding of the Scl2 protein in vitro. Here, recombinant V_sp domain alone is shown to form trimers with a significant α‐helix content and to have a thermal stability of T_m = 45°C. Examination of a new construct shows that the V_sp domain facilitates efficient in vitro refolding only when it is located N‐terminal to the triple‐helix domain but not when C‐terminal to the triple‐helix domain. Fusion of the V_sp domain N‐terminal to a heterologous (Gly‐Xaa‐Yaa)_n sequence from Clostridium perfringens led to correct folding and refolding of this triple‐helix, which was unable to fold into a triple‐helical, soluble protein on its own. These results suggest that placement of a functional trimerization module adjacent to a heterologous Gly‐Xaa‐Yaa repeating sequence can lead to proper folding in some cases but also shows specificity in the relative location of the trimerization and triple‐helix domains. This information about their modular nature can be used in the production of novel types of bacterial collagen for biomaterial applications.     

Mutational approaches to improve the biophysical properties of human single-domain antibodies

Monoclonal antibodies are a remarkably successful class of therapeutics used to treat a wide range of indications. There has been growing interest in smaller antibody fragments such as Fabs, scFvs and domain antibodies in recent years. In particular, the development of human V_H and V_L single-domain antibody therapeutics, as stand-alone affinity reagents or as “warheads” for larger molecules, are favored over other sources of antibodies due to their perceived lack of immunogenicity in humans. However, unlike camelid heavy-chain antibody variable domains (V_HHs) which almost unanimously resist aggregation and are highly stable, human V_Hs and V_Ls are prone to aggregation and exhibit poor solubility. Approaches to reduce V_H and V_L aggregation and increase solubility are therefore very active areas of research within the antibody engineering community. Here we extensively chronicle the various mutational approaches that have been applied to human V_Hs and V_Ls to improve their biophysical properties such as expression yield, thermal stability, reversible unfolding and aggregation resistance. In addition, we describe stages of the V_H and V_L development process where these mutations could best be implemented. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Characterization of neutralizing mouse‐human chimeric and shuffling antibodies against botulinum neurotoxin A

Mouse‐human chimeric monoclonal antibodies that could neutralize botulinum neurotoxins were developed and an attempt was made to establish mouse hybridoma cell clones that produced monoclonal antibodies that neutralized botulinum neurotoxin serotype A (BoNT/A). Four clones (2–4, 2–5, 9–4 and B1) were selected for chimerization on the basis of their neutralizing activity against BoNT/A and the cDNA of the variable regions of their heavy (V_H) and light chains (V_L) were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO‐DG44 cells were transfected with these plasmids and mouse‐human chimeric antibodies (AC24, AC25, AC94 and ACB1) purified to examine their binding and neutralizing activities. Each chimeric antibody exhibited almost the same capability as each parent mouse mAb to bind and neutralize activities against BoNT/A. From the chimeric antibodies against BoNT/A, shuffling chimeric antibodies designed with replacement of their V_H or V_L domains were constructed. A shuffling antibody (AC2494) that derived its V_H and V_L domains from chimeric antibodies AC24 and AC94, respectively, showed much higher neutralizing activity than did other shuffling antibodies and parent counterparts. This result indicates that it is possible to build high‐potency neutralizing chimeric antibodies by selecting and shuffling V_H and V_L domains from a variety of repertoires. A shuffling chimeric antibody might be the best candidate for replacing horse antitoxin for inducing passive immunotherapy against botulism.     

Generation of a phage‐display library of single‐domain camelid VHH antibodies directed against Chlamydomonas reinhardtii antigens,and characterization of VHHs binding cell‐surface antigens

Single‐domain antibodies (sdAbs) are powerful tools for the detection, quantification, purification and subcellular localization of proteins of interest in biological research. We have generated camelid (Lama pacos) heavy chain‐only variable V_H domain (V_HH) libraries against antigens in total cell lysates from Chlamydomonas reinhardtii. The sdAbs in the sera from immunized animals and V_HH antibody domains isolated from the library show specificity to C. reinhardtii and lack of reactivity to antigens from four other algae: Chlorella variabilis, Coccomyxa subellipsoidea, Nannochloropsis oceanica and Thalassiosira pseudonana. Antibodies were produced against a diverse representation of antigens as evidenced by sera ELISA and protein‐blot analyses. A phage‐display library consisting of the V_HH region contained at least 10⁶ individual transformants, and thus should represent a wide range of C. reinhardtii antigens. The utility of the phage library was demonstrated by using live C. reinhardtii cells to pan for V_HH clones with specific recognition of cell‐surface epitopes. The lead candidate V_HH clones (designated B11 and H10) bound to C. reinhardtii with EC₅₀ values ≤0.5 nm . Treatment of cells with V_HH B11 fused to the mCherry or green fluorescent proteins allowed brilliant and specific staining of the C. reinhardtii cell wall and analysis of cell‐wall genesis during cell division. Such high‐complexity V_HH antibody libraries for algae will be valuable tools for algal researchers and biotechnologists.     

Structure of Fab fragment of malaria transmission blocking antibody 2A8 against P. vivax P25 protein

Understanding the structural basis of recognition between antigen and antibody requires the structural comparison of free and complexed components. Previously, we have reported the crystal structure of the complex between Fab fragment of murine monoclonal antibody 2A8 (Fab2A8) and Plasmodium vivax P25 protein (Pvs25) at 3.2 Å resolution. We report here the crystallization and X-ray structure of native Fab2A8 at 4.0 Å resolution. The 2A8 antibody generated against Pvs25 prevents the formation of P. vivax oocysts in the mosquito, when assayed in membrane feeding experiment.Comparison of native Fab2A8 structure with antigen bound Fab2A8 structure indicates the significant conformational changes in CDR-H1 and CDR-H3 regions of V_H domain and CDR-L3 region of V_L domain of Fab2A8. Upon complex formation, the relative orientation between V_L and V_H domains of Fab2A8 is conserved, while significant differences are observed in elbow angles of heavy and light chains. The combing site residues of complexed Fab2A8 exhibited the reduced temperature factor compared to native Fab2A8, suggesting a loss of conformational entropy upon antigen binding.     

Malachite Green Mediates Homodimerization of Antibody VL Domains to Form a Fluorescent Ternary Complex with Singular Symmetric Interfaces

We report that a symmetric small-molecule ligand mediates the assembly of antibody light chain variable domains (V_Ls) into a correspondent symmetric ternary complex with novel interfaces. The L5* fluorogen activating protein is a V_L domain that binds malachite green (MG) dye to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two V_L domains. Unliganded L5* V_L domains crystallize as a similar antiparallel V_L/V_L homodimer. The complementarity-determining regions are spatially oriented to form novel V_L/V_L and V_L/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable V_L/MG/V_L complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two V_L domains into a single protein tightens MG binding over 1000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high-affinity protein tags and capture reagents.